Dextran sulfate sodium enhances interleukin-1β release via activation of p38 MAPK and ERK1/2 pathways in murine peritoneal macrophages

Abstract

Interleukin (IL)-1β is a pro-inflammatory cytokine that has been shown to play a pivotal role in the onset of inflammatory bowel disease (IBD), however, the molecular mechanisms underlying the production of IL-1β in IBD are not fully understood. We investigated dextran sulfate sodium (DSS)-induced IL-1β production and caspase-1 activities in murine peritoneal macrophages (pMϕ). Further, the activation status of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase 1/2 (ERK1/2), and c-Jun NH₂-terminal kinase (JNK1/2), as well as their upstream target kinases, were examined by Western blotting. In addition, mRNA expression was assessed by RT-PCR and CXC chemokine ligand 16 (CXCL16) protein was detected by immunocytochemistry. DSS-treated pMϕ released IL-1β protein in a time-dependent manner without affecting mRNA levels during 3–24 h, and caspase-1 activity peaked at 5 min (29-fold). IL-1β release and caspase-1 activity induced by DSS were significantly inhibited by a MAPK kinase 1/2 inhibitor, a p38 MAPK inhibitor, and NAC, however, not by JNK1/2 or a protein kinase C inhibitor. In addition, DSS strikingly induced the phosphorylation of p38 MAPK and ERK1/2 within 2 and 10 min, respectively. DSS also induced intracellular generation of reactive oxygen species (ROS). Pre-treatment with anti-CXCL16 for 24 h, but not anti-scavenger receptor-A, anti-CD36, or anti-CD68 antibodies, significantly suppressed DSS-induced IL-1β production. Our results suggest that DSS triggers the release of IL-1β protein from murine pMϕ at a post-translational level through binding with CXCL16, ROS generation, and resultant activation of both p38 MAPK and ERK1/2 pathways, and finally caspase-1 activation.

Introduction

Inflammatory bowel disease (IBD), which includes ulcerative colitis (UC) and Crohn's disease (CD), is a chronic, relapsing, and remitting inflammatory condition of unknown origin that afflicts individuals of both sexes throughout life (Podolsky, 1991, Strober et al., 1998). The disease is characterized by a pronounced infiltration of neutrophils into colonic lesions, accompanied by epithelial cell necrosis and ulceration. Although infection, environmental factors, heredity, and immunologic abnormalities have been proposed as causes (Sartor, 1997, Shanahan, 2001), the precise pathogenesis of IBD is poorly understood. Several models of experimental colitis have been developed to investigate the molecular and cellular mechanisms of inflammation and immunological disorders (Elson et al., 1995), with a dextran sulfate sodium (DSS)-induced colitis animal model the most widely used in studies of IBD. That DSS model exhibits many symptoms similar to those seen in human UC, i.e., diarrhea, bloody feces, body weight loss, mucosal ulceration, and shortening of the colorectum (Okayasu et al., 1990), therefore, it is considered to be reliable for studying the pathogenesis of UC and testing drugs for treatment (Dieleman et al., 1994, Elson et al., 1995, Murthy et al., 1993, Okayasu et al., 1990). However, the precise mechanism of colitis induction by DSS remains unclear.

Increased levels of several pro-inflammatory cytokines during active states of IBD have been implicated (Brynskov et al., 1992). For example, tissue levels of interleukin (IL)-1β, IL-6, IL-8, and granulocyte macrophage-colony stimulating factor are elevated in active UC and CD, and correlate with the severity of inflammation (Ishiguro, 1999, Sartor, 1994). These cytokines may play some important roles in the induction and amplification of inflammatory responses, as well as the healing of intestinal tissue injury. Dieleman et al. have reported that chronic experimental colitis induced by DSS is characterized by Th1 and Th2 cytokines (Dieleman et al., 1998). A number of studies have strongly suggested that IL-1β is activated by IL-1β-converting enzyme (ICE, also known as caspase-1) at the early stage of the cascade of events, resulting in intestinal inflammation characteristic of experimental colitis (Cominelli and Dinarello, 1989). IL-1β is a pro-inflammatory cytokine that is produced primarily by activated monocytes and macrophages, as well as other cell types, including fibroblasts, smooth muscle cells, and endothelial cells (March et al., 1985). The cytokine possesses a wide spectrum of biological activities and has been demonstrated to be involved in the regulation of immune responses, as well as the pathogenesis of several acute and chronic inflammatory diseases (Dinarello, 1991). Enhanced production of IL-1β has been detected at both the mRNA and protein levels in human IBD (Cappello et al., 1992), and in DSS-induced colitis (Savendahl et al., 1997, Tsune et al., 2003). In our previous study, we found that IL-1β levels were profoundly increased in both colonic mucosa and murine peritoneal macrophages (pMϕ) in DSS-induced colitis (Kwon et al., 2005). Therefore, pMϕ-derived IL-1β may be closely and critically associated with the pathogenesis of the disease.

Mitogen-activated protein kinases (MAPKs) are conserved among all eukaryotes and participate in multiple cellular processes (Widmann et al., 1999). Three groups of MAPKs have been identified in mammalian cells, extracellular signal-regulated kinases (ERKs), c-Jun NH₂-terminal kinases (JNKs) or stress-activated protein kinases, and p38 MAPKs (Chang and Karin, 2001, Robinson and Cobb, 1997), each of which are activated by specific upstream MAPK kinases, leading to phosphorylation of both their tyrosine and threonine residues (Kunnimalaiyaan and Chen, 2006). p38 MAPK and JNK are activated by inflammatory stimuli and environmental stress, while ERK is stimulated mainly by growth factors and tumor promoters (Robinson and Cobb, 1997, Takenobu et al., 2003). Each MAPK cascade cooperates in the orchestration of inflammatory responses, while extensive cross-talk with other inflammatory pathways, such as nuclear factor-κB and Janus kinase/STAT signaling, has been described (Kyriakis and Avruch, 2001, Tibbles and Woodgett, 1999). IL-1β is regulated by p38 MAPK and ERKs in human smooth muscle cells (Jung et al., 2001). Most recently, aggregated ursolic acid, a natural triterpenoid, induces IL-1β release from murine peritoneal macrophages via activation of p38 MAPK and ERK1/2 pathways (Ikeda et al., 2007). However, the molecular mechanism by which DSS induces IL-1β production in pMϕ remains to be elucidated.

In the present study, we investigated DSS-induced IL-1β production by examining DSS-induced caspase-1 activity, as well as the status of MAPKs and reactive oxygen species (ROS) generation, in pMϕ. Our results highlighted CXC chemokine ligand 16 (CXCL16) as a possible receptor for DSS.