Human mast cells stimulate fibroblast proliferation, collagen synthesis and lattice contraction: a direct role for mast cells in skin fibrosis.

BACKGROUND: Mast cells, the key cells of immediate hypersensitivity type reactions, have also been postulated to have a central role in influencing tissue remodelling and fibrosis occurring in the skin. OBJECTIVE: Our aim was to investigate the direct role of human mast cells (HMC) in skin fibrotic processes, by assessing the effects of the addition of the human mast cell line HMC-1 to human skin fibroblasts, and to identify the responsible mediators. METHODS: HMC-1 sonicates were added to human skin fibroblasts and the following parameters were evaluated: proliferation ([3H]-thymidine), collagen synthesis ([3H] proline), activity of matrix metalloproteinases (MMPs) (zymography) and tissue inhibitors of metalloproteinases (TIMPs) (reverse zymography), and collagen gel contraction. RESULTS: HMC-1 sonicate increased significantly both proliferation and collagen production in the human skin fibroblasts and these properties were not affected by heating of the sonicate (56 degrees C, 30 min, or 100 degrees C, 3 min). Two main mast cell mediators, histamine and tryptase, were found to be responsible for the increase in fibroblast proliferation and collagen production. HMC-1 sonicate did not display any pre-formed gelatinase activity, and its addition to the fibroblasts did not change their pro-MMP-2 and MMP-2 activity. On the other hand, HMC-1 were found to possess TIMP-1 and TIMP-2. Addition of HMC-1 had no effect on fibroblasts TIMP-1 but induced a dose-dependent increase of TIMP-2 activity. In addition, HMC-1 sonicate seeded together with the fibroblasts in tri-dimensional collagen gel significantly enhanced their contraction. CONCLUSION: We have shown that human mast cells, by granule-stored and therefore quickly releasable mediators, increase human skin fibroblast proliferation, collagen synthesis, TIMP-2 and collagen gel contraction. Therefore, mast cells have a direct and potentiating role in skin remodelling and fibrosis.

Human eosinophils induce histamine release from antigen-activated rat peritoneal mast cells: a possible role for mast cells in late-phase allergic reactions.

BACKGROUND: Mast cells and eosinophils are believed to interact during the late and the chronic stages of allergic inflammation. OBJECTIVE: In this study we investigated whether eosinophils can cause activation and consequent histamine release of already challenged mast cells, a situation likely to take place during the allergic late-phase reaction. METHODS: Rat peritoneal mast cells presensitized with IgE anti-dinitrophenol-human serum albumin and challenged by dinitrophenol-human serum albumin or compound 48/80 were incubated with either eosinophil sonicate or major basic protein (MBP). Eosinophils were purified from the peripheral (>98%) blood of mildly allergic patients. Heparin and pertussis toxin and different extracellular Ca(2+) concentrations were used to modulate mast cell reactivation by MBP. Histamine release was assessed as a marker of mast cell activation. RESULTS: IgE-challenged mast cells were sensitive to reactivation induced by eosinophil sonicate and MBP. Reactivation was not cytotoxic for the mast cells. Mast cells previously challenged with compound 48/80 did not respond to subsequent MBP activation. Furthermore, heparin and pertussis toxin both inhibited mast cell reactivation induced by MBP. The ability of eosinophil sonicate and MBP to activate mast cells was not significantly affected at the different Ca(2+) concentrations. CONCLUSIONS: In summary, we have shown a direct activating activity of eosinophils, partially due to MBP, toward IgE-challenged and immunologically desensitized mast cells. This suggests that in vivo mast cells can be reactivated during a late-phase reaction to release histamine by a non-IgE-dependent mechanism.

Nerve growth factor displays stimulatory effects on human skin and lung fibroblasts, demonstrating a direct role for this factor in tissue repair.

Nerve growth factor (NGF) is a polypeptide which, in addition to its effect on nerve cells, is believed to play a role in inflammatory responses and in tissue repair. Because fibroblasts represent the main target and effector cells in these processes, to investigate whether NGF is involved in lung and skin tissue repair, we studied the effect of NGF on fibroblast migration, proliferation, collagen metabolism, modulation into myofibroblasts, and contraction of collagen gel. Both skin and lung fibroblasts were found to produce NGF and to express tyrosine kinase receptor (trkA) under basal conditions, whereas the low-affinity p75 receptor was expressed only after prolonged NGF exposure. NGF significantly induced skin and lung fibroblast migration in an in vitro model of wounded fibroblast and skin migration in Boyden chambers. Nevertheless NGF did not influence either skin or lung fibroblast proliferation, collagen production, or metalloproteinase production or activation. In contrast, culture of both lung and skin fibroblasts with NGF modulated their phenotype into myofibroblasts. Moreover, addition of NGF to both fibroblast types embedded in collagen gel increased their contraction. Fibrotic human lung or skin tissues displayed immunoreactivity for NGF, trkA, and p75. These data show a direct pro-fibrogenic effect of NGF on skin and lung fibroblasts and therefore indicate a role for NGF in tissue repair and fibrosis.

Activation and inhibition of mast cells degranulation affect their morphometric parameters.

Activation of mast cells, the key cells of allergic inflammation, causes typical morphological changes associated with an increase in volume, that is a function of area and perimeter. The purpose of this study was to evaluate the effect of mast cell activation to degranulate, carried out by the secretagogue Compound 48/80, and of inhibition of this activation carried out by Nedocromil sodium, a mast cell stabilizing drug, on mast cell area, perimeter and shape factor by a computerized image analyzer. Mast cells were isolated and purified by peritoneal lavage of rats (purity >98%) and co-cultured with mouse 3T3 fibroblasts to which they adhere. Cultures were incubated for 10 min at 37 degrees C with culture medium alone (Enriched Medium) or Enriched Medium containing either Nedocromil (10(-4) M) or Compound 48/80 (0.3 microg/ml) or Compound 48/80 and Nedocromil (0.3 microg/ml and 10(-4) M respectively). Supernatants were then assessed for histamine release, as a marker of mast cell activation and the cell monolayers were fixed and stained with an alcoholic-acidic toluidine blue solution and examined with a computerized image analyzer connected with a light microscope. Mast cells incubated in Enriched Medium or Nedocromil possessed similar morphometric parameters. Mast cells activated with Compound 48/80 (70% histamine release) had a significant increase in area and perimeter and a decrease in shape factor in comparison to mast cells in Enriched Medium alone. Simultaneous incubation of mast cells with Compound 48/80 and Nedocromil significantly inhibited their histamine release (36% histamine release) and the increase in area and perimeter, but did not affect significantly their shape factor, in comparison with mast cells incubated with Compound 48/80 alone. These data clearly show that there is a relationship between mast cell activation, consequent histamine release and changes in cell area, perimeter and shape factor and that Nedocromil not only inhibits mast cell histamine release but also the activation induced morphometric changes in mast cells.