Mast cells enhance contraction of three-dimensional collagen lattices by fibroblasts by cell-cell interaction: role of stem cell factor/c-kit

Abstract

Reorganization of the extracellular matrix is important in many biological and pathophysiological processes, including tissue remodelling, wound healing, or cancer metastasis. The ability of cultured fibroblasts to reorganize and contract three-dimensional type I collagen gels is regarded as an in vitro model for this process. In tissue fibrosis, complex interactions among fibroblasts, inflammatory cells and the extracellular matrix are taking place. Mast cells have often been discussed to play a role in several fibrotic conditions including scleroderma, scar formation, or wound healing. In this study, we examined the effects of mast cells on contraction of collagen lattices. The results demonstrate that co-culture of dermal fibroblasts with a human mast cell line (HMC-1) significantly enhanced contraction of the three-dimensional collagen lattices, whereas mast cells alone failed to contract the gel. Addition of culture supernatants of mast cells did not enhance the speed of gel contraction, indicating the importance of cell-cell contact. Morphological analysis showed that mast cells were incorporated into the lattices. Histological examination also demonstrated that within the lattices, mast cells were localized in close contact to, or attached to, fibroblasts. As fibroblasts and mast cells are known to attach via stem cell factor (SCF)/c-kit interaction when co-cultured in monolayers, we also examined the effect of antibodies against SCF and c-kit in this system. Addition of both antibodies inhibited gel contraction up to 70%. In contrast, antibodies against interleukin-4 (IL-4) and IL-4 receptor did not affect gel contraction. These results indicate that mast cells enhance fibroblast-mediated contraction of collagen lattices via direct cell-cell contact, mediated in part by SCF/c-kit interactions.

Figure 1

Kinetics of collagen lattice contraction by human skin fibroblasts, co-cultured with or without human mast cells (HMC-1). Lattices contained 5 × 10⁶ fibroblasts (F) and mast cells at 1 : 1 (F/M 1 : 1) or in fivefold excess (F/M 1 : 5). Cultures were photographed at 6 hr after culture initiation: (a) fibroblasts alone; (b) fibroblasts co-cultured with mast cells at a 1 : 1 ratio; and (c) at a 1 : 5 ratio. Contraction of type I collagen matrices by fibroblasts was assessed by measuring gel diameters (graph). *P < 0·01, **P < 0·005, compared with fibroblasts alone. Representative data of the five independent experiments are shown.

Figure 2

Morphology of fibroblasts and mast cells, co-cultured in three-dimensional lattices by phase contrast microscopy (a–c): (a) fibroblasts alone, (b) fibroblasts and mast cells at a 1 : 1 ratio, (c) fibroblasts and mast cells at a 1 : 5 ratio. Mast cells are indicated by arrows. Histopathological analysis of three-dimensional lattices 24 hr after the gel casting (d–f): H & E stain of fibroblasts alone (d), and fibroblasts and mast cell (m) co-cultures (1 : 1 ratio) (e), (f) toluidine blue stain of fibroblasts (f) and mast cell co-cultures (1 : 5 ratio). Magnifications: a–c, ×40; d, ×400; e,f, ×200.

Figure 3

Inhibitory effect of antibodies against SCF and c-kit on collagen lattice gel contraction. Fibroblasts and mast cells were seeded at a 1 : 5 ratio. Antibodies (Ab) against SCF (0·1–10 µg/ml) and c-kit (0·05–1 µg/ml) were added singly or in combination prior to gel casting. As gel contraction was maximally enhanced at 4–6 hr after gel casting, gel diameters were determined at 6 hr and compared with control (fibroblasts only). Inhibition of gel contraction was assessed by the comparison of the alteration of gel diameter between those with and without antibodies.