Comparison of select innate immune mechanisms of fish and mammals

Invited Review Article

Comparison of select innate immunemechanisms of fish and mammals

Introduction

Prior to the emergence of the genes encoding thehallmark components of the adaptive immuneresponse [T- and B-cell receptors and the majorhistocompatibility complex (MHC) molecules],organisms relied exclusively on the diversity ofnon-specific defenses [1]. Fish represent the earliestclass of vertebrates possessing the elements of bothinnate and acquired immunity, although it is clearthat their adaptive immune response is less devel-oped than that of higher vertebrates [2,3]. Despitethis apparent deficiency, fish comprise the greatestgroup of vertebrate species and its members can befound in most extreme aquatic habitats. In addi-tion, fish are in constant contact with an environ-ment containing potential pathogenic organismsand have evolved a number of constitutive andinducible innate immune responses to defendagainst infection.This review will focus on the information

obtained from the study of the innate immuneresponses of different fish groups, and whereverpossible highlight the differences in the innate

immune mechanisms of lower and higher verte-brates.

Soluble mediators of innate immunity in fish

Antimicrobial peptides

Antimicrobial peptides are small (12 to 50 aminoacids) molecules that have a broad spectrum ofactivity against bacterial, fungal, viral, and proto-zoan pathogens [4]. The localization of antimicro-bial peptides to host epithelial cells and mucosalsurfaces is a testament to their important role inthe first line of defense against invading pathogens[5,6]. In addition to their direct microbicidaleffects, antimicrobial peptides have other roles ininflammatory responses, including recruitment ofneutrophils and fibroblasts, promotion of mast celldegranulation, enhancement of phagocytosis, anddecreasing fibrinolysis. In order to prevent tissueinjury associated with chronic inflammatoryresponses, antimicrobial peptides stimulate apop-tosis of activated/infected cells, decrease cytokineproduction, and some have the ability to bind and

Plouffe DA, Hanington PC, Walsh JG, Wilson EC, Belosevic M.Comparison of select innate immune mechanisms of fish and mammals.Xenotransplantation 2005; 12: 266–277. � Blackwell Munksgaard, 2005

Abstract: The study of innate immunity has become increasinglypopular since the discovery of homologs of many of the innate immunesystem components and pathways in lower organisms including inver-tebrates. As fish occupy a key position in the evolution of the innate andadaptive immune responses, there has been a great deal of interestregarding similarities and differences between their defense mechanismsand those of higher vertebrates. This review focuses on describing selectmechanisms of the innate immune responses of fish and the implicationsfor evolution of immunity in higher vertebrates.

Debbie A. Plouffe,1 PatrickC. Hanington,1 John G. Walsh,1

Elaine C. Wilson1 and MiodragBelosevic1,21Department of Biological Sciences and 2MedicalMicrobiology and Immunology, University of Alberta,Edmonton, Alberta, Canada

Key words: antimicrobial peptides – cytotoxic cells– fish – inflammation – innate immunity –mammals – phagocytes – teleosts –xenotransplantation

Address reprint requests to Dr M. Belosevic,Department of Biological Sciences, CW-405Biological Sciences Building, University ofAlberta, Edmonton, Alberta, Canada T6G 2E9(E-mail: [email protected])

Received 28 February 2005;Accepted 4 March 2005

Xenotransplantation 2005: 12: 266–277Printed in Singapore. All rights reserveddoi: 10.1111/j.1399-3089.2005.00227.x

Copyright � Blackwell Munksgaard 2005

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neutralize bacterial lipopolysaccharide (LPS), pre-venting endotoxin-induced damage [4,6–8].Compared with other classes of vertebrates, the

quantity and diversity of mature antimicrobialpeptides identified in fish is relatively small. Itshould be noted, however, that many species of fishpossess a number of other innate defense moleculesthat can be found atmucosal/epithelial surfaces thatare also important in innate defense. These mole-cules include natural antibodies, apolipoproteins,lysozyme (of which a number of different isotypeshave been identified), non-peptide antimicrobialcompounds such as squalamine (a 655-Da cationicsteroidal compound from the dogfish shark), andmost recently cationic steroidal derivatives fromcatfish peripheral blood leukocytes [9–14].Fish antimicrobial peptides have broad-spec-

