IL-17A potentiates TNFα-induced secretion from human endothelial cells and alters barrier functions controlling neutrophils rights of passage

Abstract

Interleukin-17A (IL-17A) is an important pro-inflammatory cytokine that regulates leukocyte mobilization and recruitment. To better understand how IL-17A controls leukocyte trafficking across capillaries in the peripheral blood circulation, we used primary human dermal microvascular endothelial cells (HDMEC) to investigate their secretory potential and barrier function when activated with IL-17A and TNFα. Activation by TNFα and IL-17A causes phosphorylation of p38 as well as IκBα whereby NFκB subsequently becomes phosphorylated, a mechanism that initiates transcription of adhesion molecules such as E-selectin. Members of the neutrophil-specific GRO-family chemokines were significantly up-regulated upon IL-17A stimulation on the mRNA and protein level, whereas all tested non-neutrophil-specific chemokines remained unchanged in comparison. Moreover, a striking synergistic effect in the induction of granulocyte colony-stimulating factors (G-CSF) was elicited when IL-17A was used in combination with TNFα, and IL-17A was able to significantly augment the levels of TNFα-induced E-selectin and ICAM-1. In accordance with this observation, IL-17A was able to markedly increase TNFα-induced neutrophil adherence to HDMEC monolayers in an in vitro adhesion assay. Using a trans-well migration assay with an HDMEC monolayer as a barrier, we here show that pre-stimulating the endothelial cells with TNFα and IL-17A together enhances the rate of neutrophil transmigration compared to TNFα or IL-17A alone. These results show that IL-17A and TNFα act in cooperation to facilitate neutrophil migration across the endothelial cell barrier. In addition, the synergistic actions of IL-17A with TNFα to secrete G-CSF appear to be important for mobilizing neutrophils from the bone marrow to the blood stream.

Fig. 1

Human microvascular endothelial cells express and signal through the IL-17A receptor. a Agarose-gel photos showing PCR products for IL-17RA (548 bp) and IL-17RC (416 bp) at the predicted band sizes loaded next to a DNA ladder for band size determination. b Expression of IL-17 receptor A (IL-17RA) on human dermal microvascular endothelial cells (HDMECs) as shown by surface binding of IL-17RA antibody (black curve) compared to control IgG1 antibody (grey curve) using a BD FACSArray. The mean fluorescent intensity (MFI) is 99 for control and 689 for IL-17RA antibody. One representative experiment out of three. c Western blotting showing phosphorylation of p38 (P-p38), IκBα (P- IκBα) and NFκB (P-NFκB) after stimulation with IL-17A (50 ng/ml) and TNFα (10 ng/ml) alone or together. d Measurements of grey values from the Western blotting of P-p38 and P-NFκB

Fig. 2

IL-17A cooperates with TNFα in the induction of G-CSF and GRO-family chemokines. a Relative mRNA levels of G-CSF in HDMEC challenged with IL-17A (50 ng/ml), TNFα (2 ng/ml) or both for 4 h (means of technical triplicates). b Protein levels of secreted G-CSF from HDMEC challenged with IL-17A (50 ng/ml), TNFα (2 ng/ml) or both for 24 h. c Relative mRNA levels of GROα in HDMEC challenged with IL-17A (50 ng/ml), TNFα (10 ng/ml) or both for 4 h (means of technical triplicates). d Protein levels of secreted GROα from HDMEC challenged with IL-17A (50 ng/ml), TNFα (10 ng/ml) or both for 12 h. e Protein levels of secreted IL-8 from HDMEC challenged with IL-17A (50 ng/ml), TNFα (10 ng/ml) or both for 24 h. f Protein levels of secreted CXCL10 from HDMEC challenged with IL-17A (50 ng/ml), TNFα (10 ng/ml) or both for 24 h. CXCL10 was undetectable in the CTR- and IL-17A-treated samples. b, d, e and f are representative of two independent experiments

Fig. 3

IL-17A modulates TNFα-induced E-selectin and ICAM-1 expression. a ELISA data showing cell-associated (lysate) protein levels of E-selectin in HDMEC when challenged with TNFα (2 ng/ml) or TNFα in combination with IL-17A (100 ng/ml) for 2–32 h. The effect of IL-17A was statistically significant at all time-points (P < 0.05) except at “4 h”. b Same as in a only for VCAM-1 protein. The effect of IL-17A was statistically significant at all time-points (P < 0.05) except at “4” and “32 h”. c Same as in a only for ICAM-1 protein. The effect of IL-17A was only statistically significant at “24 h” (P < 0.05). a, b and c are representative of two independent experiments

Fig. 4

IL-17A modulates TNFα-induced neutrophil adhesion and transmigration. a ELISA data showing cell-associated (lysate) protein levels of E-selectin in HDMEC when challenged with IL-17A (50 ng/ml), TNFα (1 ng/ml) or both for 24 h. b Relative number of neutrophils adhering to a confluent HDMEC monolayer pre-treated with either IL-17A (100 ng/ml), TNFα (2 ng/ml) or both for 30 h. c Relative number of neutrophils that had transmigrated through a confluent HDMEC monolayer pre-treated with either IL-17A (100 ng/ml), TNFα (2 ng/ml) or both for 30 h. a, b and c are representative of two independent experiments. d Relative number of neutrophils adhering to a confluent HDMEC monolayer pre-treated with either IL-17A (100 ng/ml), TNFα (2 ng/ml) or both for 24 h. The cells were pre-treated with 25 μg/ml E-selectin or 1 μg/ml P-selectin blocking antibodies for 1 h before the addition of neutrophils. e Photos of transmigrated neutrophils from c revealing the shape changes induced by TNFα with IL-17A

Fig. 5

IL-17A does not directly affect neutrophil adherence and function. a Data showing neutrophil adherence to a fibrinogen substratum when challenged with IL-17A (50 ng/ml) or fMLP (1 μM) for 30 min. b Data showing neutrophil superoxide radical generation, as measured by reduction of ferricytochrome C, when challenged with IL-17A (50 ng/ml), fMLP (1 μM), PMA (10 ng/ml) or combinations of these