IL-32-dependent effects of IL-1beta on endothelial cell functions

Abstract

Increasing evidence demonstrates that interleukin (IL)-32 is a pro-inflammatory cytokine, inducing IL-1alpha, IL-1beta, IL-6, tumor necrosis factor (TNF)-alpha, and chemokines via nuclear factor (NF)-kappaB, p38 mitogen-activated protein kinase (MAPK), and activating protein (AP)-1 activation. Here we report that IL-32 is expressed and is also functional in human vascular endothelial cells (EC) of various origins. Compared with primary blood monocytes, high levels of IL-32 are constitutively produced in human umbilical vein EC (HUVEC), aortic macrovascular EC, and cardiac as well as pulmonary microvascular EC. At concentrations as low as 0.1 ng/ml, IL-1beta stimulated IL-32 up to 15-fold over constitutive levels, whereas 10 ng/ml of TNFalpha or 100 ng/ml of lipopolysaccharide (LPS) were required to induce similar quantities of IL-32. IL-1beta-induced IL-32 was reduced by inhibition of the IkappaB kinase-beta/NF-kappaB and ERK pathways. In addition to IL-1beta, pro-coagulant concentrations of thrombin or fresh platelets increased IL-32 protein up to 6-fold. IL-1beta and thrombin induced an isoform-switch in steady-state mRNA levels from IL-32alpha/gamma to beta/epsilon. Adult EC responded in a similar fashion. To prove functionality, we silenced endogenous IL-32 with siRNA, decreasing intracellular IL-32 protein levels by 86%. The knockdown of IL-32 resulted in reduction of constitutive as well as IL-1beta-induced intercellular adhesion molecule-1 (ICAM-1) (of 55% and 54%, respectively), IL-1alpha (of 62% and 43%), IL-6 (of 53% and 43%), and IL-8 (of 46% and 42%). In contrast, the anti-inflammatory/anti-coagulant CD141/thrombomodulin increased markedly when IL-32 was silenced. This study introduces IL-32 as a critical regulator of endothelial function, expanding the properties of this cytokine relevant to coagulation, endothelial inflammation, and atherosclerosis.

Fig. 1.

Expression and regulation of IL-32 in HUVEC. HUVEC were incubated with the indicated stimuli and/or inhibitors for 20 hours (A, B, D, E) or for the periods indicated (C). Concentrations (in ng/ml) were as follows: LPS, 100; TGF-β₁, 20; IFNγ, 25; VEGF, 25; IL-1Ra, 10000; IL-18BP, 1000; sTNFR, 10000. (B) Mean fold-changes in IL-32 protein levels normalized to total protein (in mg) ± SEM; n = 9; *P < 0.05; **P < 0.01; and ***P < 0.001 for stimulated vs. untreated cultures. Normalized absolute IL-32 concentrations ranged from 28 to 269 pg/mg in controls and from 581 to 4025 pg/mg in IL-1β– (10 ng/ml) stimulated cells. (A) One representative of five independently performed Western blots of HUVEC cell lysates is shown. (C) HUVEC were cultured with different concentrations of FCS. Normalized absolute IL-32 levels ± SEM are shown; n = 5; *P < 0.05; and **P < 0.01 for 2% vs. 10 or 20%; ##P < 0.01 for 5% vs. 20%; oP < 0.05 for 10% vs. 20%; ♦, 20% at 24 hours vs. 20% at 72 hours. (D) IL-32 protein after incubation with the indicated concentrations of thrombin, mean ± SEM; n = 5; *P < 0.05; and **P < 0.01 for constitutive vs. thrombin. (E) Effect of incubation of HUVEC with 50 × 10⁶ platelets on IL-32 is depicted. n = 3; *P < 0.05 for constitutive vs. platelets.

Fig. 2.

IL-32 production in EC of different origins. AEC (A, B), CMEC (C, D), and PMEC (E, F) were treated with the indicated stimuli (concentrations as in Fig. 1) or IL-1Ra (10 μg/ml) for 20 hours in the presence of 2% FCS. “Control” indicates constitutive production of IL-32. IL-1β concentrations are displayed in ng/ml. Cell lysates were harvested and IL-32 protein levels were determined. (A, C, E) The absolute IL-32 concentrations normalized to total protein are shown. Mean ± SEM; n = 4 for AEC and n = 8 for CMEC and PMEC; *P < 0.05; **P < 0.01; and ***P < 0.001 for treated cells vs. controls. (B, D, F) One of four blots resulting from independent experiments from each cell type is shown.

Fig. 3.

Expression and regulation of IL-32 mRNA isoforms in EC. Two hours after changing from growth- to stimulation medium, HUVEC (A, C) or CMEC (B, D) were stimulated with 10 ng/ml IL-1β or 7.5 U/ml thrombin for the indicated periods of time or were left untreated. Relative mRNA quantities were calculated using the ΔΔC_T method. (A, B) Constitutive mRNA levels at 1 hour are defined as background. Mean fold increases ± SEM in all isoforms of IL-32 mRNA over background are shown; n = 4; *P < 0.05; **P < 0.01; and ***P < 0.001 for treated cells vs. controls. (C, D) The fractions of IL-32α/γ, β/ε, and other isoforms are depicted for each stimulation condition. The levels of all isoforms for each sample were set at 100%; n = 4.

Fig. 4.

Knockdown of IL-32 and its effect on ICAM-1 and thrombomodulin/CD141. (A) After transfection and stimulation with 10 ng/ml IL-1β, EC were cultured for the indicated time periods and IL-32 was measured in cell lysates. The graph depicts mean percent changes in IL-32 protein levels comparing siIL-32-transfected to scrambled-transfected cells ± SEM; n = 3. (B, C) HUVEC transfected with 100 nmol/l of either siIL-32 or scrambled were stimulated with 10 ng/ml IL-1β for 20 hours (B) or 30 minutes (C) or left untreated. (B) ICAM-1 was assayed by ELISA in cell lysates. Means of normalized protein concentration ± SEM in ng/mg is shown; n = 6; *P < 0.05 for siIL-32 compared with scrambled. (C) One representative of four Western blots of HUVEC lysates is depicted. The degree of silencing in these lysates is shown in Fig. S1 A and B.

Fig. 5.

The effects of siIL-32 on cytokine levels in HUVEC. Cell lysates (A, B) or supernatants (C–F) from unstimulated (Left column) or IL-1β-treated (10 ng/ml, Right column) HUVEC transfected with concentration-matched pairs of either siIL-32 or scrambled siRNA were analyzed for protein levels of IL-1α (A, B), IL-6 (C, D), and IL-8 (E, F) after a 20-hour incubation period. IL-32 levels in these cultures are shown in Fig. S1 A and B. Means of normalized absolute cytokine concentrations ± SEM are shown; n = 10; *P < 0.05; and **P < 0.01 for siIL-32 compared with scrambled.