1. Introduction
It is customary to divide the immune system into the innate (non-specific) and the acquired (specific) immune system. However, an increasing body of evidence, both from fish and mammalian immunology, shows that these are combinational systems. Innate response generally precedes the adaptive response, activates and determines the nature of the adaptive response and co-operates in the maintenance of homeostasis [1,2].
By definition the innate immune system’s recognition of non-self is mediated by germline-encoded pattern recognition proteins/receptors that identify molecular patterns, which are characteristic of microbes. These molecules include polysaccharides, lipopolysaccharide (LPS), peptidoglycans, bacterial DNA and double stranded viral RNA and other molecules not normally found on the surface of multicellular organisms [3e6].
This characteristic of the innate system is fundamentally different from the recognition arrangement of the acquired immune system and has its roots in ancient evolutionary history, or as far back as the early metazoa (porifera), which evolved around a billion years ago. The origin of the acquired immune system is traced back to the origin of the jawed vertebrates about 450 million years ago with the advent of the RAG genes. Their appearance signalled the rearrangement of the Ig super-family genes, the B- and T-cell receptors and MHC classes, leading to the possible recognition of practically unlimited antigenic epitopes [7e10].
Increased diversity and advanced affinity maturation and memory perfected the evolution of the acquired system as seen in mammals. There are several examples of the innate immune parameters of fish being more active and showing more diversity than comparable components of mammalian species. An example of this is the diversity of certain complement components like C3 and Bf, and high spontaneous activity of the alternative pathway [11e13].
However, the innate immune system is far from redundant in mammalian species, as many studies of man and mice have shown. In fact the apparent down regulation of the activity of the innate system in mammals may be seen as an evolutionary shift in its function, in which communication with the acquired immune system and co-operation with its components in maintaining homeostasis become increasingly important [2,14,15].
In recent years many review articles have been published about the innate immune system of fish [8,10,16e20]. Also a special issue of Developmental & Comparative Immunology (DCI) was recently devoted to the innate immune system (2001, volume 25 [8e9]). In addition, several papers have examined the function and modulation of different innate parameters with reference to disease resistance, prophylactic measures, environmental changes and genetic traits. Reflecting this renewed interest in the innate system of fish is the number of papers in the present issue that deal with both cellular and humoral innate components, like the activity of macrophages and cytotoxic cells, complement components, interferon and antibacterial peptides. This overview will describe the different roles played by the innate system in the immune defence of fish (teleosts) and its mode of non-self recognition. The main parameters of the innate system will be listed, concentrating on some widely distributed humoral innate components of fish, most of which are shared by both invertebrates and mammalian species. Parameters include, for example, lysozyme, haemolytic activity, pentraxins, and natural antibodies. The constitutive or induced intervention of these mechanisms will be discussed and how their activity may be influenced by genetic attributes, external conditions or immune stimuli. Some ontogenic data will also be presented. When relevant, examples based on studies of the innate immune system of cod will be given.
2. The different roles of the innate immune system
In spite of limited pathogen recognition machinery, the strength of innate defence against pathogens is impressive. This is demonstrated by the very efficient immune defence of invertebrates, which exclusively rely on innate parameters for coping with a large variety of pathogens in diverse environmental conditions. The innate immune system is also of primary importance in combating infections in fish. The reason is basically the intrinsic inefficiency of the acquired immune response of fish due to its evolutionary status and poikilothermic nature. This results in a limited antibody repertoire, affinity maturation and memory and a slow lymphocyte proliferation. The acquired immune response of fish is therefore sluggish (up to 12 weeks) compared to the instant and relatively temperature independent innate immune response [16,20,21]. The innate immune system is also important in activating an acquired immune response. In recent years this communication between the innate and the acquired system has received increased attention in mammalian research. Several studies, for example, of different ‘‘knock-out’’ mice and of acute phase response, have shown that the innate immune system is essential to the function of acquired immunity and determines the nature of the acquired response [1,2,22,23]. The activation of innate recognition components, through the stimulation of phagocytes, production of cytokines and chemokines and activation of the complement system and various cell receptors, stimulates T- and B-cells and antigen presenting cells [14]. Although less studied in fish a similar communication probably takes place between the innate and the acquired system in fish [24]. Participation of the innate system in homeostasis has also been mostly studied in mammalian species. Studies of ‘‘knock-out’’ mice lacking different innate machinery like complement components or natural antibodies have demonstrated the vital role of the innate immune system in the maintaining cellular and molecular equilibrium. This applies to both natural processes of cell death (apoptosis) and renewal and maintenance following injury or during acute phase and inflammatory reactions associated with infections.
