**4. Concluding remarks**

following LPS injection. The identification of a carp IL-1R1 was also described and the re‐

The trout IL-1RII was identified through a selective subtractive hybridization of genes upregulated following immune stimulation [164]. This receptor displayed low sequence identi‐ ty with the mammalian IL-1RII, but exhibited surprisingly similar overall gene organization including a very short intracellular domain. The gene expression of the identified sea bream IL-1RII increased in stimulated macrophages to levels 15 times greater than IL-1β expression [108], suggesting a conservation in the roles of the sea bream IL-1RII as a "decoy" receptor. Furthermore, sea bream IL-1RII expressed on HEK 293 cells bound the recombinant IL-1β,

Enhanced understanding of the evolutionary mechanisms responsible for shaping the verte‐ brate IL-1 biological pathways will be achieved through studies that examine the IL-1 recep‐

Using the selective subtractive hybridization technique, a carp IL-1β was identified [53] and the C-terminus of the cytokine produced as a recombinant protein [206]. This recombinant carp IL-1β dimerized and enhanced carp antibody responses to *Aeromonas hydrophila*, where sera from carp co-injected with the IL-1β and *A. hydrophila* had a greater agglutinating ca‐

A number of studies have since utilized recombinant technology to investigate the functions of the fish IL-1β. A recombinant form of the mature trout IL-1β (rtIL-1β) was produced by Hong and co-workers [80] and shown to enhance the expression of the MHCII β chain, IL-1β and COX-2 genes in trout head kidney leukocytes and the macrophage cell line, RTS-11. Functionally, rtIL-1β elicited the proliferation of trout head kidney cells as well as the prolif‐ eration of a murine cell line, D10.G4.1, known for its dependence on the mammalian IL-1β [80]. Also, rtIL-1β enhanced the phagocytosis of yeast particles by trout head kidney cells [80] while peritoneal admininstrations of rtIL-1β induced migration of trout leukocytes to the site of injection, enhanced phagocytosis of peritoneal cells and increased the systemic ex‐ pression of IL-1β, COX-2 and lysozyme II [77]. The injection of fish with rtIL-1β also en‐ hanced trout resistance to infection with the fish pathogen *A. salmonicida*. Additionally, the rtIL-1β and the recombinant sea bass IL-1β were demonstrated to induce Ca2+ mediated downstream signaling events, abrogated by leukocyte trypsin-treaments and indicating a re‐ quirement for receptor engagement [9]. Interestingly, these authors reported that IL-1β of trout, sea bass and humans were highly species specific, which is in contradiction of the ear‐ ly fish IL-1β carp and catfish work, where cross-reactivity was observed [9, 47, 74, 175, 195].

The distinct biological roles of individual sub-domains of the trout IL-1β were also exam‐ ined by generating appropriate peptides [78, 144, 145]. While a control scrambled peptide (P2) had no effect and the peptide corresponding to the putative trout IL-1β receptor bind‐ ing region (P1) had little effect on its own, a peptide (P3) corresponding to an alternative re‐ ceptor binding area of the mammalian IL-1β [5, 196], had chemotactic properties towards

tor-ligand relationships and biological outcomes of these interactions in teleosts.

ceptor characterized in the context of acute stress conditions [121].

confirming the specificity of this receptor-ligand pair.

*3.3.6. Inflammatory roles of the fish IL-1β*

74 New Advances and Contributions to Fish Biology

pacity than the respective controls.

A successful inflammatory response is defined by the presence and proficient coordination of cytokine networks consisting of hallmark mediators such as TNFα, IFNγ and IL-1β. The synchronized involvement of these pleiotropic yet functionally distinct agents in the recruit‐ ment, regulation and functional polarization of inflammatory cells dictates the outcome of the mounted response. Thus, it can be argued that the inflammatory processes are largely defined by the efficacy of the individual and interdependent cytokine pathways.

The regulation of the vertebrate inflammatory response is complex, involving numerous mechanisms, some of which are poorly understood while others remain to be identified. This is particularly true for the teleost model systems, where lack of specific reagents for dif‐ ferent fish species hampers our ability to examine different aspects of the regulation of in‐ flammation at a mechanistic level. However, there is growing evidence that the key immune components required for effective inflammatory responses are present in teleosts. Notably, certain fish species possess additional pathways that regulate inflammatory processes (for example IFNγrel and its receptor IFNGR1-1, novel chemokines and PRRs) that are distinct from those reported in mammals. The elucidation of the coordination of inflammatory re‐ sponses by these factors may shed new light on the evolution of innate host defense mecha‐ nisms in lower vertebrates.

