Increased substance P responses in dorsal root ganglia and intestinal macrophages duringClostridium difficiletoxin A enteritis in rats

Abstract

Previously we reported that pretreatment of rats with the substance P (SP) antagonist CP-96,345 inhibits the enterotoxic responses following administration of toxin A from Clostridium difficile into ileal loops, indicating that SP participates in the intestinal responses to this toxin. We now report that injection of toxin A into rat ileum causes a rapid increase in SP content in lumbar dorsal root ganglia (DRG) and mucosal scrapings 30-60 min after toxin A administration. Toxin A-mediated f luid secretion, mannitol permeability, and ileal histologic damage is significantly increased only after 2 hr. Toxin A also causes an increase in the abundance of SP mRNA in lumbar DRG and ileal mucosa as measured by reverse transcription-PCR. Lamina propria macrophages (LPMs) obtained from toxin A-injected loops release greater amounts of tumor necrosis factor ␣ (TNF␣) and SP as compared with LPMs isolated from buffer-injected loops (P < 0.01). Pretreatment of rats with the SP antagonist CP-96,345 inhibits toxin A-mediated TNF␣ release from isolated LPMs, whereas an inactive enantiomer (CP-96,344) of the SP antagonist has no effect. LPMs obtained from toxin A-injected ileal loops incubated in vitro with SP (10 ؊8 to 10 ؊9 M) show enhanced TNF␣ secretion, whereas LPMs isolated from buffer-injected loops do not respond to SP. In addition, LPMs obtained from toxin A-injected ileal loops incubated in vitro with CP-96,345 showed a diminished TNF␣ release. Our results indicate that activated LPMs secrete SP during toxin A enteritis that can lead to secretion of cytokines, suggesting an autocrine͞paracrine regulation of cytokine secretion by SP from LPMs during intestinal inf lammation.Substance P (SP), an 11-aa peptide member of the tachykinin family originally isolated by Chang and Leeman (1), is a peptide distributed throughout the central and peripheral nervous system. In the intestine, SP has been found in capsaicin-sensitive sensory neurons (2), enteric neurons (3), as well as intestinal enteroendocrine cells (4). SP is also synthesized in the cell bodies of the dorsal root ganglia (DRG) (5), and evidence indicates that release of SP from DRG to spinal cord mediates nociceptive and inflammatory stimuli (6-8). Recently, Reinshagen et al. (9) reported decreased levels of SP in the DRG of rabbits 48 hr after induction of colitis, suggesting that SP may be released from sensory neurons during the acute phase of inflammation. However, there are no studies specifically evaluating the SP content in the DRG during acute intestinal inflammation mediated by bacterial enterotoxins.In addition to its role as a neurotransmitter, SP also participates in immune and inflammatory responses. Mast cells (10), polymorphonuclear leukocytes (11), T lymphocytes (12, 13), and macrophages (14) can respond to SP, indicating that the effects of SP during inflammation may be mediated by direct activation of these cells. SP levels are also elevated in inflamed tissues (15-17), and increased binding sites for SP have been demonstrated at sites o...

Corticotropin-releasing hormone (CRH) requirement inClostridium difficiletoxin A-mediated intestinal inflammation

“…4B). In line with prior observations (25,40), toxin A administration in Crh ϩ/ϩ mice results in increased SP content (Fig. 4A) and immunostaining (Fig.…”

“…Injection of toxin A into ileal or colonic loops of anesthetized animals triggers mucosal neutrophil infiltration and increases fluid secretion and mucosal permeability 1-4 h after toxin A administration (22)(23)(24)(25). Although the in vivo mechanism(s) leading to acute enterocolitis are not completely understood, activation of sensory nerves (26,27) and release of sensory neuropeptides, such as substance P (SP) (25,28,29), are pivotal in the mediation and amplification of the inflammatory signal in response to toxin A.The development of CRH-deficient mice (30, 31) allows us to directly evaluate the contribution of CRH in C. difficile toxin A-induced ileal fluid secretion and intestinal inflammation. Crh Ϫ/Ϫ mice have impaired hypothalamic-pituitary-adrenal axis activity as shown by their lower plasma corticosterone levels after physiological or psychological stimuli (30, 32), or carrageenin-induced acute inflammation (33).…”

“…C. difficile causes diarrhea and colitis by releasing two high-molecular-mass protein exotoxins, toxin A and B, with potent cytotoxic and enterotoxic properties in animal and human intestine (21). Injection of toxin A into ileal or colonic loops of anesthetized animals triggers mucosal neutrophil infiltration and increases fluid secretion and mucosal permeability 1-4 h after toxin A administration (22)(23)(24)(25). Although the in vivo mechanism(s) leading to acute enterocolitis are not completely understood, activation of sensory nerves (26,27) and release of sensory neuropeptides, such as substance P (SP) (25,28,29), are pivotal in the mediation and amplification of the inflammatory signal in response to toxin A.…”

Substance P antagonist (CP-96,345) inhibits HIV-1 replication in human mononuclear phagocytes

“…The pretreatment of rats with the SP antagonist CP-96,345 inhibited Clostridium difficile toxin A-mediated TNF-␣ release from isolated lamina propria macrophages. Furthermore, lamina propria macrophages obtained from the toxin A-injected ileal loops incubated in vitro with CP-96,345 had reduced TNF-␣ release, indicating an autocrine stimulation of SP in the cytokine secretion (9). Autocrine regulation of SP also has been suggested in other cell types (10)(11)(12).…”

“…SP has an autocrine regulation role in macrophage function (8,9). For example, in the macrophage cell line P388D1, anti-SP antibody depletion of macrophage-secreted SP from the culture resulted in abrogation of SP-increased IL-1 production (8).…”