3. The innate immune system’s recognition of non-self
The innate immune system of all multicellular organisms is served by a variety of germline-encoded pattern recognition receptors (PRR) or pattern recognition proteins (PRP) [3,6,25]. Unlike the recognition molecules of the acquired resistance, the recognition receptors of the innate system are relatively few and vertically transmitted, reflecting the evolutionary defence battles of the species and their adaptation to specific environmental conditions. Two categories of molecular patterns are believed to induce an immune response: Foreign or pathogen associated molecular patterns and molecular patterns exposed through damage of the host’s own tissues due to infection, necrotic changes and natural cell death, signalling danger to the immune system. The molecular patterns that are recognised by these parameters are for example peptidoglycans and lipopolysaccharides (LPS) in bacterial cell wall, fungal b1,3-glucan, viral double stranded RNA and bacterial DNA. Pathogen associated molecular patterns (PAMP) is the collective term used for these highly conserved molecules not generally expressed in multicellular organisms [6]. The danger signals, on the other hand, are molecules which are released or exposed through injury, infection, inflammation or normal cell apoptosis but are not normally expressed on the cell surface [26]. These include molecules like the host’s DNA, RNA, heat shock proteins and other chaperons and oligomannose of pre-secreted glycoproteins. Likewise, the surface carbohydrates of apoptotic cells are known to undergo a subtle change in the terminal sialic acid content identified by certain cell receptors [6]. The PRR can be soluble components like the complement protein C3, lectins and various other humoral innate components or they can be expressed as receptors on phagocytes and other cells of the immune system. There is evidence for b1,3-glucan receptors on salmon macrophages [27] and on catfish neutrophils [28]. Receptors with LPS binding activity have also been described in rainbow trout and seabream macrophages [29,30]. The Toll-like PRRs have received considerable attention in recent years. Toll was first described in the fruit fly Drosophilia melanogaster [31] and Toll-like genes have since been demonstrated in vertebrates and shown to be involved in the recognition of non-self [32,33]. Putative homologues of the TLR family have also been described in fish [34,35]. It has been suggested that some self-cells express molecular patterns like sialic acid, and are recognised by protective ligands like the siglec lectin, which, when bound to sialic acid, protects the cells from, for example, the complement cascade. If these molecular patterns change as occurs during apoptosis, a danger signal induces a PRR response [6]. Some pathogenic bacteria have surface sialic acid, which may facilitate their entry into the host and avoid normal innate attack [36]. Once activated the recognition molecules can induce osponization and phagocytosis of the pathogen, stimulate natural cytotoxic cells or activate different signalling/executive processes like the complement system and the lytic pathway or an acute phase response. Recognition molecules, like lysozyme or a2 macroglobulin, can also partake in direct elimination.
4. The main parameters of the innate immune system
The innate immune parameters (PRRs and implementers of innate response) have been extensively studied in fish, both with respect to practical immunoprophylatic measures and in comparative or evolutionary immunology. Most of the parameters of the innate immune system of fish are shared by both invertebrates and higher vertebrates. The components of the innate immune system are commonly divided into physical parameters, cellular and humoral factors. The humoral parameters can be both cell associated receptors or soluble molecules of plasma and other body fluids.
4.1. Physical parameters
Fish scales, mucous surfaces of skin and gills and the epidermis act as the first barrier against infection [20,37,38]. The important defence role of the mucus is well known and has been studied in several fish species [39e43]. Apart from efficient trapping and sloughing of pathogens, fish mucus contains immune parameters like lectins, pentraxins, lysozyme, complement proteins, antibacterial peptides and IgM [16,40,41]. Variations in disease resistance between fish species can at least in some instances be attributed to genetic differences in the protective element of the mucus. This is demonstrated by similar susceptibility of, for example, salmonids to an intraperitoneal injection of a particular pathogen but a different susceptibility to an immersion challenge [44].
