Role of stromal cells and macrophages in fibronectin biosynthesis and matrix assembly in human long-term marrow cultures

Abstract

Fibronectin is a major component of the extracellular matrix of adherent layers of human long-term marrow cultures where it may stabilize the extracellular matrix network and provide adhesion sites for primitive hemopoietic cells. This study was devised to analyze the role of adherent cell populations in fibronectin synthesis, matrix assembly, and degradation. In cultures performed under the conditions described by Gartner and Kaplan, immunoprecipitation after metabolic labeling showed that adherent cells synthesized a fibronectin variant comprising the EDa domain and lacking the EDb one. Vascular smooth muscle-like stromal cells were the cell subset responsible for this synthesis. Once synthesized by stromal cells, EDa+fibronectin was secreted into the supernatant and incorporated into the extracellular matrix. The cumulation in the extracellular matrix was predominant by weeks 5 and 6 of culture, when a decrease in the stromal cell intracytoplasmic content of fibronectin was observed. Stromal cells from a transformed cell line, L2Ori-, were also able to synthesize the EDa+fibronectin variant, although for these cells the assembly into the extracellular matrix was partly impaired. Besides stromal cells, other cell types participated in fibronectin synthesis: early-adhering granulomonocytic cells and macrophages appearing later in culture were able to synthesize an EDa-, EDb- fibronectin variant, clearly distinct from the EDa+ variant produced by stromal cells. Studies on cultures in which macrophage growth was stimulated at the expense of stromal cells by adding granulocyte-macrophage colony-stimulating factor (50 ng/mL) to the culture medium showed a striking decrease in amounts of fibronectin measured in the adherent layer. This decrease was caused by a lack of incorporation of fibronectin in the extracellular matrix, disclosing a major difference between stromal cells and macrophages in terms of matrix assembly. This study confirms the similarity between stromal cells and vascular smooth muscle cells, because in vivo subendothelial intimal aortic smooth muscle cells and cultured smooth muscle cells from the aortic media express the EDa+, EDb- fibronectin variant. Furthermore, our results suggest that the level of fibronectin in adherent layers is regulated by stromal cells and macrophages. The balance between these two cell populations may therefore be crucial for the local control of hemopoiesis by regulating the extracellular fibronectin available for the adhesion of hematopoietic cells. Our data indicate that it may be essential to study the adhesion of stem cells to EDa+, EDb- fibronectin instead of EDa-, EDb- soluble fibronectin, as found in human plasma.

Cytokines active on granulomonopoiesis: release and consumption by human marrow myeloid stromal cells

“…The adherent layer of human LTBMC contains several cell types known to produce cytokines: macrophages, stromal cells (Singer et al, 1985) and some resident T lymphocytes (Berneman et al, 1989). However, the two major types are macrophages and stromal cells whose ratio was approximately 1:3 in our culture conditions (Lerat et al, 1993). Stromal cells are mesenchymal cells with vascular smooth muscle cell characteristics whose in vivo counterpart might be that of myeloid cells (Galmiche et al, 1993;Charbord et al, 1996).…”

“…Taken together, these data suggest that TGFb1 was synthesized by stromal cells from CDCL, although the low release rate might not reflect the intensity of synthesis. TGFb1 is known to bind to fibronectin (Hynes, 1990), an extracellular matrix glycoprotein heavily expressed by vascular smooth muscle-like stromal cells (Lerat et al, 1993;Li et al, 1995). Moreover, TGFb1 exists in a biologically inactive form not detected unless media or cell extracts are acidified (Pircher et al, 1986).…”

Glucocorticoids down‐regulate the extracellular matrix proteins fibronectin, fibulin‐1 and fibulin‐2 in bone marrow stroma

“…In bone marrow, extracellular matrix molecules are synthesised both by haematopoietic and stromal cells. Fibronectin is synthesised by ®broblasts (40), neutrophils (41), monocytes, macrophages and vascular smooth muscle like cells (42). Fibulin-1 is expressed in ®broblasts, smooth muscle cell lines, but not in endothelial cells, monocytes, or lymphoid or myeloid cell lines (43).…”

Human macrophages synthesize type VIII collagenin vitroand in the atherosclerotic plaque

“…E-mail: cullen@ uni-muenster.de 3 Abbreviations: APAAP, alkaline phosphatase, anti-alkaline phosphatase; CD, cluster of differentiation; DIG, digoxin; EDTA, ethylene diamine tetraacetic acid; ELF, enzyme-linked fluorescence; FACS, fluorescence-activated cell scanning; FITC, fluorescein isothiocyanate; GAPDH, glyceraldehyde-3phosphate dehydrogenase; IgG, immunoglobulin G; LPS, lipopolysaccharide; MMP, matrix metalloproteinase; PBS, phosphate-buffered saline; RPMI, Roswell Park Memorial Institute; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; SMC, smooth muscle cell; SSC, standard saline citrate; TGF, transforming growth factor; TRITC, tetramethyl rhodamine B isothiocyanate; UTP, uridyl triphosphate. ation of fibronectin that not only functions as a component of the extracellular matrix, but has also been implicated in phagocytosis ('opsonic protein') (13)(14)(15)(16). Macrophages have also been shown to produce proteoglycans (17) and components of the basement membrane such as laminin (18).…”