trum activity at sub-micromolar concentrationsagainst a wide range of human and fish pathogens,including bacteria, fungi, viruses, and protozoa.From the point of view of evolution of thevertebrate immune system, the identification ofantimicrobial peptides from the intestinal sub-mucosa of the Atlantic hagfish (Myxine glutinosa)is perhaps the most intriguing as they seem torepresent the evolutionary precursors of the cath-elicidin family of peptides thus far found only inmammals [15]. In the bony fish (teleosts), agrowing number of cationic peptides have beenisolated from a variety of species. Most of thesemolecules have been isolated from the epidermalcells or secretions of the skin, gills, and intestine.Many of the fish antimicrobial peptides have highsequence homology to segments of other proteins(particularly histone or histone-like molecules)indicating that they may in fact be cleavageproducts of larger molecules. Cleavage peptideshave been isolated from the channel catfish,rainbow trout, hybrid-striped bass, as well as Cohoand Atlantic salmon [16,17]. A number of specificantimicrobial molecules characterized in rainbowtrout, called onchorhyncins, have been found to bevery similar to chromosomal proteins [12,18,19].The remaining antimicrobial peptides isolatedfrom fish are a heterogenous group of compounds;the majority of which are known to form amphi-pathic a-helices. They include the pardaxins [20],misgurin [21], the pleurocidins [22], the piscidinsand moronocidins [23,24], the chrysophsins [25],and two hydrophobic, pore-forming peptides fromcarp [26].

Pro-inflammatory cytokines

The recent sequencing of the pufferfish and zebra-fish genomes, and the numerous fish-expressed

sequence tag databases appearing online, has beenof central importance for the identification andcharacterization of fish cytokines. In general, fishpossess a repertoire of cytokines similar to that ofmammals. At present, the most well-characterizedfish cytokines are TNF-a and IL-1b, which sharefunctional similarities with their mammalian coun-terparts.

Tumor necrosis factor-aTumor necrosis factor (TNF)-like genes have beenidentified in a number of fish species. The Japaneseflounder TNF was identified using an expressedsequence tag screen of flounder immune tissues[27]. Subsequently, TNF-like genes were discov-ered in rainbow trout [28], brook trout [29],gilthead sea bream [30], carp and channel catfish[31]. Genomic analysis of the rainbow trout TNFgene revealed an organization similar to mamma-lian TNF-a, with the coding sequences of troutTNF and mammalian TNF-a both spanning fourexons of similar size [28].Mammalian TNF-a is produced by macroph-

ages, monocytes, neutrophils, natural killer (NK)cells, and T-cells after stimulation with LPS.Similarly, the mRNA expression of TNF-a inperipheral blood leukocytes and macrophages ofrainbow trout, brook trout, channel catfish, carp,and Japanese flounder has been shown to increasefollowing stimulation with LPS [28,31–33].Recombinant trout TNF-a has been shown toincrease the phagocytosis and chemotaxis ofrainbow trout anterior kidney leukocytes, andinduce the expression of a number of genesinvolved in the immune response including IL-1b, IL-8, and COX2 [31].Tumor necrosis factor receptors have been

identified in zebrafish and Japanese flounder. Thezebrafish TNF receptor, named OTR for ovarianTNF receptor, was identified from cDNA ofzebrafish ovaries and encodes a 438 amino acidprotein that contains the signature cysteine-richdomains, transmembrane domain, and a deathdomain. Furthermore, the residues in the deathdomain required for the receptor to induce killingare conserved between ovarian TNF receptors andother apoptosis-inducing TNF receptors. Theexpression of the ovarian TNF receptor transcriptappears to be highest in the ovary with lowerexpression observed in the gills, heart, intestine,kidney, muscle, and testis [29].

