STAT-6-mediated control of P-selectin by substance P and interleukin-4 in human dermal endothelial cells

Abstract

P-Selectin expressed on endothelial cells contributes to acute and chronic inflammation by promoting leukocyte tethering/rolling. Despite increasing evidence of P-selectin expression on human umbilical vein endothelial cells in vitro, the regulatory mechanisms of P-selectin expression on dermal endothelial cells in skin diseases are not fully understood. Here, we demonstrate increased expression of P-selectin in dermal vessels of regional skin in urticaria and atopic dermatitis. The present in vitro analyses with human dermal microvascular endothelial cells (HDMECs) revealed that histamine rapidly induced P-selectin expression. Interleukin (IL)-4 and IL-13 induced prolonged expression of surface P-selectin by HDMECs. A combination of tumor necrosis factor-alpha and IL-4 inhibited P-selectin expression. Pretreatment of HDMECs with tumor necrosis factor-alpha followed by incubation with IL-4 markedly increased P-selectin expression. Notably, incubation with substance P alone induced prolonged P-selectin expression. Activation of STAT6 appears to be a key factor in P-selectin expression induced by substance P and IL-4 because treatment with STAT6 decoy oligodeoxynucleotides significantly inhibited P-selectin expression. The present results indicate that novel, complex mechanisms are involved in endothelial P-selectin expression in the skin. STAT6 in dermal endothelial cells appears to be a potent target for controlling cellular infiltrate in allergic and/or neuroinflammatory skin diseases.

Figure 1-6931

Location of STAT6 and NF-κB consensus sequences in the P-selectin promoter. The two STAT6-binding sequences, site 1 and site 2, are located in the proximal P-selectin gene promoter (bold italic letters). NF-κB site (underlined) is located adjacent to STAT6 site 2.

Figure 2-6931

P-selectin expression on dermal vessels in human skin diseases. P-selectin is constitutively expressed in normal skin. We observed high-level expression in the lesional skin of atopic dermatitis and urticaria. E, Endothelial cells; L, lumen.

Figure 3-6931

Flow cytometric analysis of P-selectin on HDMECs. A: Basal expression of P-selectin was minimal or undetectable. When HDMECs were incubated with histamine (10⁻⁵ mol/L), transient expression was observed 5 and 10 minutes after histamine treatment. B: HDMECs were incubated with IL-1α (10 ng/ml), TNF-α (10 ng/ml), interferon-γ (10 ng/ml), IL-4 (10 ng/ml), IL-13 (10 ng/ml), or substance P (100 nmol/L) for 24 hours. Whereas IL-4, IL-13, and substance P increased P-selectin expression, TNF-α had no effect on P-selectin expression. The concentration of each cytokine that induced maximal response was determined in preliminary experiments. C and D: Time course changes in P-selectin expression by HDMECs (C) and HUVECs (D) after treatment with IL-4. Surface P-selectin expression in HDMECs peaked 24 hours after treatment with IL-4. Black line, control; green line, treated with stimulants. Results are from a single representative of three separate experiments.

Figure 4-6931

Laser-scanning microscopic features of P-selectin on HDMECs. A: HDMECs incubated with histamine expressed P-selectin on their surface 5 and 10 minutes after treatment. At 20 minutes, cells exhibited shape changes. B: P-selectin expression was detected on HDMECs incubated with IL-4, IL-13, or substance P. C: Mean fluorescence intensity of surface P-selectin analyzed by Scion Image for Windows software (Scion). Error bars indicate SD. AU, arbitrary units. *P < 0.05. Original magnifications, ×400.

Figure 5-6931

Effect of TNF-α on IL-4-induced P-selectin expression assessed by cell-based ELISA. A: Dose-dependent P-selectin expression induced by IL-4. B: HDMECs were incubated with a combination of IL-4 (20 ng/ml) and TNF-α (0.1 to 10.0 ng/ml) for 24 hours. Surface P-selectin expression was significantly inhibited by TNF-α. C: HDMECs were precultured with TNF-α (0.2 to 20 ng/ml) for 24 hours. They were then incubated with IL-4 (20 ng/ml) for an additional 24 hours. TNF-α pretreatment synergistically increased P-selectin expression, whereas TNF-α alone (20 ng/ml) had no effect. Experiments were repeated at least three times. *P < 0.05.

Figure 6-6931

TNF-α pretreatment followed by IL-4 incubation increases P-selectin expression via increased expression of IL-4R. A: HDMECs weakly, constitutively express IL-4R on their surface (gray line). Incubation with TNF-α (20 ng/ml) for 24 hours significantly increased IL-4R expression (black line). B: TNF-α-pretreated HDMECs were incubated with IL-4 (20 ng/ml). IL-4R-blocking antibody (1 μg/ml) inhibited the increase in P-selectin expression induced by TNF-α pretreatment. *P < 0.05.

Figure 7-6931

Substance P induces P-selectin expression via activation of STAT6. A: Confocal laser-scanning microscopic features and cell-based ELISA for P-selectin expression induced by substance P. Surface P-selectin expression was dose dependently induced by substance P. *P < 0.05, compared with control. B: EMSA for STAT6 in HDMECs incubated with substance P. The substance P-inducible factors specifically bound to the conventional probe (Santa Cruz Biotechnology) and probes specific to site 1 and site 2 STAT6-binding sequences (Figure 1). Addition of anti-STAT6 antibody to the binding reaction inhibited complex formation. An oligonucleotide probe with a mutation in the STAT6 consensus sequence (see Materials and Methods) did not compete for binding to the STAT6 probes (data not shown). M, medium alone; SP, substance P. C: Confocal laser-scanning microscopic features of STAT6 protein in HDMECs. IL-4 (20 ng/ml) and substance P (100 nmol/L) induced translocation of STAT6 into the nucleus. Original magnifications, ×400.

Figure 8-6931

Blocking binding of STAT6 to promoter regions using decoy oligodeoxynucleotides (ODNs) inhibits IL-4- and substance P-induced P-selectin expression. HDMECs were treated with STAT6 decoy ODNs (20 μmol/L) for 8 hours, followed by incubation with IL-4 or substance P for 24 hours. Surface P-selectin expression was evaluated by cell-based ELISA. A:In vitro transfection of STAT6 decoy ODNs into HDMECs markedly inhibited IL-4- and substance P-induced P-selectin expression, but scramble decoy ODNs had no effect. B: NF-κB decoy ODNs had no effect on P-selectin expression. Results are from a single representative of two separate experiments. *P < 0.05, compared with medium alone.