Copyright Blackwell Munksgaard 2005

Xenotransplantation 2005: 12: 266277

Printed in Singapore. All rights reserved
doi: 10.1111/j.1399-3089.2005.00227.x

XENOTRANSPLANTATION

Invited Review Article

Comparison of select innate immune

mechanisms of sh and mammals
Plouffe DA, Hanington PC, Walsh JG, Wilson EC, Belosevic M.
Comparison of select innate immune mechanisms of sh and mammals.
Xenotransplantation 2005; 12: 266277. Blackwell Munksgaard, 2005
Abstract: The study of innate immunity has become increasingly
popular since the discovery of homologs of many of the innate immune
system components and pathways in lower organisms including invertebrates. As sh occupy a key position in the evolution of the innate and
adaptive immune responses, there has been a great deal of interest
regarding similarities and dierences between their defense mechanisms
and those of higher vertebrates. This review focuses on describing select
mechanisms of the innate immune responses of sh and the implications
for evolution of immunity in higher vertebrates.

Debbie A. Plouffe,1 Patrick

C. Hanington,1 John G. Walsh,1
Elaine C. Wilson1 and Miodrag
Belosevic1,2
1
Department of Biological Sciences and 2Medical
Microbiology 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-405
Biological Sciences Building, University of
Alberta, Edmonton, Alberta, Canada T6G 2E9
(E-mail: mike.belosevic@ualberta.ca)
Received 28 February 2005;
Accepted 4 March 2005

Introduction

Prior to the emergence of the genes encoding the

hallmark components of the adaptive immune
response [T- and B-cell receptors and the major
histocompatibility complex (MHC) molecules],
organisms relied exclusively on the diversity of
non-specic defenses [1]. Fish represent the earliest
class of vertebrates possessing the elements of both
innate and acquired immunity, although it is clear
that their adaptive immune response is less developed than that of higher vertebrates [2,3]. Despite
this apparent deciency, sh comprise the greatest
group of vertebrate species and its members can be
found in most extreme aquatic habitats. In addition, sh are in constant contact with an environment containing potential pathogenic organisms
and have evolved a number of constitutive and
inducible innate immune responses to defend
against infection.
This review will focus on the information
obtained from the study of the innate immune
responses of dierent sh groups, and wherever
possible highlight the dierences in the innate
266

immune mechanisms of lower and higher vertebrates.

Soluble mediators of innate immunity in fish

Antimicrobial peptides

Antimicrobial peptides are small (12 to 50 amino

acids) molecules that have a broad spectrum of
activity against bacterial, fungal, viral, and protozoan pathogens [4]. The localization of antimicrobial peptides to host epithelial cells and mucosal
surfaces is a testament to their important role in
the rst line of defense against invading pathogens
[5,6]. In addition to their direct microbicidal
eects, antimicrobial peptides have other roles in
inammatory responses, including recruitment of
neutrophils and broblasts, promotion of mast cell
degranulation, enhancement of phagocytosis, and
decreasing brinolysis. In order to prevent tissue
injury associated with chronic inammatory
responses, antimicrobial peptides stimulate apoptosis of activated/infected cells, decrease cytokine
production, and some have the ability to bind and