Interleukin-1bInterleukin-1 (IL-1) is a pro-inflammatory cytokinethat is involved in the process of inflammation aswell as the induction of other immunomodulatory

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cytokines. In mammals, the IL-1 family is com-posed of 10 ligands and 10 receptors [34].Interleukin-1b has been identified in 13 different

species of fish, and has been shown to be function-ally active in many of the species where it has beenfound. Phylogenetic analysis of the teleost IL-1bmolecules groups them in a cluster separate fromthe molecules isolated from amphibians, birds, andmammals [35]. One of the most striking differencesin the amino acid sequences of fish IL-1b, whencompared with mammalian IL-1b, is the absence ofa clear caspase 1 cut site [36]. However, in the RTS-11 trout macrophage cell line, it has been shownthat the 29 kDa precursor IL-1b was cleaved into a24-kDa molecule detectable in culture superna-tants. These findings suggest that trout IL-1b mustbe cleaved to become activated and secreted [37].Functional assays have demonstrated that tele-

ost IL-1b shares many of the same characteristicsas mammalian IL-1b. Sequence analysis leading tothe identification of a putative IL-1b cut sitefacilitated the production of a recombinant, bio-active, IL-1b molecule [38]. Stimulation with IL-1bupregulated the expression of other transcriptsrelated to the immune response such as COX2 andMHC class II. The incubation of trout anteriorkidney leukocytes with recombinant IL-1b inducesphagocytosis and chemotaxis [38,39]. Carp IL-1binduces the proliferation of carp leukocytes, andthe addition of carp IL-1b to a vaccine againstAeromonas hydrophila yielded a significant increasein the agglutinating antibody titer [40].Interleukin-1b exerts its biologic effects by acti-

vating its receptor. To date, IL-1-like receptorshave been identified in Atlantic salmon (Salmosalar) and rainbow trout [41,42]. The expression ofsalmon IL-1R appears to be constitutive in alltissues tested, and was upregulated in the anteriorkidney, spleen, liver, and gills following LPStreatment [42].

Interleukin-18Interleukin-18 is a member of the IL-1 family ofcytokines and, like IL-1b, IL-18 is synthesized asan inactive precursor peptide that is stored intra-cellularly. The active form of IL-18 is released aftercleavage of the mature peptide by caspase 1. It isthe cleavage of the mature peptide from theinactive precursor form that is thought to be theprimary regulatory mechanism of IL-18 activity[43–45]. In mammals, IL-18 is produced by macro-phages, dendritic cells, T cells, and B cells, and itcan also act synergistically with IL-12 to induceIFN-c production by Th1 T-cells and NK cells.Zou et al. [46] identified a partial IL-18-like

sequence in trout by searching salmon EST

databases. The trout IL-18 gene has a similarorganization to the IL-18 gene of humans (con-taining six exons and five introns). Constitutiveexpression of trout IL-18 was observed in brain,gill, gut, heart, kidney, liver, muscle, skin, andspleen, with the highest levels being found in thespleen and kidney.

InterferonsIn mammals, there are two types of interferons(IFNs), type I and type II. The type I IFNs areprimarily antiviral cytokines that are produced inresponse to viral challenge or exposure to double-stranded RNA [reviewed in 47]. The type II IFNsconsist of only IFN-c that is produced mainly byTh1 T-cells and NK cells. IFN-c activates phago-cytes, T-cells, and NK-cells [48].Although the presence of IFN-like activity in

crude fish cell supernatants has been known fordecades, an IFN-like molecule had not beenidentified in fish [49,50]. To date, IFN genes havebeen identified in catfish [51], pufferfish [50],Atlantic salmon [52], and rainbow trout (GenbankAJ582754 and AJ580911).Evidence suggesting that fish IFN genes are

functionally similar to those found in mammals isderived from the observation of mRNA expressionduring viral infection. Catfish IFN expression wasincreased 2 h after exposure to a double-strandedRNA retrovirus that had been UV-inactivated. Itsexpression was also elevated 2 h after exposure topoly-inosinic:cytidilic acid (poly I:C) [51]. poly I:Cinduced the expression of zebrafish IFN dramatic-ally [53]. In addition, zebrafish and rainbow troutIFN transcripts activate the IFN-inducible Mxpromoter [49,54]. Current research has focused onthe cloning of other IFN-inducible genes as well asfactors involved in IFN signaling [53,55].