4.2. The cells of the innate system
The functions of fish macrophages as well as the activity of inflammatory cells and of cytotoxic cells is the subject of other papers in this issue. This subject will therefore not be addressed here except to mention the key cells of the innate immune system: the phagocytic cells (granulocytes (neutrophils) and monocytes/ macrophages) and the non-specific cytotoxic cells [45e47]. Epithelial and dendritic cells also participate in the innate defence in fish [48e50].
4.3. Humoral parameters
The classification of humoral parameters is commonly based on their pattern recognition specificities or effector functions. Transferrin acts as a growth inhibitor of bacteria by chelating available iron essential for the bacterial maintenance [51]. Pathogenic bacteria may produce chelating agents to overcome this defence mechanism and hypoferraemic activity acting as a counter response has been demonstrated in some fish species [52]. Transferrin is also an acute phase protein invoked during an inflammatory response to remove iron from damaged tissue [53] and an activator of fish macrophages [54,55]. Interferon is another growth inhibitor, which induces the expression of Mx and other antiviral proteins [20,56,57]. Interferon-like activity has been demonstrated in several fish species and recently interferon genes were cloned from Atlantic salmon [58] (also addressed in this issue). Various protease inhibitors are present in fish serum and other body fluids [59e62]. The primary role of these inhibitors is in homeostasis of the body fluids. They are also involved in acute phase reactions [53] and in defence against pathogens, which secrete proteolytic enzymes [63e65]. Most widely studied is the a2- macroglobulin (a2-M). a2-M has a broad specificity, inhibition involving the physical encapsulation of the protease [16,66]. There is evidence that a2-M and complement component C3 have a common molecular ancestor in an invertebrate lineage. These proteins show significant amino acid sequence homology, both have interchain thioester bond and both can have multiple isoforms in fish with variable functional activity [67e69]. The total serum anti-protease activity has been measured in several groups of cod. The activity was generally high and seemed unaffected by immunization or infection, both conditions that might involve acute phase reaction, but was adversely affected by prolonged elevated environmental temperature [70e72]. Various lytic enzymes, acting either singly or in a cascade, are important defence elements especially against bacteria. These are hydrolases like lysozyme and chitinase, the cathepsins, the lytic pathway of the complement system and other bacteriolytic/haemolytic enzymes found in tissues and body fluids of fish [16]. Lysozyme is an important parameter in the immune defence of both invertebrates and vertebrates. Lysozyme is bactericidal, hydrolysing b-[1,4] linked glycoside bonds of bacterial cell wall peptidoglycans resulting in lysis. Although primarily associated with defence against Gram positive bacteria, Gram negative bacteria can also be lysed by this enzyme. Lysozyme is also known to be an opsonin and activate the complement system and phagocytes [73,74]. It is present in mucus, lymphoid tissue, plasma and other body fluids of most fish species [74e77]. Cod and several other marine species like haddock, pollack and wolffish show very little or no lysozyme activity in their tissues or body fluids. These species on the other hand show high chitinase activity in their plasma and various organs [78]. Chitinase is a hydrolase, which may be involved in the defence against bacterial and fungal pathogens but such a role in immune defence of fish has still to be proven [79,80]. Other natural lysins in fish serum, commonly detected by their spontaneous haemolytic effect on heterologous erythrocytes, are usually, but not always, attributed to the activation of the alternative pathway of the complement system [16]. Very high spontaneous haemolytic activity was detected in several groups of wild and cultured cod in the late 1990s [70,71,81]. This activity was unusual in being heat insensitive and enhanced by a low concentration of EDTA, the traditional inhibitor of both the alternative and classical pathways. Other features, like the inhibition by zymosan and bacterial LPS, point to a complement-like nature [82]. In the past 4e5 years however, all sera collected from cultured cod in Iceland has shown very low or no haemolytic activity. This low activity appears to be heat sensitive and inhibited by EDTA. The reason for this change in the cultured cod and the true nature of this lytic pathway is not known. However, a distinguishing feature between these sets of cod sera was shown to be a strong, hydrophobic association of the complement factor C3 and apolipoprotein