Altered IL-32 Signaling in Abdominal Aortic Aneurysm

Abstract

Introduction and objective: Interleukin (IL)-32 is a pro-inflammatory cytokine not previously studied in relation to abdominal aortic aneurysm (AAA). The aim of this study was to elucidate the expression and localization of IL-32 in AAA.

Methods: Expression and localization of IL-32 in human aortic tissue was studied with immunohistochemical analysis and Western blot (AAA: n = 5; controls: n = 4). ELISA was used to measure IL-32 in human plasma samples (AAA: n = 140; controls: n = 37) and in media from cultured peripheral blood mononuclear cells (PBMCs) from 3 healthy donors. IL-32 mRNA in PBMCs, endothelial cells, aortic smooth muscle cells (SMCs), and aortic tissue samples of AAA (n = 16) and control aortas (n = 9) was measured with qPCR.

Results: IL-32 was predominantly expressed in SMCs and T-cell-rich areas. Highest mRNA expression was observed in the intima/media layer of the AAA. A weaker protein expression was detected in non-aneurysmal aortas. Expression of IL-32 was confirmed in isolated T cells, macrophages, endothelial cells, and SMCs, where expression was also inducible by cytokines such as interferon-γ. There was no difference in IL-32 expression in plasma between patients and controls.

Conclusion: IL-32 signaling is altered locally in AAA and could potentially play an important role in aneurysm development. Further studies using animal models would be helpful to study its potential role in AAA disease.

Keywords: Abdominal aortic aneurysm; Cytokines; Inflammation; Leukocytes; T cells.

Fig. 1

Representative immunohistochemical localization of IL-32 in the tunica adventitia of the aneurysmal tissue from a 65-year-old man with an aneurysm of 60 mm. Expression of IL-32 (a, b), α-actin (c, d), von Willebrand (e, f), CD3ε (g, h), and isotypic IgG control for IL-32 (i, j). Scale bar = 200 μm. IL-32, interleukin-32.

Fig. 2

Representative immunohistochemical localization of IL-32 in the tunica intima and media of the aneurysmal tissue from a 65-year-old man with an aneurysm of 60 mm. Expression of IL-32 (a, b), CD3ε (c, d), CD66b (e, f), CD68 (g, h), and isotypic IgG control for IL-32 (i, j). Scale bar = 200 μm. IL-32, interleukin-32.

Fig. 3

IL-32 expression in aneurysmal aortas. Protein expression of IL-32 in non-aneurysmal control aortas (n = 4, <30 mm) and AAA (n = 5, 70 ± 2.2 mm) (a). β-Tubulin used as loading control. Quantification of IL-32 expression (b) by densitometry analysis. Expression of IL-32 mRNA (c) in non-aneurysmal control aortas (n = 9), AAA (n = 8), and AAA divided into intima/media layer (n = 7), adventitia layer (n = 10), and PVAT layer (n = 6). Statistical analysis of the data from the qPCR of patient samples was performed with an ANOVA and Bonferroni corrections. Determination of the significance of the results of the Western blot was done with Student's t test. Data were considered statistically significant at p < 0.05. * indicates p value <0.05. IL-32, interleukin-32; AAA, abdominal aortic aneurysm; PVAT, perivascular aortic tissue.

Fig. 4

Expression of IL-32 mRNA in cultured HUVECs (a) and AoSMC (b) from 3 separate experiments. Statistical analysis of the data from the qPCR was performed with an ANOVA and Bonferroni corrections. * indicates p value <0.05, ** p values <0.01, and *** p values <0.001, versus respective control and time point. IL, interleukin; HUVEC, human vascular endothelial cell; AoSMC, aortic smooth muscle cell; INF-γ, interferon-γ; TNF-α, tumor necrosis factor-α; LPS, lipopolysaccharide.

Fig. 5

Expression of IL-32 mRNA in PBMCs, unstimulated or stimulated with IFN-γ for 40 h, from 3 donors. Determination of the significance of the results was done with Student's t test. * indicates significant difference between untreated monocytes at p < 0.05. IL, interleukin; PBMC, peripheral blood mononuclear cell; INF-γ, interferon-γ; Mon, monocytes; TBP, TATA box-binding protein.