Acute ileal inflammatory cytokine response induced by irradiation is modulated by subdiaphragmatic vagotomy

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Abstract

Neural involvement plays a role in the genesis of the peripheral inflammatory process that contributes to the irradiation intestinal disorders. However, little is known about the role of vagus nerve in modulating inflammatory process in rat. Here, we have shown that the NF-κB activation was consistent with the acute overexpression of pro-inflammatory cytokines (IL- 1β, TNF-α, IL-6) at 3, 6, and 12 h induced by whole-body irradiation (8 Gy). Subdiaphragmatic vagotomy reduced NF-κB activation and cytokine transcription in the early period post-irradiation. In contrast, vagotomy amplified overexpression of irradiation-induced anti-cytokines (IL-10, IL-1Ra) and of receptors involved in anti-inflammatory effects (IL- 1RII, TNFRII). These results show that the vagus nerve is a pro-inflammatory pathway in early irradiation-induced intestinal inflammation.

Introduction

The intestine is a dose-limiting organ during radiotherapy, it is characterized by diarrhea when acute and leads to progressively reduced motility in cases of chronic enteropathy. Detailed data analysis indicates that the severity of the acute phase is correlated with the manifestation of late effects (Donner, 1998, Dörr and Hendry, 2001).

Recent reports show that radiation injury appears to involve cytokine pathways (Rubin et al., 1995, Linard et al., 2003) and clinical symptoms (diarrhea, dismotility) may be caused by the early stage of this inflammatory process (Dörr and Hendry, 2001). In this way, many cytokines, including IL-1β, TNF-α, and IL-6, are closely associated with radiation injury and contribute significantly to the disorders associated with radiotherapy in lymphoid tissues and lungs (Hong-Hong et al., 1999, Rubin et al., 1995). We previously reported that irradiation exposure (10 Gy) induces overexpression of IL-1β, TNF-α, and IL-6 and thereby modifies the balance between pro- and anti-inflammatory cytokines in the intestine (Linard et al., 2004). Although most reports of radiation-induced inflammatory processes focus on later development of tissue damage rather than the initial phase — in the first 24 h after irradiation — it has recently been proposed that the early acute phase may determine the late phase (Dörr and Hendry, 2001, Denham and Hauer-Jensen, 2002). In this way, it was important to consider the mechanism generate the early “acute phase response” to radiation.

There is now incontrovertible evidence of neural involvement in the genesis and development of the peripheral inflammatory process (Watkins et al., 1995, Widdicombe, 2003) that causes functional damage; the nervous and immune systems interact bidirectionally. An increasing number of reports describe the heterogeneous effects that vagotomy has on the pathological consequences of inflammatory processes (induced, for example, by IL-1β and lipopolysaccharide (LPS)) (Bluthé et al., 1994, Watkins et al., 1995, Goehler et al., 1995).

Peripheral cytokines may act locally and communicate with the brain by both neural and blood-borne routes (Watkins et al., 1995, Goehler et al., 1995), while the brain provides information to the periphery through both the autonomic nervous system, via direct neural influences, and the neuroendocrine humoral outflow, via the hypothalamic–pituitary–adrenal (HPA) axis (Sanders and Straub, 2002). At the intestinal level, nerves are intimately associated with inflammatory cells (Collins, 1996, Cornet et al., 2001) and local increases in tissue levels of such inflammatory mediators as neuropeptides and cytokines would be sufficient to send signals to the brain. Activation of sensory neurons like unmyelinated C fibers participate in the local inflammatory reaction, in response to infection or toxins or trauma (Shanahan, 1998, Lundgren, 2002). This process, called neurogenic inflammation, includes local release by the fibers peripheral endings of sensory neuropeptides, called Substance P (SP) (Maggi, 1995) and causes immune cell activation (Shanahan, 1998). We previously reported that sensory neurons containing SP play a role in the early intestinal motor alterations induced by irradiation, but that pretreatment with capsaicin, a neurotoxic agent that depletes the sensory afferent fibers, does not prevent pro-inflammatory cytokine concentrations from rising (Linard et al., 2003). This finding suggests that the irradiation induced increase in local cytokines depends on another stimulation pathway.

Complex interactions exist between activation of sensory neurons present in the enteric nervous system and extrinsic primary afferents nerves. The vagus nerve contains, besides these unmyelinated afferent fibers, mixed afferent/efferent sensory and motor nerves that play an important role in intestinal immune function (Lindgren et al., 1993). Sectioning the abdominal vagus nerve abrogates most brain-mediated illness responses and, in particular, inhibits a variety of acute phase responses (hyperthermic, feeding, hyperalgesic, and adrenocortical responses) that are induced by intraperitoneal administration of IL-1β or LPS (Watkins et al., 1995, Goehler et al., 1995). Vagal signaling of immune activation occurs fairly rapidly (Watkins et al., 1995) and the inflammatory process induced by irradiation is detectable within the same time period (Linard et al., 2004).

Many inflammatory responses, particularly in the gut, are mediated by the activation of transcription factors such as nuclear transcription factor κB (NF-κB) and activator protein-1 (AP-1), which is composed of the proto-oncogenes c-Fos and c-Jun. NF-κB is a key mediator of the immune response. It plays a pivotal role in the inducible expression of many genes, including cytokines in gut immune and inflammatory responses (Neurath et al., 1998). The temporal relations that we recently observed between irradiation-induced activation of NF-κB and increased expression of TNF-α, IL-1β, and IL-6 (Linard et al., 2004) suggest that this activation plays a key role in the induction of these cytokines in vivo. Hypothesizing that the intestinal muscle layer, including mesenchymal tissue, fibroblasts, myofibroblasts, and muscle cells, may be the source of the inflammatory mediators that account for acute inflammation-induced changes in motor function (Collins, 1996), we investigated the involvement of the vagus nerve in early acute post-irradiation inflammatory molecular events (the cytokines IL-1β, TNF-α, Il-6, and IFN-γ, and neuropeptides, i.e., SP and cytokine receptors) and in NF-κB activation within the muscularis layer of the ileum.

Section snippets

Subjects

Adult male Sprague–Dawley rats (Janvier, France) were housed on a 12:12 h light/dark cycle (light on at 20:00) with food and water available ad libitum. The reverse light cycle permitted to obtain the period of rat activity during the day. Thus, irradiation has been delivered in the morning (during the activity period). In this way, neurophysiology tests may be added in the same conditions than biological measures. All experiments were conducted in accordance with the French regulations for

Effects of vagotomy on irradiation-induced pro-inflammatory cytokine expression

The mRNA levels of the pro-inflammatory cytokines IL-1β, TNF-α, IFN-γ, and IL-6 were quantified by real-time PCR and expressed as a ratio to a reference gene, hypoxanthineguanine phosphoribosyltransferase (HPRT) in the ileal muscularis layer (Fig. 1A–D). The HPRT mRNA level in the ileal tissue was unchanged after an 8-Gy whole-body irradiation of non-vagotomized rats (data not shown). Within 3 h of this irradiation, we observed significantly increased mRNA levels of IL-1β (6-fold, P < 0.005, t =  

Discussion

The concept of an interaction between immune cells and intestinal smooth muscle cells may be particularly pertinent in the external neuromuscular layers of the intestine have been reported by Collins (1996), where inflammatory processes are known to cause functional and structural damage and disturb intestinal physiology; they thus provide indirect evidence of altered neural function in the inflamed gut (Collins, 1996). In this way, the motor system (neuromuscular tissue) may play an active

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