Innate immune mechanisms

neutralize bacterial lipopolysaccharide (LPS), preventing endotoxin-induced damage [4,68].
Compared with other classes of vertebrates, the
quantity and diversity of mature antimicrobial
peptides identied in sh is relatively small. It
should be noted, however, that many species of sh
possess a number of other innate defense molecules
that can be found at mucosal/epithelial surfaces that
are also important in innate defense. These molecules include natural antibodies, apolipoproteins,
lysozyme (of which a number of dierent isotypes
have been identied), non-peptide antimicrobial
compounds such as squalamine (a 655-Da cationic
steroidal compound from the dogsh shark), and
most recently cationic steroidal derivatives from
catsh peripheral blood leukocytes [914].
Fish antimicrobial peptides have broad-spectrum activity at sub-micromolar concentrations
against a wide range of human and sh pathogens,
including bacteria, fungi, viruses, and protozoa.
From the point of view of evolution of the
vertebrate immune system, the identication of
antimicrobial peptides from the intestinal submucosa of the Atlantic hagsh (Myxine glutinosa)
is perhaps the most intriguing as they seem to
represent the evolutionary precursors of the cathelicidin family of peptides thus far found only in
mammals [15]. In the bony sh (teleosts), a
growing number of cationic peptides have been
isolated from a variety of species. Most of these
molecules have been isolated from the epidermal
cells or secretions of the skin, gills, and intestine.
Many of the sh antimicrobial peptides have high
sequence homology to segments of other proteins
(particularly histone or histone-like molecules)
indicating that they may in fact be cleavage
products of larger molecules. Cleavage peptides
have been isolated from the channel catsh,
rainbow trout, hybrid-striped bass, as well as Coho
and Atlantic salmon [16,17]. A number of specic
antimicrobial molecules characterized in rainbow
trout, called onchorhyncins, have been found to be
very similar to chromosomal proteins [12,18,19].
The remaining antimicrobial peptides isolated
from sh are a heterogenous group of compounds;
the majority of which are known to form amphipathic a-helices. They include the pardaxins [20],
misgurin [21], the pleurocidins [22], the piscidins
and moronocidins [23,24], the chrysophsins [25],
and two hydrophobic, pore-forming peptides from
carp [26].
Pro-inflammatory cytokines

The recent sequencing of the puersh and zebrash genomes, and the numerous sh-expressed

sequence tag databases appearing online, has been

of central importance for the identication and
characterization of sh cytokines. In general, sh
possess a repertoire of cytokines similar to that of
mammals. At present, the most well-characterized
sh cytokines are TNF-a and IL-1b, which share
functional similarities with their mammalian counterparts.
Tumor necrosis factor-a
Tumor necrosis factor (TNF)-like genes have been
identied in a number of sh species. The Japanese
ounder TNF was identied using an expressed
sequence tag screen of ounder immune tissues
[27]. Subsequently, TNF-like genes were discovered in rainbow trout [28], brook trout [29],
gilthead sea bream [30], carp and channel catsh
[31]. Genomic analysis of the rainbow trout TNF
gene revealed an organization similar to mammalian TNF-a, with the coding sequences of trout
TNF and mammalian TNF-a both spanning four
exons of similar size [28].
Mammalian TNF-a is produced by macrophages, monocytes, neutrophils, natural killer (NK)
cells, and T-cells after stimulation with LPS.
Similarly, the mRNA expression of TNF-a in
peripheral blood leukocytes and macrophages of
rainbow trout, brook trout, channel catsh, carp,
and Japanese ounder has been shown to increase
following stimulation with LPS [28,3133].
Recombinant trout TNF-a has been shown to
increase the phagocytosis and chemotaxis of
rainbow trout anterior kidney leukocytes, and
induce the expression of a number of genes
involved in the immune response including IL1b, IL-8, and COX2 [31].
Tumor necrosis factor receptors have been
identied in zebrash and Japanese ounder. The
zebrash TNF receptor, named OTR for ovarian
TNF receptor, was identied from cDNA of
zebrash ovaries and encodes a 438 amino acid
protein that contains the signature cysteine-rich
domains, transmembrane domain, and a death
domain. Furthermore, the residues in the death
domain required for the receptor to induce killing
are conserved between ovarian TNF receptors and
other apoptosis-inducing TNF receptors. The
expression of the ovarian TNF receptor transcript
appears to be highest in the ovary with lower
expression observed in the gills, heart, intestine,
kidney, muscle, and testis [29].
Interleukin-1b
Interleukin-1 (IL-1) is a pro-inammatory cytokine
that is involved in the process of inammation as
well as the induction of other immunomodulatory

