Fish Immune System

A E Ellis, SOAEFD Marine Laboratory, Aberdeen, UK

It is important to realize that among the Vertebrata, the class Pisces has within it the oldest and most numerous species. The most recent and evol-utionarily advanced group of fish, and the one of most commercial importance, is the teleosts (bony fish), which first appeared in the late Devonian Age, 300 million years ago, and rose to prominence 70 million years ago. Now they are the most numerous with over 20 000 extant species (compared with the 4500 species of mammals) occupying very diverse aquatic habitats. It is to be expectcd, therefore, that interspecies differences will exist in the immune system of fish. Most of the available information appertains to the teleosts, and in particular to a few species of commercial importance in aquaculture. Differences among these species are not considerable and this entry presents a generalized picture of the teleosts only, there being no attempt to draw attention to anatomical, cellular or molecular differences which exist between teleosts and other groups of fish for which data exist, e.g. the hagfish and lamprey (Agnatha) or the sharks and rays (Chondrichthyes).

Functional anatomy of the fish immune system

The primary and secondary lymphoid tissues of fish are located in the thymus, kidney and spleen (Figure 1). Of course, the whole integument offish, including the skin, is a mucous membrane and contains immune cells and secretions. The gills are important in antigen uptake (see Vaccination of Fish below). Fish do not possess lymph nodes or organized gut-associated lymphoid tissues, although the gut and gill do contain lymphoid and antibody secreting cells.

The thymus is a paired organ located dorsolateral^ in the gill chamber. In ontogeny it is the first organ to become lymphoid and arises within the epithelium, external to the basement membrane, between the gill arches. In young fish it is covered externally only by a single layer of epithelial cells. With age, the thymus becomes surrounded by a thin connective tissue capsule which gradually invades the lymphoid tissue in a slow process of involution. In most species the thymus retains a very superficial position.

In structure and function the fish thymus is similar to that of mammals except in some details. It is composed mainly of lymphocytes in various stages of development and division and a few epithelial-type cells and macrophages arc also present. However, differentiation into cortex and medulla and presence of Hassell's corpuscles are not marked. Blood vessels within the organ possess a specialized endothelium

Figure 1 Lymphoid organs of teleost fish. (A) The thymus insert is that of Atlantic salmon eight days prehatch. The thymocytes develop within the pharyngeal epithelium, external to the basement membrane (bm). The organ is covered externally by a single layer of squamous epithelium, gb, primary gill bars. (B) The kidney insert is that of the plaice, a flatfish, showing the melanomacroph-age centers and kidney tubules surrounded by hematopoietic tissue. (C) The spleen depicts the branching ellipsoid system within the splenic pulp.

Thymus

Kidney

Spleen

Thymus

Kidney

Fish Immune Organ

Figure 1 Lymphoid organs of teleost fish. (A) The thymus insert is that of Atlantic salmon eight days prehatch. The thymocytes develop within the pharyngeal epithelium, external to the basement membrane (bm). The organ is covered externally by a single layer of squamous epithelium, gb, primary gill bars. (B) The kidney insert is that of the plaice, a flatfish, showing the melanomacroph-age centers and kidney tubules surrounded by hematopoietic tissue. (C) The spleen depicts the branching ellipsoid system within the splenic pulp.

with tight junctions. There is considerable evidence to indicate that the fish thymus, as in higher vertebrates, is a primary lymphoid organ. Such evidence includes the exclusion of foreign particles and anti gens present in the circulation, the exclusion of circulating lymphocytes, the high mitotic rate of thymocytes, which is presumably antigen independent, the absence of antibody-producing cells, the migration of thymocytes to other lymphoid organs and the fact that the relative size of the organ is greatest in young fish. Data appertaining to lymphocyte subpopulations indicate that the thymocytes of fish are similar in many respects to the T cell lineage in mammals (see below). Unfortunately the crucial experiments of early thymectomy to show ablation of T cell function have proved difficult to perform in fish as the organ contains lymphocytes at or soon after hatching.

The kidney is the major hematopoietic and immunologically reactive organ in teleost fish. The organ is located ventral to the spinal chord and extends the full length of the peritoneal cavity. The anterior tip (so-called 'head kidney' or pronephros) is constituted entirely of hematopoietic and lympho-reticular tissues, which, in the rest of the organ (opisthonephros), occupy the intertubular spaces. The hematopoietic tissue bears close resemblance to the bone marrow of higher vertebrates but differs in having a highly active reticuloendothelial system and is the major antibody-producing tissue of fish. In this respect it has functional similarity to mammalian lymph nodes. However, there is no anatomical differentiation of the lymphoid tissue and no primary or secondary lymphoid follicles in fish. Within the hematopoietic tissue are many pigment-containing cells, termed melanomacrophages. These macrophages contain melanosomes and to a greater or lesser degree ceroid and lipofuscin.

