IL-33 augments substance P-induced VEGF secretion from human mast cells and is increased in psoriatic skin

Abstract

The peptide substance P (SP) has been implicated in inflammatory conditions, such as psoriasis, where mast cells and VEGF are increased. A relationship between SP and VEGF has not been well studied, nor has any interaction with the proinflammatory cytokines, especially IL-33. Here we report that SP (0.1-10 microM) induces gene expression and secretion of VEGF from human LAD2 mast cells and human umbilical core blood-derived cultured mast cells (hCBMCs). This effect is significantly increased by coadministration of IL-33 (5-100 ng/mL) in both cell types. The effect of SP on VEGF release is inhibited by treatment with the NK-1 receptor antagonist 733,060. SP rapidly increases cytosolic calcium, and so does IL-33 to a smaller extent; the addition of IL-33 augments the calcium increase. SP-induced VEGF production involves calcium-dependent PKC isoforms, as well as the ERK and JNK MAPKs. Gene expression of IL-33 and histidine decarboxylase (HDC), an indicator of mast cell presence/activation, is significantly increased in affected and unaffected (at least 15 cm away from the lesion) psoriatic skin, as compared with normal control skin. Immunohistochemistry indicates that IL-33 is associated with endothelial cells in both the unaffected and affected sites, but is stronger and also associated with immune cells in the affected site. These results imply that functional interactions among SP, IL-33, and mast cells leading to VEGF release contribute to inflammatory conditions, such as the psoriasis, a nonallergic hyperproliferative skin inflammatory disorder with a neurogenic component.

Fig. 1.

SP stimulates VEGF production in human mast cells. LAD2 cells (A) and hCBMCs (B) were stimulated with the indicated concentration of SP (0–10 μM) for 24 h, and supernatant VEGF was measured by ELISA. (C) Time-dependent secretion of VEGF. Cells were stimulated with SP (1 μM) or vehicle for the indicated times and supernatant VEGF was measured by ELISA. (D) SP induces VEGF mRNA. LAD2 cells were stimulated with SP (1 μM) for the indicated times, RNA was extracted, and relative VEGF mRNA levels were determined by real-time PCR. Data are mean ± SD of three separate experiments performed in triplicate (*P < 0.05 vs. unstimulated cells).

Fig. 2.

IL-33 augments SP in inducing (A) VEGF protein secretion from LAD2 cells, and (B) VEGF mRNA expression from LAD2 cells, or (C) VEGF protein secretion from hCBMCs. Cells were treated for 6 h with IL-33 (100 ng/mL) or IL-1 (10 ng/mL) alone or together with SP as shown (n = 3). *P < 0.05.

Fig. 3.

NK-1 receptor antagonist inhibits SP-induced VEGF release from LAD2 cells. LAD2 cells were pretreated with NK-1 receptor antagonist (NK1RAntag) L-733,060 (10 μM) for 30 min and were then retained throughout stimulation with SP (1 μM) for 24 h. VEGF was measured in the supernatant fluid by ELISA (n = 3). *P < 0.05.

Fig. 4.

Effect of SP and IL-33 on LAD2 cytosolic calcium levels. Cytosolic calcium was measured in LAD2 cells using Fura-2 AM (1 mM; Invitrogen). Cells were stimulated with IL-33 (100 ng/mL) or SP (1 μM) or both for the time indicated. Results were processed according to the Invitrogen Fura-2 protocol. Stimulation was carried out in the presence of extracellular calcium (1 mM). Results of one experiment, representative of three equivalent experiments, are shown.

Fig. 5.

Increased gene expression in psoriatic affected (lesional) skin, psoriatic unaffected (at least 15 cm away from the lesion) skin, and normal skin from healthy controls. (A) IL-33 (control n = 7; unaffected, n = 6; affected n = 9); (B) HDC (control n = 5; unaffected n = 6; affected n = 5); and (C) TACI. Relative quantities of mRNA expression were measured by quantitative RT-PCR and normalized to 18S. TaqMan was performed with cDNA reverse transcribed from 100 ng RNA from each sample. The number of samples was less for HDC because the amount of cDNA from some samples had been exhausted (*P < 0.05 vs. control).

Fig. 6.

Photomicrographs of skin biopsy samples from patients with psoriasis (A and B) lesional, affected skin; (C and D) unaffected skin; and (E and F) control without primary antibody. Immunohistochemical staining was performed using the LSAB+ system kit (DAKO). Incubation with the primary antibody (mouse monoclonal anti–human-IL-33 antibody, at 1:100 dilution; Abcam) was performed for 30 min; secondary antibody was provided in the DAKO kit and was also used for 30 min, followed by appropriate washes. Magnification, ×200; Red rectangle indicates blood vessel; asterisk indicates sweat gland; solid arrow indicates inflammatory cells.