A-I (apo A-I) in sera showing low haemolytic activity and the absence of such an association in the sera showing the high, unusual haemolytic activity ([83] and unpublished data). Apo A-I is the protein component of the high density lipoprotein (HDL) of serum. In human plasma, apo A-I, possibly in association with clusterin, can bind to membrane sites exposed upon C9 polymerization and thus interfere with the assembly of the membrane attack complex and inhibit lysis [84,85]. Mucosal or serum agglutinins and preciptins are lectins like C-type lectins and pentraxins. C-type lectins show binding specificity for different carbohydrates like mannose, N-acetyl glucosamine or fucose in the presence of Ca ions. The interaction of these carbohydrate binding proteins and carbohydrate leads to opsonization, phagocytosis and activation of the complement system [86]. Most widely studied is the mannose binding lectin (MBL), which shows specificity for mannose, N-acetyl glucosamine, fucose and glucose. MBL mediates the lectin pathway of the complement system in association with a serine protease (MASP) in mammals [13,87,88]. Lectins, with various carbohydrate specificities, have been isolated from the serum of several fish species [89,90]. Some show similar carbohydrate affinity, molecular structure and function to mammalian MBL [91,92], while others, also implicated in innate immune defence, have different carbohydrate binding specificity or heterologous sequence structure [93e95]. Calcium dependent LPSbinding protein had been isolated from some fish sera [96,97]. A lectin with binding affinity for N-acetyl-glucosamine and mannose, has recently been isolated from cod serum. Proteomic analysis of this lectin showed homology with ficolin (Sigrun Lange, unpublished data). Ficolin is believed to play a role in host defence through opsonization and complement activation in a similar manner to MBL [98e100]. Pentraxins (C-reactive protein, CRP and serum amyloid protein, SAP) are lectins, which are present in the body fluids of both invertebrates and vertebrates and are commonly associated with the acute phase response [53,101e104]. As well as showing significantly increased serum levels following tissue injury, trauma or infection (acute phase response), the pentraxins take part in innate immune defence through their lectin type binding role (PRR) [105,106], activate the complement pathways [107,108] and play a role in the recognition and clearance of apoptotic cells [109]. By definition CRP binds to the phosphorylcholine moiety of the bacterial cell wall in the presence of Ca ions whereas SAP shows affinity for phosphoryl-ethanolamine and is also known to bind LPS of Gram negative bacteria [110,111]. Some fish species appear to have either the CRP pentraxin (like cod and channel catfish) or the SAP pentraxin (like salmon, wolffish and halibut) whereas others (like plaice and rainbow trout) have both types like higher vertebrates [110,112]. The level of pentraxin is normally high in fish compared to mammals and may or may not be elevated during an acute phase response [111]. Cod has CRP type lectin and the normal (resting) level in cod serum is comparatively high (O300 mg/ ml). The molecular weight of CRP analysed at our laboratory was about 125 kDa, which under nonreducing conditions in polyacrylamide electrophoresis (SDS-PAGE) split into a trimer (72 kDa) and a dimer (50 kDa) and under reducing conditions into 1e4 monomeric units, 24e28 kDa in size. The monomeric units showed considerable variations between individuals and groups of cod, both in numbers and relative density, suggesting a structural diversity as shown by several other innate components of fish (Magnado´ttir, unpublished data). Although antibodies (immunoglobulins) are an acquired immune parameter natural antibodies can also be classified as components of the innate system. Natural antibodies are produced in the absence of generearrangement and without any apparent specific antigen stimulation. Natural antibodies are found in the serum of most healthy vertebrates but are commonly dismissed as background reaction in serological tests. In recent years natural antibodies have received further attention, which stems basically from work carried out on knock-out mice lacking IgM [22,113,114]. In mammals (man and mouse) natural antibodies are produced by long living B1 (CD5C) lymphocytes that are active during embryonic and early developmental stages. These antibodies are polyreactive and show low affinity for various self epitopes like nucleic acids, myosin, thyroglobulin and heat shock proteins. They also show reactivity for non-self associated molecular patterns like LPS, viral and parasitic products [115e119]. The specificity of the natural antibody repertoire against self-associated patterns is characteristic of individuals or groups and reflects