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Plouffe et al.
cytokines. In mammals, the IL-1 family is composed of 10 ligands and 10 receptors [34].
Interleukin-1b has been identied in 13 dierent
species of sh, and has been shown to be functionally active in many of the species where it has been
found. Phylogenetic analysis of the teleost IL-1b
molecules groups them in a cluster separate from
the molecules isolated from amphibians, birds, and
mammals [35]. One of the most striking dierences
in the amino acid sequences of sh IL-1b, when
compared with mammalian IL-1b, is the absence of
a clear caspase 1 cut site [36]. However, in the RTS11 trout macrophage cell line, it has been shown
that the 29 kDa precursor IL-1b was cleaved into a
24-kDa molecule detectable in culture supernatants. These ndings suggest that trout IL-1b must
be cleaved to become activated and secreted [37].
Functional assays have demonstrated that teleost IL-1b shares many of the same characteristics
as mammalian IL-1b. Sequence analysis leading to
the identication of a putative IL-1b cut site
facilitated the production of a recombinant, bioactive, IL-1b molecule [38]. Stimulation with IL-1b
upregulated the expression of other transcripts
related to the immune response such as COX2 and
MHC class II. The incubation of trout anterior
kidney leukocytes with recombinant IL-1b induces
phagocytosis and chemotaxis [38,39]. Carp IL-1b
induces the proliferation of carp leukocytes, and
the addition of carp IL-1b to a vaccine against
Aeromonas hydrophila yielded a signicant increase
in the agglutinating antibody titer [40].
Interleukin-1b exerts its biologic eects by activating its receptor. To date, IL-1-like receptors
have been identied in Atlantic salmon (Salmo
salar) and rainbow trout [41,42]. The expression of
salmon IL-1R appears to be constitutive in all
tissues tested, and was upregulated in the anterior
kidney, spleen, liver, and gills following LPS
treatment [42].
Interleukin-18
Interleukin-18 is a member of the IL-1 family of
cytokines and, like IL-1b, IL-18 is synthesized as
an inactive precursor peptide that is stored intracellularly. The active form of IL-18 is released after
cleavage of the mature peptide by caspase 1. It is
the cleavage of the mature peptide from the
inactive precursor form that is thought to be the
primary regulatory mechanism of IL-18 activity
[4345]. In mammals, IL-18 is produced by macrophages, dendritic cells, T cells, and B cells, and it
can also act synergistically with IL-12 to induce
IFN-c production by Th1 T-cells and NK cells.
Zou et al. [46] identied a partial IL-18-like
sequence in trout by searching salmon EST

268

databases. The trout IL-18 gene has a similar

organization to the IL-18 gene of humans (containing six exons and ve introns). Constitutive
expression of trout IL-18 was observed in brain,
gill, gut, heart, kidney, liver, muscle, skin, and
spleen, with the highest levels being found in the
spleen and kidney.
Interferons
In mammals, there are two types of interferons
(IFNs), type I and type II. The type I IFNs are
primarily antiviral cytokines that are produced in
response to viral challenge or exposure to doublestranded RNA [reviewed in 47]. The type II IFNs
consist of only IFN-c that is produced mainly by
Th1 T-cells and NK cells. IFN-c activates phagocytes, T-cells, and NK-cells [48].
Although the presence of IFN-like activity in
crude sh cell supernatants has been known for
decades, an IFN-like molecule had not been
identied in sh [49,50]. To date, IFN genes have
been identied in catsh [51], puersh [50],
Atlantic salmon [52], and rainbow trout (Genbank
AJ582754 and AJ580911).
Evidence suggesting that sh IFN genes are
functionally similar to those found in mammals is
derived from the observation of mRNA expression
during viral infection. Catsh IFN expression was
increased 2 h after exposure to a double-stranded
RNA retrovirus that had been UV-inactivated. Its
expression was also elevated 2 h after exposure to
poly-inosinic:cytidilic acid (poly I:C) [51]. poly I:C
induced the expression of zebrash IFN dramatically [53]. In addition, zebrash and rainbow trout
IFN transcripts activate the IFN-inducible Mx
promoter [49,54]. Current research has focused on
the cloning of other IFN-inducible genes as well as
factors involved in IFN signaling [53,55].
Chemokines and interleukin-8
Although mammalian chemokines have been well
characterized, chemokines of lower vertebrates
have only recently become a focus of immunological research. The rst sh chemokine, CK1, was
identied in rainbow trout [56]. To date, chemokines have been identied in carp [57], the silver
chimera (Chimaera phantasma) [58], catsh [59],
the banded dogsh [60], ounder (Paralichthys
olivaceous) [61] lamprey [62], the catshark [63], and
rainbow trout [64,65].
Among the sh chemokines, most is known
about IL-8. The main functions of IL-8 are to
stimulate neutrophil activation, migration, and
degranulation [6668]. It is produced by a variety
of cells, primarily endothelial cells and macrophages, in response to activating stimuli including

