Neurotensin decreases the proinflammatory status of human skin fibroblasts and increases epidermal growth factor expression

Abstract

Fibroblasts colonization into injured areas during wound healing (WH) is responsible for skin remodelling and is also involved in the modulation of inflammation, as fibroblasts are immunologically active. Herein, we aimed to determine neurotensin effect on the immunomodulatory profile of fibroblasts, both in homeostatic and inflammatory conditions. Neurotensin mediated responses occurred through NTR1 or NTR3 receptors, while under inflammatory conditions NTR1 expression increase seemed to modulate neurotensin responses. Among different immunomodulatory genes, CCL11, IL-8, and IL-6 were the most expressed genes, while CCL4 and EGF were the less expressed genes. After neurotensin exposure, IL-8 mRNA expression was increased while CCL11 was decreased, suggesting a proinflammatory upregulation and chemoattractant ability downregulation of fibroblasts. Under inflammatory conditions, gene expression was significantly increased. After neurotensin exposure, CCL4 and IL-6 mRNA expression were decreased while CCL11 was increased, suggesting again a decrease in the chemoattractant capacity of fibroblasts and in their proinflammatory status. Furthermore, the expression of EGF, a crucial growth factor for skin cells proliferation and WH, was increased in all conditions. Overall, neurotensin, released by nerve fibers or skin cells, may be involved in the decrease of the chemotaxis and the proinflammatory status in the proliferation and remodelling phases of WH.

Figures

Figure 1

Neurotensin receptor mRNA and protein expression. Cells were maintained in medium (A, B) or treated with 1 μg/mL of LPS for 6 h (C), at 37°C, with 5% CO2. Total RNA was isolated and retrotranscribed as indicated in Section 2. The mRNA levels were assessed by quantitative real-time RT-PCR. Gene expression is indicated as genes studied/10,000 molecules of the reference gene HPRT1 (A) or mean log2 values of fold changes relative to the control (C). Each value represents the mean ± SD from three independent experiments (*P < 0.05; Kruskall-Wallis test followed by Dunn's multiple comparison posttest). Cells extracts from untreated BJ cells (BJ) and from the murine cortical cortex (CC) were subjected to Western blot analysis (B) using NTR1, NTR2, and NTR3 antibodies, with normalization to β-actin. The blot shown is representative of 3 independent experiments yielding similar results.

Figure 2

Neurotensin receptor localization. Cells were subjected to immunocytochemistry analysis as described in Section 2 using NTR1, NTR2, and NTR3 antibodies.

Figure 3

Expression of cytokines, chemokines, and growth factors in BJ cells, under homeostatic and inflammatory conditions. Cells were plated at 8 × 105 cells/dish in 60 mm dishes in a final volume of 6 mL of medium and treated with 1 μg of LPS during 6 h (LPS), or left untreated (Ctrl), at 37°C, with 5% CO2. Total RNA was isolated and retrotranscribed as indicated in Section 2. The mRNA levels were assessed by quantitative real-time RT-PCR. Gene expression is indicated as genes studied/10 000 molecules of the reference gene HPRT1. Values represent the mean ± SD from four independent experiments. Kruskall-Wallis test followed by Dunn's posttest statistical analysis was performed between cytokines, chemokines, and growth factor expression under homeostatic and inflammatory conditions (**P < 0.01; ***P < 0.001) and among cytokines, chemokines, and growth factor expression under homeostatic conditions (## < 0.01; ### < 0.001). IL: interleukin; EGF: epidermal growth factor.

Figure 4

Modulation of gene expression by neurotensin under homeostatic and inflammatory conditions in BJ cells. Cells were maintained in culture medium (Ctrl) and treated with 10 nM of NT (white bars) or 100 nM of NT (black bars) during 30 h (a) or pretreated with 10 nM (white bars) and 100 nM (black bars) of NT during 24 h and stimulated with 1 μg of LPS during 6 h (b), at 37°C, with 5% CO2. Total RNA was isolated and retrotranscribed as indicated in Section 2. The mRNA levels were assessed by quantitative real-time RT-PCR. Gene expression is indicated as mean log2 values of fold changes relative to the control. Each value represents the mean ± SD from four independent experiments (*P < 0.05; Kruskall-Wallis test followed by Dunn's posttest statistical analysis between 10 and 100 nM of NT, 10, and 100 nM of NT with the corresponding control, under homeostatic and inflammatory conditions).