3T3 fibroblasts induce cloned interleukin 3-dependent mouse mast cells to resemble connective tissue mast cells in granular constituency.

Abstract

As assessed by ultrastructure, histochemical staining, and T-cell dependency, in vitro-differentiated interleukin 3-dependent mouse mast cells are comparable to the mast cells that reside in the gastrointestinal mucosa but not in the skin or the serosal cavity of the mouse. We now demonstrate that when cloned interleukin 3-dependent mast cells are cocultured with mouse skin-derived 3T3 fibroblasts in the presence of WEHI-3 conditioned medium for 28 days, the mast cells acquire the ability to stain with safranin, increase their histamine content :50-fold and their carboxypeptidase A content =lOO-fold, and augment =45-fold their biosynthesis of proteoglycans bearing 35S-labeled heparin relative to 35S-labeled chondroitin sulfate glycosaminoglycans. Thus, fibroblasts induce interleukin 3-dependent mouse mast cells to change phenotype from mucosal-like to connective tissue-like, indicating that the biochemical and functional characteristics of this mast cell type are strongly influenced by the connective tissue microenvironment.Cloned and noncloned mouse mast cells have been obtained by culturing progenitors from bone marrow, spleen, lymph node, or fetal liver in conditioned medium that contains interleukin 3 (IL-3) (1-6). The IL-3-dependent mast cells obtained in vitro from these different tissue sources are characterized by failure to stain with safranin, preferential synthesis of 35S-labeled chondroitin sulfate E proteoglycans (7,8), low content of histamine (1-6), and low content of carboxypeptidase A (see below).The connective tissue mast cell, represented by the mouse or rat serosal mast cell, is safranin+, preferentially synthesizes 35S-labeled heparin proteoglycans (7, 9), has high levels of granular histamine (10) and carboxypeptidase A (11), and can be maintained ex vivo by coculture with mouse 3T3 fibroblasts (12). Recently we demonstrated that noncloned, IL-3-dependent bone marrow-derived mast cells cocultured with fibroblasts for 7-14 days changed their histochemical properties from safranin-to safranin+, increased their histamine content =15-fold, and augmented their rate of synthesis of 35S-labeled heparin proteoglycans (13). Because these mast cells were not cloned, whether fibroblasts selectively promoted expansion of a minor subpopulation of safranin+ mast cells or altered the phenotype of the predominant population of cells could not be decided. By using cloned mast cells, we establish in the present study that coculture with fibroblasts induces changes in the entire population of safranin-mast cells. Phenotypic changes were seen in all three major classes of constituents of the secretory granule-namely, histamine, proteoglycan, and neutral protease. MATERIALS AND METHODSCulture of Mast Cells with and without Fibroblasts. Spleen cells (2 x 105) from 10-to 15-wk-old female BDF1 mice (Simonsen Laboratories, Gilroy, CA) were suspended in 1 ml of alpha-modified medium (Flow Laboratories, Rockville, MD) containing 30% fetal calf serum (Flow Laboratories), 1% deionized bovine serum albumin (Calb...

Serglycin Is Essential for Maturation of Mast Cell Secretory Granule

“…In contrast to the mature connective tissue-type MCs in the peritoneum and ear tissue, for example, MCs derived by in vitro differentiation of bone marrow stem cells predominantly express GAG chains of CS type, although it has been reported that SCF stimulation of BMMCs can induce an increase in heparin synthesis (42). Consequently, the inactivation of SG is expected to have a much more profound effect on BMMCs than what was caused by the inactivation of NDST-2, because the lack of SG should affect both CS and heparin biosynthesis.…”

“…BMMCs are readily generated by culturing bone marrow stem cells in the presence of IL-3 and can be induced to mature further by addition of SCF (41)(42)(43). Bone marrow cells were prepared from SG ϩ/ϩ and SG ϩ/Ϫ animals as well as from littermate SG Ϫ/Ϫ animals and were cultured together with WEHI-3B-conditioned medium as a source of IL-3.…”

Synovial Fibroblasts Promote the Expression and Granule Accumulation of Tryptase via Interleukin-33 and Its Receptor ST-2 (IL1RL1)

“…During co-culture with fibroblasts, immature mBMMCs develop a histochemical phenotype that is more similar to that of the mature MCs in the arthritic joint, due in part to increased expression of heparin-containing serglycin proteoglycans (30). The co-cultured mBMMCs also undergo granule maturation as evidenced by their marked increased granule accumulation of histamine and the exopeptidase carboxypeptidase A3 (32,33). In support of these data, the cell granules become electrondense at the ultrastructural level (34).…”

Histamine synthesis is required for granule maturation in murine mast cells

“…Maturation of BMMCs were evaluated by the surface expression levels of FcεRI and c-kit and by granule maturation based on the acidic Toluidine blue staining. A CTMC culture model was originally established through coculture of BMMCs with Swiss 3T3 fibroblasts [33,34]. We modified this culture method and obtained CTMC-like cultured mast cells using mitomycin C-treated Swiss 3T3 fibroblasts in the presence of Kitl (30 ng/mL) as previously described [12].…”