Innate immune mechanisms

other cytokines and LPS [69]. The gene encoding
IL-8 has been identied in lamprey [62], trout
[65,70], catsh [59], and the silver chimera [60]. In
the RTS-11 trout macrophage cell line, IL-8
expression was induced by exposure to LPS and
poly I:C [65].
Chemokine receptors have been identied in
lamprey [63], trout [71], and sterlet [72]. None of
the receptors have been functionally characterized,
nor have expression studies been performed to
determine whether they exhibit expression patterns
suggesting sensitivity to cellular activation.
Complement

In mammals, the complement system consists of

more than 30 serum and membrane-associated
proteins that function in activation and regulation
[73]. Among the lower vertebrates, the complement
system has been intensively studied in the three
main classes of sh, including the agnathans
(hagsh and lamprey), the cartilaginous sh
(sharks and rays), and the bony sh (teleosts). As
such, sh provide an excellent model in which to
study the complement system in terms of its
evolution and function. The complement system
of teleost sh is particularly unique in that they
possess multiple isoforms of some of the important
complement 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/or
cloning of genes for most of the complement
components has provided strong support for the
existence of all three pathways of activation as well
as a functional lytic pathway. The most intriguing
dierence between the teleost complement system
and the complement systems of other vertebrates is
the structural and functional diversity of some of
its components. Teleosts possess a large repertoire
of genes encoding the complement components,
and some of these genes also demonstrate allelic
polymorphism. The presence of multiple copies of
some of the central complement proteins, such as
C3 and factor B, has generated a great deal of
interest in determining the functional purpose of
these duplications [74].
Multiple genes encoding structurally and functionally dierent C3 molecules have been identied
in rainbow trout, carp, gilthead sea bream, medaka, and zebrash. Some of the earliest studies
involved the isolation of four dierent C3 proteins
from the serum of rainbow trout [74,75]. The
rainbow trout C3 isoforms were found to bind
dierentially to various complement-activating

surfaces (zymosan, sheep and rabbit erythrocytes)

suggesting that the evolution of multiple C3
molecules may aid in the recognition of a variety
of pathogenic organisms [74].
Homologs to mammalian C5, the initiating
molecule of the terminal lytic cascade, have been
identied in trout [76], sea bream [77], and carp
[78]. In rainbow trout, a membrane attack complex
(MAC)-forming C5 protein was puried from
plasma, and a cDNA clone representing a single
C5 gene was cloned from the liver [76]. In carp, two
distinct, full-length, C5 cDNA clones (C5-I and
C5-II) in addition to a truncated form of C5-I were
isolated from the liver. The remainder of the
terminal components (C6, C7, C8, and C9) have
also been identied from a variety of sh species,
including carp [79], Japanese ounder [80], and
rainbow trout [81,82].
One of the most interesting implications for the
existence of functionally distinct isoforms of many
of the complement components in teleosts, particularly the C3/4/5 molecules, is the possibility that
the cleavage products of these molecules may
possess their own diverse functions. For example,
as complement components are known to have a
role in linking innate and adaptive immunity, there
is the potential for the dierent sh proteins to
have a number of eects on both branches of the
immune response. The C3a, C4a, and C5a molecules are conserved in teleost sh, and studies have
shown that activation of serum using LPS or
zymosan can induce chemotaxis, respiratory burst,
and phagocytosis [8385]. Recently C3a and C5a
receptors have been identied in carp and rainbow
trout [86,87]. In trout, the C5a receptor appears to
be primarily expressed on granulocytes and macrophages [87].