In primitive teleosts (e.g. salmonids) the pigment cells are scattered throughout the tissue, while in advanced species (e.g. flatfish and cyprinids) they form aggregates, termed melanomacrophage centers, which possess a delicate reticulin fiber capsule and contain other cell types, particularly lymphocytes and pyroninophilic cells. Foreign materials (e.g. carbon particles) in the circulation are actively phagocytosed by cells of the reticuloendothelial system lining the hematopoietic sinuses. These phagocytes then migrate and aggregate within melanomacrophage centers.

The spleen of fish is mainly composed of red pulp sinuses, while the white pulp is poorly differentiated, forming a cuff of tissue surrounding melanomacrophage centers, similar to those in the kidney and the extensive ellipsoid system. The latter comprises the whole of the splenic arterial capillary network, the vessels of which are surrounded by a thick sheath of reticulin fibres, embedded in which are numerous macrophages which phagocytose foreign particulate matter in the blood and, like the reticuloendothelial cells of the kidney, then migrate and accumulate in the melanomacrophage centers. Following antigen stimulation, antibody-producing cells appear in the spleen but are less numerous than in the kidney tissue.

The origin and function of melanomacrophage centers in fish kidney and spleen is far from clear but the available information suggests they may be primitive analogs of germinal centers in higher vertebrates. Following antigen stimulation, clusters of pyroninophilic cells appear in the kidney hematopoietic tissue and the splenic ellipsoid walls and it is thought these clusters develop into melanomacrophage centers. The latter are also focal points for the aggregation of replete macrophages, and studies on lymphocyte recirculation in fish have demonstrated that small lymphocytes also migrate to the centers. Furthermore, immune complexes are trapped by reticulin fibers in the spleen and kidney, especially within the splenic ellipsoids and melanomacrophage centers, where they are retained for several months. The function of germinal centers in mammals is concerned with immunologic memory and the finding that priming fish with immune complexes, besides resulting in immediate trapping within ellipsoids and melanomacrophage centers, also induces stronger immunologic memory than priming with antigen alone, suggests that the ellipsoid and melanomacrophage system of fish serves a similar function.

The role of melanin associated with phagocytes in fish is not clear but a possible explanation is that as fish tissue lipids are highly unsaturated they are particularly susceptible to oxidation by free radical reactions initiated by phagocytic cells. The melanosomes present in highly phagocytic tissues in fish may function to quench the free radical chain reactions and protect the cells from severe damage.

Lymphocyte subpopulations in fish

While convincing evidence now exists for lymphocyte heterogeneity in fish, the extent to which functional diversity among lymphocyte populations have evolved in fish is still not clear. What can be said is that a T and B lymphocyte dichotomy basically similar to that of higher vertebrates has evolved in fish. The evidence supporting this view rests upon studies of surface markers, mitogen and carrier-hapten responses, and cytokine production.

The presence of surface immunoglobulin (slg) molecules, detected by labelled antisera to serum Ig, is a characteristic of mammalian B lymphocytes. In fish, such techniques stain all lymphocytes, including thymocytes. However, studies using monoclonal instead of polyclonal antibodies specific to serum Ig stain only about 30% of peripheral lymphocytes and do not stain thymocytes. The polyclonal antibody staining of fish thymocytes is thought to be due to carbo hydrate moieties on thymocyte membranes and serum Ig which cross-react. In combination with other data (see below), it seems likely that the fish B-like lymphocyte antigen receptor is slg, as in higher vertebrates. The fish T-like lymphocyte antigen receptor is still uncharacterized but recent genetic studies have indicated that gene fragments coding for molecules homologous to mammalian T cell receptor fi chains occur in the goldfish and salmonids and strongly suggest that the nature of the T cell receptor in fish is basically similar to that in all other vertebrates.

