HCl-induced inflammatory mediators in esophageal mucosa increase migration and production of H2O2by peripheral blood leukocytes

Abstract

Exposure of esophageal mucosa to hydrochloric acid (HCl) is a crucial factor in the pathogenesis of reflux disease. We examined supernatant of HCl-exposed rabbit mucosa for inflammatory mediators enhancing migration of leukocytes and production of H₂O₂ as an indicator of leukocyte activation. A tubular segment of rabbit esophageal mucosa was tied at both ends to form a sac, which was filled with HCl-acidified Krebs buffer at pH 5 (or plain Krebs buffer as control) and kept oxygenated at 37°C. The medium around the sac (supernatant) was collected after 3 h. Rabbit peripheral blood leukocytes (PBL) were isolated, and sac supernatant was used to investigate PBL migration and H₂O₂ production. HCl-exposed esophageal mucosa released substance P (SP), CGRP, platelet-activating factor (PAF), and IL-8 into the supernatant. PBL migration increased in response to IL-8 or to supernatant of the HCl-filled mucosal sac. Supernatant-induced PBL migration was inhibited by IL-8 antibodies and by antagonists for PAF (CV3988) or neurokinin 1 (i.e., SP), but not by a CGRP antagonist. Supernatant of the HCl-filled mucosal sac increased H₂O₂ release by PBL that was significantly reduced by CV3988 and by a SP antagonist but was not affected by IL-8 antibodies or by a CGRP antagonist. We conclude that IL-8, PAF, and SP are important inflammatory mediators released by esophageal mucosa in response to acid that promote PBL migration. In addition, PAF and SP induce production of H₂O₂ by PBL. These findings provide a direct link between acid exposure and recruitment and activation of immune cells in esophageal mucosa.