interleukin-8 and monodansylcadaverine

Interleukin 8 (IL-8), a neurophil-activating and chemotactic cytokine, is known to play a key role in the pathogenesis of a large number of neutrophil-driven inflammatory diseases. Although the cytokine is rapidly internalized at 37 degrees C with its receptors, there was no direct evidence for the ligand-induced endocytosis of the receptor or that of the interaction of receptor ligand complex at 37 degrees C. As a result, our understanding about the regulation of Il-8 induced biological response is very limited. In the present study, using FITC-IL-8 conjugate as a probe, we have demonstrated the time- and temperature-dependent endocytosis of IL-8 under fluorescent microscope. We have also shown that the bright fluorescent light on the surface of neutrophils gradually disappears and it becomes almost dark after 120 min of incubation. Monodansyl cadaverine (MDC, 900 microM), however, was found to retain the fluorescent light of FITC coupled with Il-8 on the cells. MDC and ouabain (2.5 mM) can inhibit the ligand induced endocytosis by 76% and 96%, respectively, compared to control. With respect to control, IL-8 induced biological responses e.g. IL-8 directed migration, intracellular Ca2+ release and superoxide release are significantly reduced by 77%, 94% and 76%, respectively, in presence of MDC. The study presents a direct visual evidence of the time and temperature-dependent receptor-mediated endocytosis of IL-8 which is inhibited by MDC and ouabain. This information is useful for understanding the ligand receptor interaction at 37 degrees C and may be useful for developing anti-inflammatory agents against IL-8.

Topics: Anions; Antigens, CD; Cadaverine; Calcium; Cell Movement; Cells, Cultured; Endocytosis; Enzyme Inhibitors; Humans; Interleukin-8; Ligands; Microscopy, Fluorescence; Neutrophils; Ouabain; Receptors, Interleukin; Receptors, Interleukin-8A; Superoxides

Interleukin-8 (IL-8), a neutrophil chemotactic agent, acts as a key mediator in a large number of acute and chronic inflammatory diseases. At 37 degrees C, the receptor for IL-8 is rapidly internalized with its ligand. But no specific inhibitor of this ligand induced internalization of the receptor has been reported so far. We have found that monodansyl cadaverine (MDC) inhibited about 70% of IL-8 induced endocytosis and caused 70% and 66% inhibition of IL-8 mediated chemotaxis and respiratory burst response, respectively, in neutrophils. The uninternalized receptor was detected by anti IL-8R antibody in MDC treated cells. The endocytosis of IL-8R was strongly inhibited under Ca2+ depleted conditions which was restored on addition of 1 mM CaCl2 indicating the critical involvement of a Ca2+ ion in the process. Absence of receptor internalisation makes the MDC treated neutrophils suitable for studying the interaction of IL-8R with potential therapeutic agents e.g. for in vitro screening of anti-inflammatory agents.

Topics: Antigens, CD; Cadaverine; Calcium; Chemotaxis; Endocytosis; Enzyme Inhibitors; Humans; Interleukin-8; Neutrophils; Receptors, Interleukin; Receptors, Interleukin-8A; Respiratory Burst

The regulation of monocyte-derived neutrophil chemotactic factor (MDNCF)/interleukin 8 (IL 8) receptor expression by the MDNCF/IL 8 ligand was examined using freshly isolated human peripheral blood neutrophils. MDNCF/IL 8 down-regulated greater than 90% of its own receptor expression within 10 min at 37 degrees C. This down-regulation was associated with internalization of the ligand. The radiolabeled MDNCF/IL 8 molecules after internalization were proteolytically degraded, and trichloroacetic acid-soluble molecules were released into the culture medium starting at 60 min. Lysosomotropic agents could inhibit this degradation of ligand suggesting the involvement of lysosomal enzymes in this proteolytic digestion. MDNCF/IL 8 receptors reappeared on the cell surface within 10 min after removal of free ligands from the culture medium. Cycloheximide did not alter the reappearance of the receptor suggesting that de novo protein synthesis of MDNCF/Il 8 receptors is not involved in this event and that receptors probably recycled. The addition of lysosomotropic agents partially inhibited the reappearance/recycling of the receptors, although none of these agents inhibited the binding of ligand to the surface receptors or ligand internalization. Ammonium chloride reduced the MDNCF/IL 8-induced neutrophil chemotactic response in a dose-dependent fashion. These data suggest that MDNCF/IL 8 receptor expression is dynamically regulated by MDNCF/IL 8 and that the rapid recycling of MDNCF/IL 8 receptors may be essential for the chemotactic response of neutrophils.

Topics: Ammonium Chloride; Cadaverine; Cell Membrane; Cells, Cultured; Chemotactic Factors; Chemotaxis, Leukocyte; Down-Regulation; Humans; Interleukin-8; Interleukins; Kinetics; Lysosomes; Neutrophils