The use of monoclonal antibodies in conjunction with mitogen or carrier-hapten responses provide compelling evidence for the presence of T and B lymphocyte types in fish. Examples of such evidence include the correlation between the presence of slg and responsiveness to lipopolysaccharides (LPS; a B cell mitogen) and the lack of slg with responsiveness to concanavalin A (Con A, a T cell mitogen). A monoclonal antibody has been produced which reacts with channel catfish slg" but not slg" blood lymphocytes. The proportions of these cell types in the blood were 46 and 38% respectively. This monoclonal stained virtually all thymocytes. The population of peripheral blood lymphocytes reacting with this monoclonal antibody was also shown to respond to con A stimulation as well as act as T helper cells but not antibody-producing cells in in vitro primary hapten-specific antibody responses. Thus, this monoclonal antibody appears to react with cells possessing typical T lymphocyte functions. Conversely, the population of peripheral lymphocytes not reacting with this monoclonal but reacting positively with a monoclonal to serum Ig were capable, after separation by panning, of in vitro antibody production to the T-independent antigen TNP-LPS, but not to the T-dcpendent antigen TNP-KLH. Responsiveness to the latter was restored by the slg population. The nature of the marker on slg cells reacting with the above monoclonal has not been characterized but molecular weight studies and the fact that it also cross-reacts with certain brain cells suggests it may be similar to mammalian Thy-2.

Studies on separated slg+ and slg" blood lymphocytes in trout have shown the latter, but not the former, to be capable of producing a factor with macrophage-activating and interferon activity.

Thus, with the use of monoclonal antibodies fish lymphocytes may be separated into distinct subpopu-lations with functions homologous to mammalian T and B cells.

Concerning the origin of T and B lymphocytes in fish, as virtually all thymocytes possess characteristics of T lymphocytes and none of B lymphocytes, it seems likely that the thymus in fish is the principal source of T lymphocytes, as in higher vertebrates. Analysis of other lymphoid organs (i.e. kidney and spleen) show there to be a mixed population of T and B cells but both organs possess large quantities of blood which contains both T and B cells. However, as the only organ containing significant amounts of lymphopoietic tissue in teleost fish is the kidney hematopoietic tissue, and this is also the most important source of antibody-producing cells, it must be more than likely that the kidney is the source of B lymphocytes.

Other leukocytes and related cell types

Monocytes and macrophages are morphologically and cytochemically similar to their mammalian counterparts. Macrophages are widespread in tissues, including the gills, where they are thought to play an important role in vaccine uptake (see below); they form an extensive reticuloendothelial system in the kidney and spleen and, in some species, in the atrium of the heart. However, teleost fish do not possess Kupffer cells in the liver. Monocytes and macrophages are characterized by their nonspecific esterase staining and avid phagocytic activity. The latter is enhanced by complement and specific antibody, indicating the presence of opsonin receptors on these cell types. Their role in immune responses in fish as antigen-presenting cells and a source of interleukins is not yet well characterized. Channel catfish monocytes have been shown to function as accessory cells required for blood T lymphocytes to respond to the mitogen Con A. Furthermore, this function of monocytes could be replaced by the supernatant obtained from monocyte cultures stimulated with LPS, indicating the production of a 'monokine'. Channel catfish monocytes also perform an essential accessory cell function in the in vitro primary antibody response of peripheral blood lymphocytes to both T-independent (TNP-LPS) and T-dependent (TNP-KLH) antigens. In these studies the role of the monocyte in stimulation of antibody-producing cells was replaceable by monocyte culture supernatants, suggesting that other cell types (possibly B lymphocytes) could effectively 'present' antigen in the presence of the monokines.

Fish macrophages can also be activated in vitro by-exposure to supernatants from mitogen-stimulated lymphocytes, indicating the responsiveness of macrophages to interleukins.

Granulocytes are numerous in the circulation of fish and play an important role in inflammatory responses. The neutrophil is the most common type of granulocyte and is well characterized, hearing close similarity in cytochemistry to mammalian neutrophils. It is phagocytic but not avidly so and its role in defense against bacterial infection may be to secrete antimicrobial enzymes rather than perform intracellular killing. Granulocytes with the morphological and staining characteristics of eosinophils and basophils are not common in teleost fishes and, where they do occur, nothing is known about their function.