**Figure 1.** Schematic representation of the NADPH oxidase complex mobilization and reactive oxygen production follow‐ ing phagocyte activation. In a resting state, the gp91phox and p22 phox components are membrane bound while the p40 phox, p47 phox and p67 phox components are located in the cytosol and the small G-protein Rac is GDP bound. Upon cell acti‐ vation, Rac is rapidly converted from a GDP- to a GTP-bound state and facilitates the translocation and assembly of the cytosolic NADPH oxidase components at the cell membrane. Following PKC activation, Rap1 is thought to serve as the fi‐ nal switch in the activation of the NADPH complex. The activated NADPH oxidase complex accepts the electrons form the reduced NADPH and transfers these to molecular oxygen, forming the superoxide anion (O2 - ). The generated O2 can sub‐ sequently be converted to other reactive oxygen species. Reviewed in references [124, 153, 154, 173].

**Figure 2.** Schematic representation of iNOS gene expression, iNOS protein synthesis and enzymatic production of ni‐ tric oxide by the iNOS dimer complex. Upon phagocyte activation, there is a substantial increase in the expression from the iNOS gene and de novo synthesis of the iNOS enzyme. The iNOS enzyme forms a dimer and associates with a Ca2+ bound calmodulin, stabilizing the structure and facilitating the function of the enzymatic complex. The iNOS dim‐ er catalyzes the transfer of electrons from a reduced NADPH, through a series of cofactors (FAD, FMN, Fe, BH4) in the

). The generated NO-

Cytokine Regulation of Teleost Inflammatory Responses

http://dx.doi.org/10.5772/53505

77

can sub‐

oxidation of L-Arginine to L-Citrulline with a concomitant production of nitric oxide (NO-

sequently be converted to other reactive nitrogen species. Reviewed in references [2, 111, 126, 172].

**Figure 2.** Schematic representation of iNOS gene expression, iNOS protein synthesis and enzymatic production of ni‐ tric oxide by the iNOS dimer complex. Upon phagocyte activation, there is a substantial increase in the expression from the iNOS gene and de novo synthesis of the iNOS enzyme. The iNOS enzyme forms a dimer and associates with a Ca2+ bound calmodulin, stabilizing the structure and facilitating the function of the enzymatic complex. The iNOS dim‐ er catalyzes the transfer of electrons from a reduced NADPH, through a series of cofactors (FAD, FMN, Fe, BH4) in the oxidation of L-Arginine to L-Citrulline with a concomitant production of nitric oxide (NO- ). The generated NO can sub‐ sequently be converted to other reactive nitrogen species. Reviewed in references [2, 111, 126, 172].

**Figure 1.** Schematic representation of the NADPH oxidase complex mobilization and reactive oxygen production follow‐ ing phagocyte activation. In a resting state, the gp91phox and p22 phox components are membrane bound while the p40 phox, p47 phox and p67 phox components are located in the cytosol and the small G-protein Rac is GDP bound. Upon cell acti‐ vation, Rac is rapidly converted from a GDP- to a GTP-bound state and facilitates the translocation and assembly of the cytosolic NADPH oxidase components at the cell membrane. Following PKC activation, Rap1 is thought to serve as the fi‐ nal switch in the activation of the NADPH complex. The activated NADPH oxidase complex accepts the electrons form the


). The generated O2

 can sub‐

reduced NADPH and transfers these to molecular oxygen, forming the superoxide anion (O2

76 New Advances and Contributions to Fish Biology

sequently be converted to other reactive oxygen species. Reviewed in references [124, 153, 154, 173].