Cells and innate immunity of fish

Fish cytotoxic cells and their receptors

The ability to recognize the presence of a dangerous pathogen is essential for the generation of an
appropriate and eective immune response. Owing
to the limitations of their adaptive response, sh
rely heavily on innate immune mechanisms to deal
with pathogens [3]. A number of similarities exist
between the cells involved in innate responses in
mammals and sh; however, novel cell populations
and receptors have been identied in sh.
Non-specic cytotoxic cells
Evans et al. [8890] reported the presence and
characterization of a novel cytotoxic cell population in the channel catsh. These cells, called

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Plouffe et al.
non-specic cytotoxic cells (NCCs), are unique in
their ability to lyse various transformed human
and mouse cell lines. The lysis of target cells
required direct cellcell contact through a mechanism thought to be similar to that of mammalian
NK cells. Morphologically these cells are similar to
monocytes. NCCs are found in the sh kidney
rather than the blood, and functional similarities to
mammalian NK cells have led researchers to
postulate that NCCs may represent the evolutionary precursor to NK cells [8890]. NCCs have
been identied in teleost species including rainbow
trout, carp, damselsh, and tilapia [9193].
In addition to their ability to lyse transformed
cell lines, NCCs appear to participate in the
immune response against protozoan parasites.
Graves et al. [94] demonstrated in vitro killing of
Tetrahymena pyriformis, an opportunistic protozoan parasite, by catsh NCCs. Through binding
deletion experiments, it was found that another
protozoan parasite, Ichthyophthirius multiliis, had
a similar target antigen. In addition, infection with
I. multiliis was found to cause a shift toward
increased percentages of NCCs in the peripheral
blood [94]. A monoclonal antibody was used to
identify a 40 to 50 kDa molecule from the lysates
of the protozoan T. pyriformis that was recognized
by catsh NCCs. The amino acid sequence of the
protein, named NKTag, has no signicant homologies with any known sequence. However, soluble
or puried NKTag was able to inhibit NCCmediated lysis of target cells. Immunouorescence
studies have revealed that a related protein was
expressed on several transformed mammalian cell
lines, including NC-37 and MOLT-4 [95,96]. These
results highlight the importance of the NKTag
epitope to the cytotoxic response of NCCs and
possibly other NK cells.
Immunoprecipitation experiments using mAb5C6
(anti-NCC antibody) resulted in the identication
of a protein of 34 kDa named NCC receptor protein
1 (NCCRP-1) [97100]. The engagement of
NCCRP-1 by NKTag was found to be responsible
for the recognition and killing of target cells by
NCCs [95,96,98,101]. Further analysis, using synthetic peptides to mimic the ligand demonstrated
that the ligand-binding region of NCCRP-1 was
located between amino acids 104 and 119 [102].
These researchers have proposed that NKTag
may be the only target molecule recognized by
NCCRP-1.
Cytotoxic NK-like cells
In addition to the NCCs, which may represent
an evolutionary precursor of mammalian NK
cells [8890], teleosts also possess NK-like cells.