Mast cells with precisely the same characteristics as in mammals do not appear to exist in fish. However, a granulocytic cell with a connective tissue distribution similar to that of mast cells in mammals does exist and it is believed to be the analog of mast cells. This cell possesses eosinophilic or PAS-positive granules and ultrastructurally resembles mast cells. They have been termed eosinophilic granular cells (EGCs). In vivo degranulation can be induced by certain bacterial toxins as well as typical mast cell degranulating agents such as compound 48/80 and Con A. Degranulation is associated with darkening in skin color of the fish, some loss of equilibrium, swelling and vasodilatation of the intestine and a return to normal by 24 hours. Degranulation is a noncytotoxic event with regeneration of granules by 48 hours. The physiologic changes associated with degranulation were blocked by prior treatment with the antihistamine drugs promethazine and cimetidine but, unexpectedly, so was the degranulation of the EGCs. In mammals, antihistamines block histamine receptors on target cells but do not inhibit degranulation of mast cells induced by 48/80 or con A.

Natural cytotoxic cells (NCCs) have been identified in a wide range of teleost species but they have not been well characterized and may represent a heterogeneous population of cells. They have cytotoxic effects on a wide range of tissue culture cells, especially those infected by virus, and activity against protozoan parasites has also been demonstrated, suggesting these cells play an important role in defense. NCCs are found in the peripheral blood and in some species have been demonstrated also in the thymus, kidney and spleen. Their activity can be enhanced in the presence of phytohemagglutinin. NCCs are nonadherent to nylon wool, and ultrastructural studies indicate the involvement of at least two cell types which resemble monocytes and lymphocytes morphologically.

The immune response

The basic elements of the humoral and cell-mediated immunity are present in fish but many parameters of these responses are not as well developed as in higher vertebrates. An important aspect of immune respon siveness in fish is temperature dependency. Different species of fish inhabit waters with very different natural temperature ranges and their physiologic responses are well adapted to these ranges. At temperatures low in the natural range a particular species of fish will respond immunologically very slowly, if at all, to an antigenic stimulus. Thus at 4°C the immune response of trout (a temperate water species I is very slow, while at 12°C the response is maximal. In contrast, the warm water carp has an optimal response at 22°C and is virtually immunosuppressed at 12°C.

Fish produce only a single class of immunoglobulin (Ig), which resembles mammalian IgM in molecular structure. However, while mammalian serum IgM is most frequently a pentameric molecule, that in the serum of teleosts is tetrameric and, in some species, monomeric. A secretory form of IgM has been characterized from the bile and mucus of skin and gut in a few species where it occurs in a dimeric form. These and other data indicate that local (secretory) and systemic responses exist in fish. Evidence for structural heterogeneity in fish IgM heavy and light chains is growing: for example, the existence of two forms of L chains which parallel the k and A chains of higher vertebrates and the different molecular masses of H chains among serum, bile and mucus Ig. Recent studies with monoclonal antibodies have revealed the existence of three forms of H chain in catfish serum Ig. Fish IgM appears to be capable of performing all of the functions for which the different mammalian Ig isotypes show specialization, e.g. complement activation and opsonization, and it is possible that these functions in fish may he carried out by different specialized subclasses of IgM. The kinetics of the antibody response in fish are similar to those of higher vertebrates but the lag phase is somewhat longer (10-15 days) and peak titers may take up to 5-6 weeks to be reached. Antibody persists for fairly long periods, particularly in primitive species such as trout, where persistence for up to 1 year has been reported for antibodies to certain bacterial antigens.

The correlates of cell-mediated immunity arc present in fish - for example, mixed leukocyte reactions, macrophage migration inhibition, interleukin production inducing macrophage activation, delayed type hypersensitivity and acute skin rejection responses. Allogeneic restriction of antigen presentation and the existence of MHC genes in fish have been demonstrated.

Evidence for lymphokine production in fish is well established and while no fish cytokines have yet been purified, evidence indicates the existence of several types, for example interferon y (IFNy), interleukin 1

(IL-1), IL-2, colony-stimulating factor (CSF), transforming growth factor (3 (TGF/3) and tumor necrosis factor.

Immune memory has been demonstrated in fish for both humoral and cell-mediated immunity. However, the magnitude of antibody titers reached in the secondary response as compared with the primary response in fish is generally lower than in mammals (about 10-fold higher, compared with 100-fold in mammals). Of course, fish differ from higher vertebrates in that there is no shift of antibody isotype from IgM to IgG. Typically, memory induction takes longer to be established in fish (3-6 months) than in mammals and persists for 8-12 months after primary stimulation. Induction of memory appears to be temperature dependent. Generally speaking, immune responses in fish which involve the stimulation of T lymphocyte functions appear to require temperatures fairly high in the natural range of a particular species, while antibody production itself can occur at lower temperatures. Some doubt has been expressed as to the ability of temperate species, such as trout, to mount a significant secondary antibody response and it may be that these species rely instead upon prolonged production of antibody to provide long-term protection.

