Inhibition of keratinocyte growth in cell culture and whole skin culture by mast cell mediators

Abstract

Mast cells are suggested to participate in regenerative processes, but their influence on epithelialization and wound healing has not been well studied. Since mast cells can be found in contact with epidermis in chronic inflammatory skin diseases and venous ulcers, the effect of mast cells on keratinocyte growth was studied. Keratinocytes were cultured in serum-free conditions with (complete medium) or without (basal medium) epidermal growth factor (EGF) and bovine pituitary extract (BPE) to reach subconfluence in a 24-well plate, and the cells were treated with different mast cell mediators histamine, heparin and tryptase, or lysate from HMC-1 cells, a human leukemic mast cell line. Whole skin cultures were used as a model for in vitro wounds to study the effect of mast cells on epithelial outgrowth from skin specimens. Histamine inhibited 3H-thymidine incorporation of keratinocytes dose-dependently by 29% at 1 mM, and 89% at 5 mM histamine. In whole skin culture, histamine inhibited epithelial outgrowth dose-dependently by 64% already at 0.1 mM histamine and maximally (91%) at 1 mM histamine. Heparin inhibited 3H-thymidine incorporation dose-dependently by up to 33% at 2 microg/ml in the absence, but not in the presence, of EGF/BPE. In contrast, in whole skin culture, heparin first inhibited the epithelial outgrowth by up to 27% at 2 microg/ml, but then reversed the inhibition to 30% stimulation at 200 microg/ml. Skin tryptase (0.0285 to 2.85 microg/ml) with or without heparin (0.5 to 20 microg/ml) did not affect thymidine incorporation in keratinocytes. Lysate from HMC-1 cells, but not that from control, neuroblastoma cells, inhibited 3H-thymidine incorporation in keratinocytes dose-dependently, and maximal (47%) inhibition was reached with 16,700 lysed HMC-1 cells/ml. In whole skin culture, HMC-1 lysate inhibited the epithelial outgrowth by up to 36% at 67,000 lysed cells/ml. The results show that mast cells and their mediators are inhibitory to keratinocyte 3H-thymidine incorporation and epithelial outgrowth in vitro, although, the inhibitory effect of histamine was seen at high concentrations suggesting a requirement for close morphologic vicinity of mast cells to keratinocytes. Thus, mast cells are assumed to control epidermal regeneration and to impair epithelialization of chronic ulcers.

Heparin modulates the growth and adherence and augments the growth‐inhibitory action of TNF‐α on cultured human keratinocytes

“…However, in the presence of complete Keratinocyte-SFM heparin first inhibited 3 H-thymidine incorporation slightly, by up to 12%, but the inhibitory effect was reversed to baseline at a higher (200 mg/ml) heparin concentration [Huttunen et al, 2001]. Similarly, in the present study, heparin reduced keratinocyte growth and DNA amount in a dose-dependent manner in incomplete Keratinocyte-SFM.…”

“…Proliferating keratinocytes were cultured from human foreskin specimens using Keratinocyte-SFM serum-free medium supplemented with epidermal growth factor (EGF), bovine pituitary extract (BPE), 100 U/ml penicillin, and 100 mg/ml streptomycin as described in detail previously Huttunen et al, 2001]. In every experiment, third to eight passage keratinocytes with a viability of over 90% were used.…”

“…The growth area of keratinocytes was measured with digital image analysis as described [Huttunen et al, 2001]. The growth area was measured in triplicate for each well and averaged.…”

“…Previously, preformed mast cell mediators, such as histamine, heparin, and TNF-a, have been found to inhibit keratinocyte growth in vitro [Pillai et al, 1989[Pillai et al, , 1994Symington, 1989;Piepkorn et al, 1994;Huttunen et al, 2001] suggesting that mast cells could control epidermal growth. TNF-a can inhibit the proliferation of cultured human keratinocytes by inducing differentiation [Pillai et al, 1989] or cytostasis [Symington, 1989].…”

Pathophysiological Role of Skin Mast Cells in Wound Healing after Scald Injury: Study with Mast Cell-Deficient W/W^V Mice

“…Previous studies showed that mast cell-derived factors, such as histamine, heparin and TNF-α, inhibited keratinocyte growth in vitro [15,16,17]. Furthermore, increased numbers of mast cells have been reported near the epithelial margin of chronic nonhealing leg ulcers, and chymase derived from mast cells impaired keratinocyte migration and adherence [18].…”