specific selection in the embryo. Greater variation occurs against non-self epitopes, which reflects the variable exposure of the individual embryo to such epitopes. Although the natural antibodies have a broad specificity it is thought that they identify similarly structured epitopes [120]. The importance of natural antibodies in maintaining homeostasis, in clearing away apoptotic cells without an immune response and in tumour defence has been demonstrated in several studies [114,118,121,122]. Natural antibodies also play a role in acquired immune defence by, for example, inducing antigen driven IgG production and by isolating a pathogen and directing it to active germinal centres [114,123]. Natural antibodies are a well known phenomenon in fish and have been shown to play an important role in their innate/acquired immune defence [124e126]. Natural antibodies of rainbow trout and goldfish take part in both viral and bacterial defence [126,127] and high non-specific antibody activity in the serum of goldfish inhibited the specific antibody response [127]. Variation in the natural antibody specificity repertoire between different fish species have been reported, activity against haptenated (TNP/DNP) proteins being relatively strongest [125]. Cod showed high natural antibody activity against haptenated proteins like TNP-BSA (bovine serum albumin), which increased with increasing age and during infection. Raised temperature enhanced the natural antibody activity in some groups while others were unaffected [70e72,81]. When immunized with protein in adjuvant or adjuvant only, no specific antibody response was detected but the natural antibody response appeared to mimic a specific antibody response. Increased levels were seen about 10 weeks after the first injection and an enhanced response 10e12 weeks after the second injection which peaked earlier in cod kept at 9 C than at 4 C [71]. There is both functional and genetic evidence that fish, like mammals, have a network of signalling molecules, cytokines and chemokines, that control and coordinate the innate and acquired immune response. These parameters have been the subject of several comprehensive papers in recent years [128e 131] including a special issue of DCI (28(5), 2004). Cecropins in insects were the first antibacterial peptides described more than 20 years ago [132]. Since then an increasing body of evidence has shown that these are important innate parameters found in vertebrates as well as in invertebrates. Studies of antibacterial peptides in fish serum or mucus has also been a growing field in the last 10e15 years [133] and will be addressed elsewhere in this issue.
5. The modification and manipulation of the innate immune system
Innate immune parameters, in particular the phagocytic, lysozyme and spontaneous haemolytic activity, and in some cases pentraxins, have been used as indicators of the effects of inherent or external factors on the immune system and the disease resistance of fish. Such factors include genetic traits [64,134,135], seasonal factors [136e138], the environmental temperature [139,140], pollution [141e143], handling and crowding stress [144e148], diets and food additives [149e151], immunostimulants and probiotics [152e154] as well as the effects of diseases and vaccination [155e158]. This work has established that all these factors can influence the innate immune system and the immune competence of fish. Genetic trait affects innate components and disease susceptibility but attempts to apply selective breeding for important parameters have been hampered by the great individual variability of different innate characters, hence the problem of obtaining a homologous group for a specific parameter and consideration of other important factors in aquaculture like the growth rate. It is commonly stated that the innate parameters are relatively temperature independent. An example of this is the wide temperature range of cod phagocytic activity, which appears to be as active at 0 C as at 12 C (Sigrı´+ur Gu+mundsdo´ttir, unpublished data). It is also the general view that innate parameters are active at the lower temperature range of the fish, whereas the acquired immune parameters like lymphocyte activity and antibody production are more effective at higher temperatures [139,159e161]. In cod the spontaneous haemolytic and antiprotease activity was more active at 1 C and 7 C than at 14 C, whereas the opposite was the case for the serum IgM concentrations [70]. There are, however, instances of low temperature having adverse effects on innate parameters. For example rainbow trout acclimatized at different temperatures showed lower phagocytic and complement activity at 5 C than at 20 C [140]. Similarly, a drop in water temperature from 22 C to 10 C had adverse effects on the protective role of channel catfish mucus [162].