## **Abbreviations**

**ConA:** concanavalin A; **GAS:** γ-IFN-activated sequence; **Jak:** janus activated kinase; **ICE:** IL-1 cleaving enzyme; **IFN:** interferon; **IFNGR:** interferon gamma receptor; **IL:** interleukin; **IL-1R:** interleukin-1 receptor; **IL-1RAcP:** IL-1R associated protein; **iNOS:** inucible nitric ox‐ ides synthase; **IRAK:** IL-1R associated kinase; **IRF:** interferon regulatory factor; **MAF:** mac‐ rophage activating factor(s); **NK:** natural killer; **NLS:** nuclear localization signal; **NO:** nitric oxide; **NTR:** neurotropin receptor; **PBL:** peripheral blood leukocyte; **PHA:** phytohemagluta‐ nin; **PKC:** protein kinase C; **PKM:** primary kidney macrophage; **PMA:** phorbol myrystate acitate; **PRR:** pattern recognition receptor; **rg:** recombinant goldfish; **RNI:** reactive nitrogen intermediates; **ROI:** reactive oxygen intermediates; **Stat:** signal transducer of activation tran‐ scription factor; **TACE:** TNFα cleaving enzyme; **TLR:** toll-like receptor; **TNF:** tumor necrosis factor; **TNFR:** tumor necrosis factor receptor.

[6] Auron, P. E., A. C. Webb, L. J. Rosenwasser, S. F. Mucci, A. Rich, S. M. Wolff, and C. A. Dinarello. 1984. Nucleotide sequence of human monocyte interleukin 1 precursor

Cytokine Regulation of Teleost Inflammatory Responses

http://dx.doi.org/10.5772/53505

79

[7] Banner, D. W., A. D'Arcy, W. Janes, R. Gentz, H. J. Schoenfeld, C. Broger, H. Loetsch‐ er, and W. Lesslauer. 1993. Crystal structure of the soluble human 55 kd TNF recep‐ tor-human TNF beta complex: implications for TNF receptor activation. Cell

[8] Belosevic, M., C. E. Davis, M. S. Meltzer, and C. A. Nacy. 1988. Regulation of activat‐ ed macrophage antimicrobial activities. Identification of lymphokines that cooperate with IFN-gamma for induction of resistance to infection. J Immunol 141:890-896.

[9] Benedetti, S., E. Randelli, F. Buonocore, J. Zou, C. J. Secombes, and G. Scapigliati. 2006. Evolution of cytokine responses: IL-1beta directly affects intracellular Ca2+ con‐ centration of teleost fish leukocytes through a receptor-mediated mechanism. Cyto‐

[10] Berton, G., L. Zeni, M. A. Cassatella, and F. Rossi. 1986. Gamma interferon is able to enhance the oxidative metabolism of human neutrophils. Biochem Biophys Res Com‐

[11] Bluyssen, H. A., R. Muzaffar, R. J. Vlieststra, A. C. van der Made, S. Leung, G. R. Stark, I. M. Kerr, J. Trapman, and D. E. Levy. 1995. Combinatorial association and abundance of components of interferon-stimulated gene factor 3 dictate the selectivi‐

[12] Bobe, J., and F. W. Goetz. 2001. Molecular cloning and expression of a TNF receptor and two TNF ligands in the fish ovary. Comp Biochem Physiol B Biochem Mol Biol

[13] Boltana, S., C. Donate, F. W. Goetz, S. MacKenzie, and J. C. Balasch. 2009. Characteri‐ zation and expression of NADPH oxidase in LPS-, poly(I:C)- and zymosan-stimulat‐ ed trout (Oncorhynchus mykiss W.) macrophages. Fish Shellfish Immunol

[14] Bosco, M. C., G. L. Gusella, I. Espinoza-Delgado, D. L. Longo, and L. Varesio. 1994. Interferon-gamma upregulates interleukin-8 gene expression in human monocytic

[15] Bujak, M., and N. G. Frangogiannis. 2009. The role of IL-1 in the pathogenesis of

[16] Buonocore, F., M. Forlenza, E. Randelli, S. Benedetti, P. Bossu, S. Meloni, C. J. Se‐ combes, M. Mazzini, and G. Scapigliati. 2005. Biological activity of sea bass (Dicen‐ trarchus labrax L.) recombinant interleukin-1beta. Mar Biotechnol (NY) 7:609-617.

[17] Buonocore, F., M. Mazzini, M. Forlenza, E. Randelli, C. J. Secombes, J. Zou, and G. Scapigliati. 2004. Expression in Escherchia coli and purification of sea bass (Dicen‐

ty of interferon responses. Proc Natl Acad Sci U S A 92:5645-5649.

cells by a posttranscriptional mechanism. Blood 83:537-542.

heart disease. Arch Immunol Ther Exp (Warsz) 57:165-176.

cDNA. Proc Natl Acad Sci U S A 81:7907-7911.

73:431-445.

kine 34:9-16.

129:475-481.

26:651-661.

mun 138:1276-1282.
