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

Abstract

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.

Figures

Figure 1

Confocal photomicrographic analysis of trkA and p75 receptors. The basal expression of trkA by lung and skin fibroblasts (green) is shown in A and C, respectively. When cultured in the presence of 100 ng/ml NGF for 6 consecutive days, both lung and skin fibroblasts also expressed p75 (green, B and D, respectively). Red in the fibroblast nuclei is propidium iodide staining. Fibroblast monolayers incubated with nonspecific purified immunoglobulins (IgG) did not display positive staining. The experiment depicted is a representative one of three. (×40.)

Figure 2

Lung and skin fibroblast migration across a wound line as a function of NGF concentration. The effect of 50 ng/ml NGF on wounded lung and skin fibroblasts after 1 day is shown in A and B, respectively. (×5.) The experiment depicted is a representative one of three. (Bottom) Quantitative evaluation of fibroblast migration beyond the wound line (*, P < 0.05 for lung fibroblasts and **, P < 0.05 for skin fibroblasts, both at 50 and 100 ng/ml NGF). Experiments (n = 3) were performed in triplicates; error bars indicate SEM.

Figure 3

Effect of NGF on α-SMA expression in lung and skin fibroblasts. Fibroblasts were cultured for 6 days with NGF or TGF-β1, and α-SMA expression by fibroblasts was evaluated by a cell surface ELISA. A and B show the effect of 100 ng/ml NGF and C and D, that of 10 ng/ml TGF-β1 on lung and skin fibroblasts, respectively. The experiment depicted is a representative one of three. (Bottom) Quantitative evaluation of α-SMA expression. α-SMA expression was found increased in both lung and skin fibroblasts (*, P < 0.05 for lung fibroblasts at 50 and 200 ng/ml NGF; **, P < 0.05 for skin fibroblasts at 50, 100, and 200 ng/ml NGF, and both for TGF-β1). Experiments (n = 3) were performed in triplicates; error bars indicate SEM.

Figure 4

Immunohistochemical analysis of NGF, trkA, and p75 in skin scar tissue (A, C, and E) and fibrotic interstitial lung disease (B, D, and F) human biopsies. NGF reactivity occurs in skin tissue (A) and lung tissue (B). The heterogeneous staining indicates that NGF reactivity is localized in structurally different cells. trkA- (C and D) and p75- (E and F, indicated with arrows) positive cells are found in skin and lung tissues. (×40.)