Chemokines and interleukin-8Although mammalian chemokines have been wellcharacterized, chemokines of lower vertebrateshave only recently become a focus of immunolo-gical research. The first fish chemokine, CK1, wasidentified in rainbow trout [56]. To date, chemok-ines have been identified in carp [57], the silverchimera (Chimaera phantasma) [58], catfish [59],the banded dogfish [60], flounder (Paralichthysolivaceous) [61] lamprey [62], the catshark [63], andrainbow trout [64,65].Among the fish chemokines, most is known

about IL-8. The main functions of IL-8 are tostimulate neutrophil activation, migration, anddegranulation [66–68]. It is produced by a varietyof cells, primarily endothelial cells and macroph-ages, in response to activating stimuli including

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other cytokines and LPS [69]. The gene encodingIL-8 has been identified in lamprey [62], trout[65,70], catfish [59], and the silver chimera [60]. Inthe RTS-11 trout macrophage cell line, IL-8expression was induced by exposure to LPS andpoly I:C [65].Chemokine receptors have been identified in

lamprey [63], trout [71], and sterlet [72]. None ofthe receptors have been functionally characterized,nor have expression studies been performed todetermine whether they exhibit expression patternssuggesting sensitivity to cellular activation.

Complement

In mammals, the complement system consists ofmore than 30 serum and membrane-associatedproteins that function in activation and regulation[73]. Among the lower vertebrates, the complementsystem has been intensively studied in the threemain classes of fish, including the agnathans(hagfish and lamprey), the cartilaginous fish(sharks and rays), and the bony fish (teleosts). Assuch, fish provide an excellent model in which tostudy the complement system in terms of itsevolution and function. The complement systemof teleost fish is particularly unique in that theypossess multiple isoforms of some of the importantcomplement components (C3/4/5 and factor B)that are structurally and functionally diverse.The complement system of teleosts is the most

intensively studied among the lower vertebrates.Functional studies as well as the isolation and/orcloning of genes for most of the complementcomponents has provided strong support for theexistence of all three pathways of activation as wellas a functional lytic pathway. The most intriguingdifference between the teleost complement systemand the complement systems of other vertebrates isthe structural and functional diversity of some ofits components. Teleosts possess a large repertoireof genes encoding the complement components,and some of these genes also demonstrate allelicpolymorphism. The presence of multiple copies ofsome of the central complement proteins, such asC3 and factor B, has generated a great deal ofinterest in determining the functional purpose ofthese duplications [74].Multiple genes encoding structurally and func-

tionally different C3 molecules have been identifiedin rainbow trout, carp, gilthead sea bream, med-aka, and zebrafish. Some of the earliest studiesinvolved the isolation of four different C3 proteinsfrom the serum of rainbow trout [74,75]. Therainbow trout C3 isoforms were found to binddifferentially to various complement-activating

surfaces (zymosan, sheep and rabbit erythrocytes)suggesting that the evolution of multiple C3molecules may aid in the recognition of a varietyof pathogenic organisms [74].Homologs to mammalian C5, the initiating

molecule of the terminal lytic cascade, have beenidentified in trout [76], sea bream [77], and carp[78]. In rainbow trout, a membrane attack complex(MAC)-forming C5 protein was purified fromplasma, and a cDNA clone representing a singleC5 gene was cloned from the liver [76]. In carp, twodistinct, full-length, C5 cDNA clones (C5-I andC5-II) in addition to a truncated form of C5-I wereisolated from the liver. The remainder of theterminal components (C6, C7, C8, and C9) havealso been identified from a variety of fish species,including carp [79], Japanese flounder [80], andrainbow trout [81,82].One of the most interesting implications for the

existence of functionally distinct isoforms of manyof the complement components in teleosts, partic-ularly the C3/4/5 molecules, is the possibility thatthe cleavage products of these molecules maypossess their own diverse functions. For example,as complement components are known to have arole in linking innate and adaptive immunity, thereis the potential for the different fish proteins tohave a number of effects on both branches of theimmune response. The C3a, C4a, and C5a mole-cules are conserved in teleost fish, and studies haveshown that activation of serum using LPS orzymosan can induce chemotaxis, respiratory burst,and phagocytosis [83–85]. Recently C3a and C5areceptors have been identified in carp and rainbowtrout [86,87]. In trout, the C5a receptor appears tobe primarily expressed on granulocytes and macro-phages [87].