270

Recently, new clonal NK-cell lines have been

identied from alloantigen-stimulated catsh
lymphocytes that share functional and morphological similarities to mammalian NK cells, but are
distinct from NCCs [103]. The catsh NK-like cells
are large and granular, which is similar to mammalian NK cells, but unlike NCCs. These NK-like
cells are able to lyse allogenic target cells but fail to
express Igl or TCR a, b, c, excluding the possibility that they are B or T-cells. In addition, these
cells do not stain with Sudan Black B and are nonspecic esterase-negative indicating that they are
not neutrophils or macrophages. Perhaps the most
signicant nding was that mAb 5C6 (anti-NCC
antibody), failed to recognize these cells, suggesting
that they are not NCCs [103]. Although all of the
NK-like cell lines investigated exhibited cytotoxicity toward allogenic targets, the specicity for
targets varied between clones. Some clones killed
several dierent allogenic cells equally, while others
demonstrated a marked specicity for a particular
cell type. Killing of target cells by catsh NK-like
cells appears to be mediated through the induction
of apoptosis [103].
Novel immune-type receptors of sh
Originally identied in the puersh, novel immune-type receptors (NITRs) are a group of NK
cell receptors that have been identied as a member
of the immunoglobulin superfamily, and have no
known homolog in mammals [104]. Although the
precise function of NITRs has not been determined,
they share many structural features with mammalian NK cell receptors and are hypothesized to be
the functional orthologs of the mammalian receptors [105,106]. NITRs have been identied in
channel catsh [107], zebrash [108], rainbow trout
[109], and puersh [104]. More than 40 NITR
genes have been identied in the zebrash genome
near the top of linkage group 7, which is an area
anking the zebrash NITR gene complex. This
area shares synteny with human chromosome19q13.3-q13.4, an area that encodes the leukocyte receptor cluster [108]. The LRC region of
humans codes a number of receptors including
killer Ig-like receptors (KIR), paired Ig-like receptors (PIR), leukocyte inhibitory receptors (LIR)
and monocyte inhibitory receptors (MIR), as well
as leukocyte-associated inhibitory receptors
(LAIR) [110114]. Unlike the genes encoded by
the leukocyte receptor cluster region, NITR genes
encode for a variable (V) domain as well as a V-like
constant domain (V/C2), whereas leukocyte receptor cluster genes do not encode V regions [107].
The NITR genes identied in the puersh,
rainbow trout, and zebrash all possess

Innate immune mechanisms

immunoreceptor tyrosine-based inhibition motifs
(ITIMs) in their cytoplasmic regions [104,108,109].
The presence of ITIMs suggests that these receptors function in the deactivation or inhibition of
the cells on which they are expressed. ITIMs
associate with SHP-1 and SHP-2 to dephosphorylate eector substrates involved in the NK lytic
pathway, especially MAPK that is involved in
regulating NK cell activity against tumor cells
[115]. The NITRs are expressed mainly in the
spleen, kidney, and intestine of the catsh, and are
expressed in a number of catsh cell lines, suggesting that these receptors play an important role
controlling cellular immune responses [107].
C-type lectins/cKLR
Natural killer cell receptors can be divided into
two broad classes that dier in their origins but
are similar in function. Killer Ig-like receptors are
type I transmembrane glycoproteins that have
one to three extracellular immunoglobulin domains. Killer cell C-type lectin receptors (KLR)
are type II transmembrane proteins that possess
an extracellular C-type lectin domain and exist as
homo- or heterodimers [116]. These receptors
monitor the expression of MHC class I or MHC
class I-like molecules on host cells. While no
KIRs have been discovered in sh, a small
number of KLRs have been identied. Two Ctype lectins (TCL-1 and TCL-2) have been
identied in rainbow trout and both molecules
are similar to known mammalian C-type lectins
[117,118]. TCL-2 diers from TCL-1 in that it
possesses two ITIMs. TCL-2 maps near the novel
immune type receptor (NITR) gene cluster; however, it is unlikely to be related to the Ly49 or
NKG2 gene families [109].
C-type lectin receptors have also been identied
in two species of cichlid sh, Paralabidochromis
chilotes and Oreochromis niloticus [119]. cKLR
was identied from an EST screen of P. chilotes
and was found to have homology with the CD94/
NKG2 subfamily of KLRs. cKLR was found to
possess a positively-charged amino acid (Arg-42)
in the transmembrane domain, a feature that is
commonly found in receptors that interact with
immunoreceptor tyrosine-based activating motifs.
The absence of an ITIM in cKLR also supports
this hypothesis. The gene encoding this receptor
in a related sh, O. niloticus, has a similar
structure to the mammalian CD94/NKG2 genes.
DNA hybridization studies of bacterial articial
chromosome clones reveal that the cKLR gene is
a member of a multi-gene family that is made up
of at least 10 copies of cKLR-like sequences
[119].