Immune tolerance has been demonstrated in several species of fish. The carp anti-BSA response has been suppressed by intravenous injection of soluble BSA. The induction of tolerance at high temperature was dependent upon the route of injection (intravenous) and not on the dose, while at low temperature it depended both on the intravenous route and high dose. Tolerance in these experiments lasted at least 16 months. Evidence also exists for induction of short-term tolerance by exposing very young fish to certain antigens.

Vaccination of fish

Since the great expansion of fish farming, which began in the early 1970s, there has been much interest in vaccinating fish against the principal bacterial and viral diseases. Effective vaccines have been developed to many fish diseases but only a few have achieved commercial production. The cost constraints on commercial application principally relate to the size of the fish requiring protection and the methods available for effective administration of a vaccine. There are three methods of vaccinating fish: injection, immersion and oral. Many vaccines are only effectively delivered by injection and this method is impractical for small fish (under 15 g). Presently there are only a few vaccines available commercially. These are against important bacterial dis eases of farmed salmonid fish, i.e. enteric redmouth (ERM) caused by Yersinia ruckeri, vibriosis caused by Vibrio anguillarum, cold-water vibriosis caused by Vibrio salmonicida, and furunculosis caused by Aeromonas salmonicida. These vaccines are effectively delivered by both injection and immersion administration.

Injection vaccination appears to be the most effective means of stimulating protective immunity. The method allows the use of adjuvants but it has a number of disadvantages: it requires fish to be handled and anaesthetized, which are stressful events; it is impractical for small fish; and it is labour and time consuming. Nevertheless, when the disease risks are high injection vaccination is cost effective and frequently practised. Recently, injection machines have been marketed.

Immersion vaccination has proved very useful for a number of bacterial vaccines. These vaccines are formalin-killed broth cultures which are diluted in water and the fish are immunized simply by immersing them, with the use of a hand net, for up to 1 mm in the vaccine. It is believed that the vaccine is taken up mainly through the gills. The technique is very-useful for vaccinating small fish with the minimum of handling.

Oral vaccination is the method of choice because it does not require the fish to be handled and in this respect is the only method which could be applied to extensive fish farming where the fish are not handled until slaughter. Unfortunately, this method has not proved to be very efficacious. Nevertheless, there is still much interest in the method because administration of vaccines by anal intubation appear to be very effective. Recent studies have demonstrated that, in carp and trout, the hind intestine is capable of antigen absorption, which results in effective stimulation of protective immunity; thus, interest is now centered upon microencapsulation of oral vaccines to protect the antigens during their passage through the anterior gut and to release them in the hind gut.

There is a growing interest in using recombinant DNA technology to achieve cheap production of viral and parasite antigens for use in vaccinating fish. There is still very little known concerning the nature of the protective antigens or the protective immune response to most vaccines used in aquaculture: the future of fish vaccination will probably depend very largely upon identifying the nature of these so as to optimize the production of vaccines and their effective administration. Fish farming is rapidly becoming a major industry in many countries of the world and there will be a growing need for vaccines to control disease in a wide range of fish species in the future.

See also: Immunoglobulin, evolution of; Innate immunity; Phyiogeny of the immune response.

Further reading

Bly JE and Clem LW (1992) Temperature and teleost immune functions. Fish and Shellfish immunology 2: 159-171.

Dixon B, van Erp SHM, Rodrigues PNS, Egberts E and Stet RJM (1995) Fish major histocompatibility complex genes: an expansion. Developmental and Comparative Immunology 19: 109-133.

Ellis AE (1995) Recent development in oral vaccine delivery systems. Fish Pathology 30: 293-300.

Ellis AF (ed) (1988) Fish Vaccination. London: Academic Press.

Matsunaga T and Andersson E (1994) Evolution of vertebrate antibody genes. Fish and Shellfish Immunology 4: 413-419.

Secombes CJ, Hardie LJ and Daniels G (1996! Cytokines in fish. Fish and Shellfish Immunology 6: 291-304.

Vallejo AB, Miller NW and Clem LW (1992) Antigen processing and presentation in teleost immune functions. Annual Review of Fish Diseases 2: 73-89.

Yoshida SH, Stuge TB, Miller NW and Clem LW (1995) Phyiogeny of lymphocyte heterogeneity: cytotoxic activity of channel catfish peripheral blood leukocytes directed against allogeneic targets. Developmental and Comparative Immunology 19: 71-77.

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