Cells and innate immunity of fish

Fish cytotoxic cells and their receptors

The ability to recognize the presence of a danger-ous pathogen is essential for the generation of anappropriate and effective immune response. Owingto the limitations of their adaptive response, fishrely heavily on innate immune mechanisms to dealwith pathogens [3]. A number of similarities existbetween the cells involved in innate responses inmammals and fish; however, novel cell populationsand receptors have been identified in fish.

Non-specific cytotoxic cellsEvans et al. [88–90] reported the presence andcharacterization of a novel cytotoxic cell popula-tion in the channel catfish. These cells, called


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non-specific cytotoxic cells (NCCs), are unique intheir ability to lyse various transformed humanand mouse cell lines. The lysis of target cellsrequired direct cell–cell contact through a mech-anism thought to be similar to that of mammalianNK cells. Morphologically these cells are similar tomonocytes. NCCs are found in the fish kidneyrather than the blood, and functional similarities tomammalian NK cells have led researchers topostulate that NCCs may represent the evolution-ary precursor to NK cells [88–90]. NCCs havebeen identified in teleost species including rainbowtrout, carp, damselfish, and tilapia [91–93].In addition to their ability to lyse transformed

cell lines, NCCs appear to participate in theimmune response against protozoan parasites.Graves et al. [94] demonstrated in vitro killing ofTetrahymena pyriformis, an opportunistic proto-zoan parasite, by catfish NCCs. Through bindingdeletion experiments, it was found that anotherprotozoan parasite, Ichthyophthirius multifiliis, hada similar target antigen. In addition, infection withI. multifiliis was found to cause a shift towardincreased percentages of NCCs in the peripheralblood [94]. A monoclonal antibody was used toidentify a 40 to 50 kDa molecule from the lysatesof the protozoan T. pyriformis that was recognizedby catfish NCCs. The amino acid sequence of theprotein, named NKTag, has no significant homol-ogies with any known sequence. However, solubleor purified NKTag was able to inhibit NCC-mediated lysis of target cells. Immunofluorescencestudies have revealed that a related protein wasexpressed on several transformed mammalian celllines, including NC-37 and MOLT-4 [95,96]. Theseresults highlight the importance of the NKTagepitope to the cytotoxic response of NCCs andpossibly other NK cells.Immunoprecipitation experiments usingmAb5C6

(anti-NCC antibody) resulted in the identificationof a protein of 34 kDa namedNCC receptor protein1 (NCCRP-1) [97–100]. The engagement ofNCCRP-1 by NKTag was found to be responsiblefor the recognition and killing of target cells byNCCs [95,96,98,101]. Further analysis, using syn-thetic peptides to mimic the ligand demonstratedthat the ligand-binding region of NCCRP-1 waslocated between amino acids 104 and 119 [102].These researchers have proposed that NKTagmay be the only target molecule recognized byNCCRP-1.

Cytotoxic NK-like cellsIn addition to the NCCs, which may representan evolutionary precursor of mammalian NKcells [88–90], teleosts also possess NK-like cells.

Recently, new clonal NK-cell lines have beenidentified from alloantigen-stimulated catfishlymphocytes that share functional and morpholo-gical similarities to mammalian NK cells, but aredistinct from NCCs [103]. The catfish NK-like cellsare large and granular, which is similar to mam-malian NK cells, but unlike NCCs. These NK-likecells are able to lyse allogenic target cells but fail toexpress Igl or TCR a, b, c, excluding the possi-bility that they are B or T-cells. In addition, thesecells do not stain with Sudan Black B and are non-specific esterase-negative indicating that they arenot neutrophils or macrophages. Perhaps the mostsignificant finding was that mAb 5C6 (anti-NCCantibody), failed to recognize these cells, suggestingthat they are not NCCs [103]. Although all of theNK-like cell lines investigated exhibited cytotoxic-ity toward allogenic targets, the specificity fortargets varied between clones. Some clones killedseveral different allogenic cells equally, while othersdemonstrated a marked specificity for a particularcell type. Killing of target cells by catfish NK-likecells appears to be mediated through the inductionof apoptosis [103].

