UNCORRECTED PROOF 1 2 Evolution of cytokine responses: IL-1b directly affects intracellular 3 Ca 2+ concentration of teleost fish leukocytes through 4 a receptor-mediated mechanism 5 S. Benedetti a , E. Randelli a , F. Buonocore a , J. Zou b , C.J. Secombes b , G. Scapigliati a, * 6 a Department of Environmental Sciences, University of Tuscia, I-01100 Viterbo, Italy 7 b Scottish Fish Immunology Research Centre, University of Aberdeen, AB24 2TZ Aberdeen, Scotland 8 Received 12 December 2005; received in revised form 6 February 2006; accepted 19 March 2006 9 10 Abstract 11 In this work we studied the biological activities of recombinant IL-1b from the teleosts sea bass (Dicentrarchus labrax) and rainbow 12 trout (Oncorhynchus mykiss) by investigating the effects induced on intracellular Ca 2+ concentrations ([Ca 2+ ] i ) of spleen leucocytes. 13 Splenocytes were loaded with the Ca 2+ -permeant Fura-2AM, and then stimulated with rIL-1b. The emitted fluorescence was read for 14 5 min at 1 min intervals on a dual excitation fluorescence fluorimeter. Results showed that rIL-1b induced in both species a rise in 15 [Ca 2+ ] i , and a subsequent decrease until 5 min after stimulation. The stimulating effect was dose-dependent in both species reaching a 16 plateau at 200 ng/ml of rIL-1b, was abolished by heat-treatment of rIL-1b, and affected in a dose-dependent fashion by treatment of 17 leucocytes with trypsin. These features suggested a functional IL-1 receptor was involved in the binding. The observed rise in [Ca 2+ ] i 18 was not detected in human PBMC and was species-specific, since rIL-1b from sea bass, trout, and human were unable to interfere each 19 other in the assay. Moreover, incubation of splenocytes with rIL-1b induced a rapid tyrosine phosphorylation of a 24 kDa polypeptide in 20 both species. This work represents the first evidence of a direct effect on [Ca 2+ ] i induced by IL-1b and suggests that in the evolution of IL- 21 1 activities, teleost fishes display a peculiar IL-1-associated behaviour that is lacking in mammals. 22 Ó 2006 Published by Elsevier Ltd. 23 Keywords: IL-1; Teleost; Intracellular Ca 2+ ; Phosphorylation; Cytokine evolution; Sea bass; Rainbow trout 24 25 1. Introduction 26 Interleukin-1b (IL-1b) is a key molecule for the regula- 27 tion of immune responses in jawed vertebrates, and its dis- 28 covery in cartilaginous and bony fish dates back cytokine 29 evolution at ca. 400 My ago [1–3]. IL-1b has a pleiotropic 30 activity, affecting in mammals nearly every cell type [4], is 31 mainly produced by activated macrophages as an inactive 32 precursor peptide, and to achieve a biological activity must 33 be cleaved intracellularly by an IL-1b converting enzyme 34 (ICE) [5]. In contrast to mammals, fish IL-1b precursors 35 cloned to date lack a clear ICE cut site [6,7]. 36 To compare the biological activity of IL-1b between 37 evolutionary-distant vertebrates, the availability of fish 38 recombinant IL-1b (rIL-1b) molecule allowed its testing 39 in ‘‘in vitro’’ and ‘‘in vivo’’ biological assays in some teleost 40 species like trout [8–10], carp [11,12], and sea bass [13,14]. 41 These studies clearly showed the activity of rIL-1b on var- 42 ious assays, suggesting that this cytokine may be endowed 43 with a pleiotropic activity also in teleosts. 44 IL-1 activity occurs as a consequence of binding to its 45 receptor complex (IL-1R) on the cell surface of target cells 46 [4,15,16], and the IL-1R transduction system in mammals 47 is extraordinarily sensitive in that just a few ligand-occu- 48 pied receptors can account for all biological activity 49 [16,17]. In teleost fish, the IL-1R has been cloned and 50 sequenced [18–20], and the 3D-structure of the IL-1/IL- 51 1R complex revealed [21]. 1043-4666/$ - see front matter Ó 2006 Published by Elsevier Ltd. doi:10.1016/j.cyto.2006.03.009 * Corresponding author. Fax: +39 0761 357179. E-mail address: scapigg@unitus.it (G. Scapigliati). www.elsevier.com/locate/issn/10434666 Cytokine xxx (2006) xxx–xxx YCYTO 51254 No. of Pages 8, Model 5+ 21 April 2006 Disk Used Jaya (CE) / Karthikeyan (TE) ARTICLE IN PRESS