Toll-like receptors
One group of receptors that has received attention
for its role in innate immunity is the Toll-like
receptor (TLR). The Toll receptor was originally
discovered in Drosophila and was identied as an
important molecule involved in larval development. It was later found to play a critical role in
fruit y defense against fungal and bacterial
infections [120]. As Toll was found to have an
additional role in the invertebrate immune system,
it was hypothesized that these receptors might
represent an evolutionarily ancient mechanism of
innate immune recognition that may have been
conserved in vertebrates. To date, there have been
10 TLRs found in mammals, each possessing a
Toll/IL-1R cytoplasmic domain responsible for
signaling, as well as an extracellular leucine-rich
repeat domain that is involved in the recognition of
specic molecular signatures of a variety of pathogens [121123].
The rst teleost TLR was characterized in our
laboratory from a goldsh-stimulated macrophage
cDNA library enriched for dierentially expressed
genes by suppressive subtractive hybridization
[124]. Shortly thereafter, TLR genes were identied
in puersh, zebrash, Japanese ounder, and the
channel catsh [125128]. As genome sequences are
available for zebrash and puersh, it was possible to identify TLR homologs present in each of
these species. Analysis of the puersh genome
resulted in the discovery of TLR homologs for
each of the known mammalian TLR genes with the
exception of TLR4 and TLR6. Interestingly, two
novel TLRs, TLR21 and TLR22, were also identied [128]. Similar results were obtained from a
survey of the zebrash genome; however, a homolog of TLR 4 was identied in addition to TLR21
and TLR22. Furthermore, it appears that a cluster
of sh-specic TLRs has been identied in zebrash, consisting of TLR 21, TLR 22, and possibly
TLR19 and TLR20a/b (possible orthologs of
TLR21) [126,127]. With the identication of what
appears to be all of the teleost TLRs, it has become
simpler to identify TLR homologs in other sh
species. The goldsh TLR was shown to have
highest homology with TLR21 of the zebrash and
puersh [126], as well as with TLR22 of the
Japanese ounder [125].
Functional analysis of the dierent teleost TLRs
has been limited to analysis of tissue expression. In
puersh, the expression of TLR2, TLR5, and
TLR22 appears to be ubiquitous amongst the
tissues analyzed, whereas TLR1 and TLR7 were
highly expressed in the kidney. TLR9 exhibits high
expression in the kidney, digestive gland, skin, and
heart, while TLR21 shows highest expression in the

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Plouffe et al.
heart and gill [128]. In contrast, TLR22 of Japanese ounder was only found to be expressed in the
anterior kidney and peripheral blood leukocytes,
while TLR2 was found in those tissues in addition
to trunk kidney, spleen, and gill [125]. The analysis
of the expression of the goldsh TLR showed
localized expression in the spleen and kidney, and
an increase in expression was observed in primary
macrophage cultures 3, 6, and 24 h after stimulation with LPS and macrophage-activation factor
(MAF) [124].

Antimicrobial response of fish phagocytes

Respiratory burst response

In mammalian systems, phagocytosis is associated

with an increase in oxygen consumption by specic
immune cells. The phenomenon is known as the
respiratory burst and has subsequently been
shown to correspond to the production of reactive
oxygen intermediates [129]. Until recently, this
response was the sole example of deliberately
produced reactive oxygen intermediates catalyzed
by the multi-component enzyme known as
NADPH oxidase [130].
The respiratory burst response has been identied in sh phagocytes, and subsequent investigation has revealed a system that possesses a great
deal of similarity to that of mammals. Direct
evidence for the presence of this enzyme in sh was
provided by Secombes et al. [131] who used
spectroscopy to identify a membrane component
that shares characteristics with cytochrome b558 in
rainbow trout macrophages. The denitive evidence for the presence of NADPH oxidase in sh
has been the molecular cloning and sequencing of
all ve subunits from the Japanese puer sh. The
two subunits, gp91phox and gp22phox, that form
the membrane-bound cytochrome b558, are highly
homologous to those of humans.
Using a goldsh macrophage model system, we
reported that crude cytokine preparations (MAF)
contain soluble factors that can activate and/or
deactivate sh macrophages [132134]. MAF was
also used to prime the respiratory burst activity of
rainbow trout macrophages in vitro [135]. The
comparison of the activation responses of monocytes and mature macrophages from goldsh has
revealed some distinct dierences in the priming of
the respiratory burst response. While monocytes
treated with MAF and LPS exhibited a rapid
respiratory burst, mature macrophages showed a
slower production of the reactive oxygen intermediates [136]. The kinetics of the respiratory burst
response of mature goldsh macrophages was
272