Novel immune-type receptors of fishOriginally identified in the pufferfish, novel im-mune-type receptors (NITRs) are a group of NKcell receptors that have been identified as a memberof the immunoglobulin superfamily, and have noknown homolog in mammals [104]. Although theprecise function of NITRs has not been determined,they share many structural features with mamma-lian NK cell receptors and are hypothesized to bethe functional orthologs of the mammalian recep-tors [105,106]. NITRs have been identified inchannel catfish [107], zebrafish [108], rainbow trout[109], and pufferfish [104]. More than 40 NITRgenes have been identified in the zebrafish genomenear the top of linkage group 7, which is an areaflanking the zebrafish NITR gene complex. Thisarea shares synteny with human chromo-some19q13.3-q13.4, an area that encodes the leu-kocyte receptor cluster [108]. The LRC region ofhumans codes a number of receptors includingkiller Ig-like receptors (KIR), paired Ig-like recep-tors (PIR), leukocyte inhibitory receptors (LIR)and monocyte inhibitory receptors (MIR), as wellas leukocyte-associated inhibitory receptors(LAIR) [110–114]. Unlike the genes encoded bythe leukocyte receptor cluster region, NITR genesencode for a variable (V) domain as well as a V-likeconstant domain (V/C2), whereas leukocyte recep-tor cluster genes do not encode V regions [107].The NITR genes identified in the pufferfish,

rainbow trout, and zebrafish all possess

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immunoreceptor tyrosine-based inhibition motifs(ITIMs) in their cytoplasmic regions [104,108,109].The presence of ITIMs suggests that these recep-tors function in the deactivation or inhibition ofthe cells on which they are expressed. ITIMsassociate with SHP-1 and SHP-2 to dephosphory-late effector substrates involved in the NK lyticpathway, especially MAPK that is involved inregulating NK cell activity against tumor cells[115]. The NITRs are expressed mainly in thespleen, kidney, and intestine of the catfish, and areexpressed in a number of catfish cell lines, suggest-ing that these receptors play an important rolecontrolling cellular immune responses [107].

C-type lectins/cKLRNatural killer cell receptors can be divided intotwo broad classes that differ in their origins butare similar in function. Killer Ig-like receptors aretype I transmembrane glycoproteins that haveone to three extracellular immunoglobulin do-mains. Killer cell C-type lectin receptors (KLR)are type II transmembrane proteins that possessan extracellular C-type lectin domain and exist ashomo- or heterodimers [116]. These receptorsmonitor the expression of MHC class I or MHCclass I-like molecules on host cells. While noKIRs have been discovered in fish, a smallnumber of KLRs have been identified. Two C-type lectins (TCL-1 and TCL-2) have beenidentified in rainbow trout and both moleculesare similar to known mammalian C-type lectins[117,118]. TCL-2 differs from TCL-1 in that itpossesses two ITIMs. TCL-2 maps near the novelimmune type receptor (NITR) gene cluster; how-ever, it is unlikely to be related to the Ly49 orNKG2 gene families [109].C-type lectin receptors have also been identified

in two species of cichlid fish, Paralabidochromischilotes and Oreochromis niloticus [119]. cKLRwas identified from an EST screen of P. chilotesand was found to have homology with the CD94/NKG2 subfamily of KLRs. cKLR was found topossess a positively-charged amino acid (Arg-42)in the transmembrane domain, a feature that iscommonly found in receptors that interact withimmunoreceptor tyrosine-based activating motifs.The absence of an ITIM in cKLR also supportsthis hypothesis. The gene encoding this receptorin a related fish, O. niloticus, has a similarstructure to the mammalian CD94/NKG2 genes.DNA hybridization studies of bacterial artificialchromosome clones reveal that the cKLR gene isa member of a multi-gene family that is made upof at least 10 copies of cKLR-like sequences[119].