The immunosuppressive effects of pollution and stress resulting in higher disease susceptibility are well known [143,146]. Choosing a universal trait or an innate component that could act as a biomarker for adverse conditions in aquaculture or nature is however problematic. This is because of the variable effects on innate parameters depending on the type and duration of adverse conditions and on the fish species [147]. Food additives like vitamins, lipids or high carbohydrate content may or may not enhance innate parameters but can still be of general benefit as regards growth and survival [147,163]. There is considerable interest in upregulating the innate immune system with the help of various immunostimulants. The immunostimulants that are commonly used are components that activate the pattern recognition mechanism of the innate system like fungal b-1,3 glucans, peptidoglycans of Gram negative and positive bacteria, LPS, common to all Gram negative bacteria and various carbohydrates like levamisol, lactoferrin, polymannuronic acid, chitosan, also bacterial or synthetic oligo-dexanucleotides (CpG motives), and herbal remedies [164e169]. Some comprehensive reviews have been published recently covering the many immunostimulants tested in aquaculture. Their mode of action and application has been described, as well as their enhancement of innate and acquired immune parameters and effects on survival and disease resistance [152,170,171]. In theory the immunostimulation of innate immune parameters using chemicals that stimulate the PRR of fish looks promising. Such an augmentation of fast responding innate components might be beneficial for adult fish under the extreme conditions of aquaculture and in particular in marine larval aquaculture where massive losses are experienced in the often long period prior to full immune competence. These losses are often attributed to infections from environmental opportunistic and pathogenic bacteria. However, in spite of some success reported in several lab-trials in recent years the aquaculture industry is generally not incorporating these measures. The reason for their scepticism is the fact that the results have been varied depending on the fish species tested and the immunostimulants used, some of which, like LPS, are not practical in aquaculture. The results are further complicated by the range of the application methods used: in the feed, with bathing or by injection or by a combination of these methods. The time of application and duration are also factors that need to be considered.
6. The ontogeny of the innate immune system
The ontogenic development of the immune system of fish has primarily involved studies of development of the different lymphoid organs of the immune system (thymus, kidney, spleen) and acquired immune parameters (B- and T-lymphocytes and expression or secretion of IgM) [172,173]. The appearance of specific immune parameters and acquired defence competence varies greatly between fish species, even between closely related species like the salmonids [174]. In general the acquired immune system develops late in marine species, which therefore depend on innate defence for the first 2e3 months after hatching [174e178]. The ontogenic studies of innate parameters have so far been mainly limited to the first appearance of macrophages and phagocytic activity. In zebrafish phagocytic activity was detected in the embryo prior to hatching [179] and at 2 days post-fertilization in the carp embryo [180]. Similarly, an early appearance of macrophages has been described in three marine species, yellowtail, sea bream and Japanese flounder [176]. The ontogeny of the complement component C3 of cod and halibut has recently been described using immunohistology [181,182]. In both instances C3 was present in several organs at hatching. A recent examination of fertilized cod eggs using both Western blotting and in situ hybridization analysis demonstrated the presence of C3 and the associated apolipoprotein A-I at the time of organogenesis, 7e9 days post-fertilization ([183] and unpublished results).
Cathepsins have been detected in embryo and larval stages of cod [183]. These are primarily involved in the proteolytic digestion of the egg vitellogenin but may have an additional defensive role, as has been demonstrated, for example, in the mucus of adult fish [184]. Lysozyme has been detected in fertilized eggs and larval stages of several fish species like sea bass [185], tilapia [186] and salmonids [77,187,188]. Studies have shown that the presence of lysozyme in eggs and embryos can prevent the mother to progeny (vertical) transfer of bacterial fish pathogens [77,188]. CRP has been isolated from the eggs of lumpfish [189]. However, CRP was not detected in cod eggs or in larvae before metamorphosis was completed (2e3 months post hatching) [183]. Agglutinating lectins, which may be involved in host defence, have been isolated from the eggs of several fish species [190e192]. With the application of genetic and proteomic methods major progress can be expected in this field in the near future.