similar to that of mammals and other sh species

[135,137,138].
Nitric oxide response

The production of reactive nitrogen intermediates

that have antimicrobial function in mammals is
controlled by an inducible form of the enzyme
known as inducible nitric oxide (NO) synthase.
The regulation of the inducible NO response is
mediated through the activity of cytokines such as
TNF, IFNc, IL-1, IL-4, and TGFb [139]. Inducible
NO synthase has been identied and sequenced in
sh [140]. Tissue expression of trout inducible NO
synthase was found to be signicantly higher in
bacterially challenged sh when compared with
controls. In addition, stimulation with LPS
induced the expression of inducible NO synthase
in trout anterior kidney macrophages [140].
We reported that LPS and MAF induced
the NO response in goldsh macrophages. This
response was inhibited by antagonists of l-arginine, indicating that the metabolic pathways for the
production of NO in sh were similar to those of
mammals [133]. Co-stimulation of cells with both
LPS and MAF also appeared to have a synergistic
eect that further enhanced the NO response
[132,133]. While immature goldsh monocytes
were unable to mount signicant NO response,
mature macrophages were potent producers of
NO, suggesting that there is a developmental
requirement for induction of this antimicrobial
response in sh [136]. Similarly, human monocytes
acquire the ability to produce NO after several
days of cultivation [141,142].
Recently, we reported that the iron-binding
molecule transferrin can induce the NO response
in goldsh macrophages [143,144]. Cleavage
products of transferrin, as well as recombinant
N- and C-lobes of the molecule, induced a potent
NO response in goldsh macrophages. Interestingly, recombinant N- and C-lobes of goldsh
transferrin also induced NO production in
mammalian macrophages, indicating that this
activation pathway may be highly conserved in
vertebrates [144].
Gilthead sea bream vaccinated against the
pathogenic bacterium Photobacterium damselae
exhibited signicantly higher levels of NO production than non-vaccinated individuals in vivo and in
vitro. This heightened response also correlated
with greater protection from subsequent challenges
[145]. In an earlier study, it was also reported that
the bactericidal activity of catsh phagocytes
toward a bacterial pathogen (A. hydrophila) was
enhanced through vaccination, and that this

Innate immune mechanisms

response was partially blocked by the addition of
NG-MMLA, another inhibitor of the NO pathway.
It was also reported that the supernatants of cells
from immunized sh that had been stimulated with
the same strain that they were vaccinated against,
triggered an NO response greater than that which
could be initiated using supernatants collected
from cells that were stimulated with a dierent
strain of bacteria [138]. These observations
demonstrate the importance of the reactive nitrogen intermediates in the resistance of sh to
pathogens.
Conclusions

In this review, we demonstrate that sh have a

highly developed innate immune response. Many
species of sh possess not only a number of innate
immune processes that are homologous to those
seen in mammals, but they also have a number of
unique ways in which they have expanded their
ability to recognize and eliminate pathogens.
Physical and chemical barriers include antimicrobial compounds that are not only constitutive but
can also be induced upon infection. Fish possess
immune cells that are able to produce soluble
mediators that regulate inammatory responses,
have a fully functional complement system, and
possess unique receptors that recognize pathogens.
Many of the genes encoding molecules associated with sh defense have counterparts in the
mammalian systems, suggesting that they represent
the evolutionary precursors of key mediators of
innate and acquired immunity.
Acknowledgments

This work was supported by a grant from the

National Science and Engineering Council of
Canada (NSERC) to M.B. D.A.P. was supported
by a postgraduate scholarship from NSERC.
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