Toll-like receptorsOne group of receptors that has received attentionfor its role in innate immunity is the Toll-likereceptor (TLR). The Toll receptor was originallydiscovered in Drosophila and was identified as animportant molecule involved in larval develop-ment. It was later found to play a critical role infruit fly defense against fungal and bacterialinfections [120]. As Toll was found to have anadditional role in the invertebrate immune system,it was hypothesized that these receptors mightrepresent an evolutionarily ancient mechanism ofinnate immune recognition that may have beenconserved in vertebrates. To date, there have been10 TLRs found in mammals, each possessing aToll/IL-1R cytoplasmic domain responsible forsignaling, as well as an extracellular leucine-richrepeat domain that is involved in the recognition ofspecific molecular signatures of a variety of patho-gens [121–123].The first teleost TLR was characterized in our

laboratory from a goldfish-stimulated macrophagecDNA library enriched for differentially expressedgenes by suppressive subtractive hybridization[124]. Shortly thereafter, TLR genes were identifiedin pufferfish, zebrafish, Japanese flounder, and thechannel catfish [125–128]. As genome sequences areavailable for zebrafish and pufferfish, it was poss-ible to identify TLR homologs present in each ofthese species. Analysis of the pufferfish genomeresulted in the discovery of TLR homologs foreach of the known mammalian TLR genes with theexception of TLR4 and TLR6. Interestingly, twonovel TLRs, TLR21 and TLR22, were also iden-tified [128]. Similar results were obtained from asurvey of the zebrafish genome; however, a homo-log of TLR 4 was identified in addition to TLR21and TLR22. Furthermore, it appears that a clusterof fish-specific TLRs has been identified in zebra-fish, consisting of TLR 21, TLR 22, and possiblyTLR19 and TLR20a/b (possible orthologs ofTLR21) [126,127]. With the identification of whatappears to be all of the teleost TLRs, it has becomesimpler to identify TLR homologs in other fishspecies. The goldfish TLR was shown to havehighest homology with TLR21 of the zebrafish andpufferfish [126], as well as with TLR22 of theJapanese flounder [125].Functional analysis of the different teleost TLRs

has been limited to analysis of tissue expression. Inpufferfish, the expression of TLR2, TLR5, andTLR22 appears to be ubiquitous amongst thetissues analyzed, whereas TLR1 and TLR7 werehighly expressed in the kidney. TLR9 exhibits highexpression in the kidney, digestive gland, skin, andheart, while TLR21 shows highest expression in the


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heart and gill [128]. In contrast, TLR22 of Japan-ese flounder was only found to be expressed in theanterior kidney and peripheral blood leukocytes,while TLR2 was found in those tissues in additionto trunk kidney, spleen, and gill [125]. The analysisof the expression of the goldfish TLR showedlocalized expression in the spleen and kidney, andan increase in expression was observed in primarymacrophage cultures 3, 6, and 24 h after stimula-tion with LPS and macrophage-activation factor(MAF) [124].

Antimicrobial response of fish phagocytes

Respiratory burst response

In mammalian systems, phagocytosis is associatedwith an increase in oxygen consumption by specificimmune cells. The phenomenon is known as the‘‘respiratory burst’’ and has subsequently beenshown to correspond to the production of reactiveoxygen intermediates [129]. Until recently, thisresponse was the sole example of deliberatelyproduced reactive oxygen intermediates catalyzedby the multi-component enzyme known asNADPH oxidase [130].The respiratory burst response has been identi-

fied in fish phagocytes, and subsequent investiga-tion has revealed a system that possesses a greatdeal of similarity to that of mammals. Directevidence for the presence of this enzyme in fish wasprovided by Secombes et al. [131] who usedspectroscopy to identify a membrane componentthat shares characteristics with cytochrome b558 inrainbow trout macrophages. The definitive evi-dence for the presence of NADPH oxidase in fishhas been the molecular cloning and sequencing ofall five subunits from the Japanese puffer fish. Thetwo subunits, gp91phox and gp22phox, that formthe membrane-bound cytochrome b558, are highlyhomologous to those of humans.Using a goldfish macrophage model system, we

reported that crude cytokine preparations (MAF)contain soluble factors that can activate and/ordeactivate fish macrophages [132–134]. MAF wasalso used to prime the respiratory burst activity ofrainbow trout macrophages in vitro [135]. Thecomparison of the activation responses of mono-cytes and mature macrophages from goldfish hasrevealed some distinct differences in the priming ofthe respiratory burst response. While monocytestreated with MAF and LPS exhibited a rapidrespiratory burst, mature macrophages showed aslower production of the reactive oxygen interme-diates [136]. The kinetics of the respiratory burstresponse of mature goldfish macrophages was

similar to that of mammals and other fish species[135,137,138].

