Ghrelin stimulates phagocytosis and superoxide production in fish leukocytes

Abstract

To clarify the role of ghrelin in the fish immune system, the in vitro effect of ghrelin was examined in phagocytic leukocytes of rainbow trout (Oncorhynchus mykiss). Administration of trout ghrelin and des-VRQ-trout ghrelin, in which three amino acids are deleted from trout ghrelin, increased superoxide production in zymosanstimulated phagocytic leukocytes from the head kidney. Gene expression of growth hormone (GH) secretagoguereceptor (GHS-R) was detected by RT-PCR in leukocytes. Pretreatment of phagocytic leukocytes with a GHS-R antagonist, [D-Lys 3 ]-GHRP-6, abolished the stimulatory effects of trout ghrelin and des-VRQ-trout ghrelin on superoxide production. Ghrelin increased mRNA levels of superoxide dismutase and GH expressed in trout phagocytic leukocytes. Immunoneutralization of GH by addition of anti-salmon GH serum to the medium blocked the stimulatory effect of ghrelin on superoxide production. These results suggest that ghrelin stimulates phagocytosis in fish leukocytes through a GHS-Rdependent pathway, and also that the effect of ghrelin is mediated, at least in part, by GH secreted by leukocytes.

A novel GH secretagogue, A233, exhibits enhanced growth activity and innate immune system stimulation in teleosts fish

“…Similarly, the stimulatory effect of the peptide, the increase in reactive oxygen species (ROS), was also evident in isolated tilapia peripheral blood leukocytes (data not shown). These results are consistent with those observed in leukocytes from the anterior kidney of rainbow trout (Oncorhynchus mykiss) stimulated in vitro with different doses of des-GRLN or GRLN VRQ -the same kind (Yada et al 2006). Additionally, this effect is similar to that reported by Acosta et al (2010), who stimulated phagocytic peripheral blood cells of tilapia (Oreochromis sp.)…”

“…HKLs were isolated as described by Yada et al (2006) with slight modifications. Fish were killed by decapitation and the head-kidney was placed in RPMI-1640 medium (Sigma).…”

“…To analyze the effect of GHSs on the immune system, the superoxide anion was measured in HKLs as the reduction of NBT (Sigma) as described by Sakai et al (1996) and Yada et al (2006). To examine whether A233 acts through the GHSR, leukocytes were exposed to the GHSR antagonist ]-GHRP6 (10 mM).…”

“…GHSR is expressed predominantly in the brain and pituitary, but it is also expressed in many peripheral organs including immune system cells (Hattori et al 2001). GRLN modulates the immune system (Dixit et al 2004) and it has been shown to stimulate phagocytosis in fish leukocytes, and this effect is mediated in part by GH secreted by leukocytes (Yada et al 2006). The molecule A233 was described by the exhaustive molecular modeling of the human GRLN receptor using combined techniques of homology modeling, molecular dynamics, and exhaustive conformational search techniques, after which a virtual library was built with several thousands of structures having such characteristics, to perform a conformational analysis, and a massive docking experiment was performed against the receptor model (Rodriguez et al 2007).…”

Molecular cloning and mRNA expression analysis of two GH secretagogue receptor transcripts in orange-spotted grouper (Epinephelus coioides)

“…Physiological roles of ghrelin in teleosts have also been widely studied, including effects on GH secretion (Riley et al 2002, Kaiya et al 2003a,b,c, Ran et al 2004, Shepherd et al 2007, appetite stimulation (Unniappan et al 2002, Matsuda et al 2006a, Jönsson et al 2007, Miura et al 2007, Shepherd et al 2007, water intake (Kozaka & Ando 2003), reproduction (Mustonen et al 2002, Parhar et al 2003, immunity (Yada et al 2006), growth (Riley et al 2005, Fox et al 2007, intestinal motility (Olsson et al 2008), and locomotor behavior (Matsuda et al 2006a,b).…”