**2. Antimicrobial responses of fish phagocytes**

It is well established that fish phagocytes possess oxidative burst responses, comparable to those of mammals. Absence of readily available fish cytokines limited the early research of fish phagocytes to employing pathogen products and/or crude activated cell supernatants, presumed to contain hallmark "activating" agents. This fundemental work of fish phago‐ cyte-mediated inflammatory processes has been comprehensively reviewed [138]. Since then the specific genes encoding the components of the fish NADPH oxidase complex (Fig. 1) have been cloned in various fish species [13, 84, 119] and their expression correlated with reactive oxygen radical production [13, 63, 141]. The priming of the fish phagocyte ROI re‐ sponses by recombinant fish cytokines such as TNFα [67, 133, 217], IFNγ [62, 63, 215] and IL-1β [54, 97, 144] has also been reported. Similar to the mammalian monocyte/macrophage paradigm [22], fish monocytes have greater ability to generate reactive oxygen intermediates (ROI) following short stimulation [67, 136, 152], whereas mature fish macrophages require relatively prolonged immune stimulations to achieve comparable magnitudes of this re‐ sponse [58, 59, 136, 171].

The last decade has yeilded significant advances in the understanding of inflammatory re‐ sponses of lower vertebrates, such as bony fish. The genes encoding hallmark cytokines have been identified and characterized in a number of fish species. Interestingly, many of these exhibit structural similarities and gene synteny organization comparable to their high‐ er vertebrate counterparts. Conversely, multiple isoforms of certain cytokines are present in

Cytokine Regulation of Teleost Inflammatory Responses

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

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Tumor necrosis factor alpha is a central inflammatory mediator, initially identifed as a se‐ rum component capable of eliciting "hemorrhagic necrosis" of certain tumors [20]. Since dis‐ covery, TNFα has been found to be produced by many cell-types and confer an increadible range of immune processess [44, 203, 209]. In the context of inflammatory responses, TNFα promotes the chemotaxis of neutrophils and monocytes/macrophages [123, 207], enhances their phagocitic capacity [95, 105, 194], primes ROI and NO reposnses [42, 135], chemoat‐

The mammaian TNFα functions as a 26 kDa type II trans-membrane protein as well as a 17 kDa soluble moiety, released by the TNFα cleaving metalloproteinase enzyme (TACE) mediated cleavage [98, 128, 148]. A homotrimerized TNFα (soluble or membrane bound) en‐ gages one of two cognate receptors, TNF-R1 or TNF-R2, which in turn trimerize around the ligand [7, 45]. Currently, there is no consensus as to the respective contribution of these re‐ ceptors to the biological effects caused by TNFα. Some evidence suggests that TNF-R1 prop‐ agates the signal from the soluble TNFα, while the membrane-bound TNFα acts exclusively through TNF-R2 [70]. Other evidence suggest that TNF-R1 is primarily involved in induc‐ tion of apoptosis while the TNF-R2 functions in proliferation and cell survival [129], while other contributions suggest cooperation between the two receptors [204]. The prevailing theory proposes that TNF-R1 confers signal propagation, while TNF-R2 binds and redistrib‐ utes TNFα to TNF-R1 in a process coined "ligand passing" [28, 43, 197]. Despite this, more recent literature suggests that TNF-R2 is directly involved in many inflammatory processes including the activation of T lymphocytes [93, 94], stimulation of myofibroblasts [190], as well as tumor suppression [212]. The TNF-R1 and TNF-R2 utilize largely non-overlapping signaling mechanisms, relying on recruitment of numerous downstream signaling mole‐ cules, the relative abundance of which ultimately dictates signaling outcomes (for a current review of TNFα signal transduction see recent review [143]). It is likely that the roles of re‐ spective TNF receptors in the biological outcomes of TNFα stimulation are cell type and cell

Presence of an endogenous bony fish TNF system was first suggested in the early 1990s where the human recombinant TNFα elicited ROI production in trout leukocytes while ad‐ ministration of a monoclonal anti-TNF-R1 antibody blocked this response [75, 87]. Hirono *et al.* [76] identified and characterized the first cDNA transcript encoding a Japanese flounder TNFα, which had only 20-30% amino acid identity with the mammalian TNFs, but had very

tracts fibroblasts [168] and elicits platelet activating factor production [19, 73, 104].

distinct fish species.

activation state dependent.

*3.1.1. Identification of TNFα in fish*

**3.1. Tumor necrosis factor alpha (TNFα)**

Phagocytes (primarily mature macrophages) also produce microbicidal/tumoricidal reactive nitrogen intermediates in a stimulus-specific manner. This response, catalyzed by the indu‐ cible nitric oxide synthase enzyme (iNOS, Fig. 2), involves the conversion of arginine to cit‐ ruline and results in the production of nitric oxide (NO) and other products including nitrite, nitrate, and nitrosamines [86, 134, 184]. The NADPH oxidase produced superoxide anion may also react with NO to form the peroxynitrite intermediate [ONOO<sup>−</sup> ] that also has potent microbiacidal activity [35, 156, 191, 213]. The biology of the iNOS enzyme has been reviewed in references [2, 111, 126].

The ability of fish phagocytes to produce microbicidal NOs has been well established (re‐ viewed in reference [138]). The iNOS gene transcript has been identified in several fish spe‐ cies [101, 102, 163, 200] and fish macrophages have been demonstrated to up-regulate iNOS expression and produce copious amounts of NO in response to a plethora of immune stimu‐ li [62-64, 66, 67, 85, 88, 140, 158, 180, 210]. The inflammatory cytokine regulation of fish iNOS and NO production is described below.