Nitric oxide response

The production of reactive nitrogen intermediatesthat have antimicrobial function in mammals iscontrolled by an inducible form of the enzymeknown as inducible nitric oxide (NO) synthase.The regulation of the inducible NO response ismediated through the activity of cytokines such asTNF, IFNc, IL-1, IL-4, and TGFb [139]. InducibleNO synthase has been identified and sequenced infish [140]. Tissue expression of trout inducible NOsynthase was found to be significantly higher inbacterially challenged fish when compared withcontrols. In addition, stimulation with LPSinduced the expression of inducible NO synthasein trout anterior kidney macrophages [140].We reported that LPS and MAF induced

the NO response in goldfish macrophages. Thisresponse was inhibited by antagonists of l-argin-ine, indicating that the metabolic pathways for theproduction of NO in fish were similar to those ofmammals [133]. Co-stimulation of cells with bothLPS and MAF also appeared to have a synergisticeffect that further enhanced the NO response[132,133]. While immature goldfish monocyteswere unable to mount significant NO response,mature macrophages were potent producers ofNO, suggesting that there is a developmentalrequirement for induction of this antimicrobialresponse in fish [136]. Similarly, human monocytesacquire the ability to produce NO after severaldays of cultivation [141,142].Recently, we reported that the iron-binding

molecule transferrin can induce the NO responsein goldfish macrophages [143,144]. Cleavageproducts of transferrin, as well as recombinantN- and C-lobes of the molecule, induced a potentNO response in goldfish macrophages. Interest-ingly, recombinant N- and C-lobes of goldfishtransferrin also induced NO production inmammalian macrophages, indicating that thisactivation pathway may be highly conserved invertebrates [144].Gilthead sea bream vaccinated against the

pathogenic bacterium Photobacterium damselaeexhibited significantly higher levels of NO produc-tion than non-vaccinated individuals in vivo and invitro. This heightened response also correlatedwith greater protection from subsequent challenges[145]. In an earlier study, it was also reported thatthe bactericidal activity of catfish phagocytestoward a bacterial pathogen (A. hydrophila) wasenhanced through vaccination, and that this

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response was partially blocked by the addition ofNG-MMLA, another inhibitor of the NO pathway.It was also reported that the supernatants of cellsfrom immunized fish that had been stimulated withthe same strain that they were vaccinated against,triggered an NO response greater than that whichcould be initiated using supernatants collectedfrom cells that were stimulated with a differentstrain of bacteria [138]. These observationsdemonstrate the importance of the reactive nitro-gen intermediates in the resistance of fish topathogens.

Conclusions

In this review, we demonstrate that fish have ahighly developed innate immune response. Manyspecies of fish possess not only a number of innateimmune processes that are homologous to thoseseen in mammals, but they also have a number ofunique ways in which they have expanded theirability to recognize and eliminate pathogens.Physical and chemical barriers include antimicro-bial compounds that are not only constitutive butcan also be induced upon infection. Fish possessimmune cells that are able to produce solublemediators that regulate inflammatory responses,have a fully functional complement system, andpossess unique receptors that recognize pathogens.Many of the genes encoding molecules associ-

ated with fish defense have counterparts in themammalian systems, suggesting that they representthe evolutionary precursors of key mediators ofinnate and acquired immunity.

Acknowledgments

This work was supported by a grant from theNational Science and Engineering Council ofCanada (NSERC) to M.B. D.A.P. was supportedby a postgraduate scholarship from NSERC.

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Comparison of select innate immune mechanisms of fish and mammals