Elsevier

Immunobiology

Volume 216, Issues 1–2, January–February 2011, Pages 32-40
Immunobiology

Interleukin-32α production is regulated by MyD88-dependent and independent pathways in IL-1β-stimulated human alveolar epithelial cells

https://doi.org/10.1016/j.imbio.2010.03.007Get rights and content

Abstract

Interleukin (IL)-32 is a recently described cytokine that appears to play a critical role in a variety of inflammatory diseases including chronic obstructive pulmonary disease (COPD). However, thus far, the regulation of IL-32 production has not been fully established. Here, we report on signaling pathways that regulate the production of IL-32α, the most abundant isoform, in the human alveolar epithelial cell line, A549. IL-32α was expressed and secreted by IL-1β. The IL-32 expression was attenuated by PP2 (a Src-family kinase [SFK] inhibitor), rottlerin (a protein kinase [PK] Cδ inhibitor), and LY294002 (a phosphatidylinositol 3-kinase [PI3K] inhibitor). Furthermore, the overexpression of Fgr rather than other SFKs upregulated IL-32α expression, while Fgr small interfering RNA (siRNA) transfection downregulated it. The suppression of Fgr with PP2 and Fgr siRNA inhibited activating phosphorylation of PKCδ and PI3K/Akt, but not IL-1 receptor-associated kinase (IRAK)1, a well-known MyD88-dependent signaling molecule, and Erk1/2, p38, and JNK. Rottlerin and PKCδ siRNA also inhibited expression of IL-32α and activation of PI3K/Akt, but not of IRAK1 and mitogen activation protein (MAP) kinases. MyD88 siRNA suppressed the expression of IL-32α and the phosphorylation of IRAK1, PI3K, and MAP kinases, but not of PKCδ. Of interest, both Fgr/PKCδ and MyD88-dependent signals regulated PI3K/Akt, suggesting that it is a crosstalk molecule. Among MyD88-dependent MAP kinases, only p38 regulated IL-32α expression and PI3K/Akt activation. With these results, we demonstrated that the expression and secretion of IL-32α are regulated by MyD88-dependent IRAK1/p38/PI3K and independent Fgr/PKCδ/PI3K pathways, and that Fgr and PKCδ are critical for the MyD88-independent IL-32α production.

Introduction

Chronic obstructive pulmonary disease (COPD) is one of major causes of morbidity and mortality worldwide, and there is evidence that it is increasing throughout the world (Lopez et al., 2006, Murray and Lopez, 1996). In patients with COPD, a chronic inflammatory process is present throughout the airways and lung parenchyma (Saetta et al., 1998, Saetta et al., 1999). However, the molecular mechanisms leading to lung inflammation are largely unknown. It has been suggested that the release of proinflammatory cytokines such as tumor necrosis factor (TNF)-α and interleukin (IL)-1β play a critical role in inducing inflammatory lung diseases (Piguer 1993). IL-1β, a proinflammatory cytokine, stimulates the production of chemokines, TNF-α, and IL-6 for neutrophil recruitment and fibroblast proliferation (Barnes 2004). The cytokines produced in pulmonary epithelial cells, including IL-1β, can recruit inflammatory cells to cause chronic lung inflammation as observed in the lungs of patients with COPD (Saetta et al., 1998, Saetta et al., 1999, Standiford et al., 1990, Standiford et al., 1991).

IL-32 is a recently described cytokine produced by T lymphocytes, natural killer (NK) cells, monocytes, and epithelial cells. Similar to other cytokines, IL-32 can be secreted as a soluble protein in several types of cells (Kim et al. 2005). Six splicing isoforms of IL-32 were currently described, namely, IL-32α, β, γ, δ, ɛ, and ζ, with IL-32α being the most abundant isoform (Goda et al., 2006, Kim et al., 2005). IL-32 acts as a proinflammatory cytokine in inflammatory disorders such as rheumatoid arthritis, Mycobacterium tuberculosis infection, and Crohn's disease as well as in COPD (Calabrese et al., 2008, Joosten et al., 2006, Mun et al., 2009, Netea et al., 2006, Shioya et al., 2007). IL-32 can also induce proinflammatory cytokines and chemokines such as IL-1β, TNF-α, and IL-8 (Kim et al. 2005). Furthermore, it was reported that IL-32 was upregulated in the lungs of patients with COPD and showed correlation with disease severity (Calabrese et al. 2008). Those reports indicated that IL-32 is closely associated with the progression of lung inflammatory diseases. However, the regulation of IL-32 production in lung alveolar epithelial cells has not been determined.

In the present study, we demonstrated that IL-32α is expressed and secreted by IL-1β in A549 cells, a human alveolar epithelial cell line. In addition, we observed that not only the MyD88-dependent IL-1 receptor-associated kinase (IRAK)1/p38/phosphatidylinositol 3-kinase (PI3K) pathway, but also the MyD88-independent Fgr/protein kinase (PK)Cδ/PI3K pathway participated in the regulation of IL-32α production.

Section snippets

Materials

Anti-IL-32α polyclonal antibody was a generous gift from Dr. S.H. Kim (Laboratory of Cytokine Immunology, Institute of Biomedical Science and Technology, Konkuk University, Korea). Antibodies against Lyn, Fgr, c-Src, Fyn, MyD88, actin, and the phosphorylated form of IRAK1 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against p38 and PKCδ, and phosphorylated forms of p38, JNK, Erk1/2, PKCδ (at Tyr311 or Thr505), PI3K, and Akt were purchased from Cell Signaling

IL-1β stimulates the expression and secretion of IL-32α

We initially evaluated whether or not IL-1β stimulates the production of IL-32α in A549 cells, the human alveolar epithelial cell line. IL-32α expression was stimulated by IL-1β in a time- and concentration-dependent manner (Fig. 1A and B). Maximal response was achieved within 8 h and then maintained thereafter (Fig. 1A). As expected, the expression of mRNA's for IL-32α, β γ, or δ was also stimulated by IL-1β (Fig. 1C), and, notably, secretion of IL-32α was also significantly enhanced by IL-1β

Discussion

IL-32 is a proinflammatory cytokine produced by T lymphocytes, NK cells, monocytes, and epithelial cells (Kim et al. 2005). IL-32 production is increased in synovial tissues (Joosten et al. 2006) and fluids (Mun et al. 2009) of patients with rheumatoid arthritis, in peripheral blood mononuclear cells infected with M. tuberculosis (Netea et al. 2006), and in inflamed mucosa of patients with Crohn's disease (Shioya et al. 2007). Recently, it was reported that elevated levels of IL-32 may be one

Acknowledgements

This work was supported by a grant from the Korean Ministry of Education, Science, and Technology (The Regional Core Research Program/Chungbuk BIT Research-Oriented University consortium and partly by the Konkuk University and the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MEST) (No. 2009–007125).

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