Attività cellulari e molecolari nei leucociti dei teleostei
Author(s)
Benedetti, Stefania
Date Issued
September 10, 2008
Type
Doctoral Thesis
Abstract
In this thesis, it was studied the biological activities of the recombinant cytokine IL-1 from sea bass (Dicentrarchus labrax) and rainbow trout (Oncorhynchus mykiss), the effects of allogeneic
recognition-derived conditioned medium, of the fish pathogens nodavirus and Photobacterium
damselae, by investigating the effects induced on intracellular Ca2+ concentration [Ca2+]i of
leucocytes from lymphoid organs. Leucocytes were loaded with the calcium-permeant Fura-2AM,
and then stimulated with the above stimulants. The emitted fluorescence was read on a dual
excitation fluorescence fluorimeter for 5 min at 1 min intervals. Results showed that rIL-1 induced
in both species a rise in [Ca2+]i and a subsequent decrease until 5 min after stimulation; conditioned medium, nodavirus and Photobacterium damselae also induced in sea bass the same rise and decrease as rIL-1, but the flux induced by nodavirus and Photobacterium damselae was dependent from days after immunization. The stimulating effect was, in all cases, dose-dependent. Regarding for rIL-1, the stimulating effect was abolished by heat-treatment of cytokine, and affected in a
dose-dependent fashion by treatment of leucocytes with trypsin, thus suggesting a functional IL-1
receptor was involved in the binding. The observed rise, induced by rIL-1, was not detected in
PBMC and was species-specific, since rIL-1 from sea bass, rainbow trout and human were unable
to interfere each other in assays. Incubation of splenocytes with rIL-1 induced a rapid tyrosine
phosphorylation of a 24kDa polypeptide in both species, a rapid serine phosphorylation of a 40 and 24 kDa in rainbow trout, and a rapid threonine phosphorylation of a 24 kDa substrate in sea bass.
By proteomic analysis, the 24 kDa polypeptide, phosphorylated on tyrosine residues, was a
thymidylate kinase. These thesis represents the first evidence of a direct effect on [Ca2+]i induced by rIL-1 in a vertebrate species, suggesting that in the evolution of IL-1 activities, teleost fishes
display a peculiar IL-1-associated behaviour that is lacking in mammals. Also, cytokine-containing
conditioned media, nodavirus and bacteria may affect fish leucocytes through the induction of
second-messenger pathways.
Additional information
Dottorato di ricerca in Evoluzione biologica e biochimica
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