The granulocytes of fish

Fish & Shellfish Immunology (1992) 2, 79-98

Review Art i cl e

The g r a n u l o c y t e s of fish P. M. HINE

Fisheries Research Centre, Ministry of Agriculture and Fisheries, P.O. Box 297, Wellington, New Zealand (Received 16June 1991, accepted in revised form 20 September 1991) The occurrence and functions of granulocytes in fishes varies between and within groups. Primitive groups (agnathans, holocephalans, elasmobranchs) all have eosinophils with homogeneous round granules. Elasmobranchs also have eosinophils with granules containing an axial crystalline rod, which is the sole eosinophil type present in lungfish. Comparative study suggests that in elasmobranchs and lungfish, heterophils and different forms of eosinophil are all of the eosinophil lineage. Agnathans, h olocephalans, dogfishes and lungfish possess fine granulocytes that may be r61ated to neutrophils of teleosts and mammalS. Holosteans and chondrosteans have eosinophiIs and neutrophils, and as in some elasmobranchs and lungfish, basophils are relatively common. Tcleosts have. neutrophils which are ultrastructurally, and possibly functionally, similar to mammalian neutrophils. More rarely they have cells with elongated granules similar t o elasmobranch and reptilian heterophils. Teleost eosinophils have large round homogeneous granules, and cytochemical and functional studies indicate that in some groups, particularly cyprinids, these cells represent an undifferentiated eosinophil[basophil lineage. Roles in inflammation, enzyme cytochemistry, function and evolutionary trends are discussed. Key words:

fish granulocytes, eosinophils, basophils, neutrophils, heterophils, ultrastructure, enzyme cytochemistry. I. I n t r o d u c t i o n

Vertebrate granulocyte nomenclature is based on the tinctorial properties of blood cells containing granules, stained with Romanovsky stains. This simple 19th c e n t u r y procedure distinguishes t h r e e cell types in humans; most numerous are neutrophils or polymorphonuclear (PMN) granulocytes t h a t contain fine mauve ' azurophil ' granules, eosinophils with large o r a n g e - p i n k granules are less abundant, and least common are basophils with large deep purple granules. Those working on non-mammalian species have had to i nt erpret their findings within this framework, with varying degrees of success. Examination of the blood of birds showed that, instead of containing neutrophils, birds have granulocytes with fine eosinophilic granules, for which the t e r m ' heterophils ' was used (Keyes, 1929 cited in Ostberg et al., 1976). This term was subsequently erroneously used to describe rabbit, guinea-pig and hystricomorph rodent neutrophils with faintly eosinophilic fine granules (Parmley 79 1050-4648[92/020079+ 20 $03.00/0 o 1992AcadcmicPress Limited

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et al., 1982; Montali, 1988). Studies on lower fish groups revealed granulocytes, particularly eosinophils, that did not resemble those of mammals, and these were given names such as' meta-eosinophils ' (Jordan & Speidel, 1931; De Laney et al., 1976), ' special eosinophils ' (Jordan & Speidel, 1931), or large and small eosinophils (Ward, 1969). This paper inter-relates granulocytes of different fish groups as far as reasonably possible, and compares them with granulocytes of higher vertebrates. A NOTE ON FISH PHYLOGENY

Agnathans (lampreys, hagfish) are regarded as the most primitive extant fish. Elasmobranchs (sharks, dogfish, rays) and holocephalans (ghost sharks) are also archaic, and there are similarities between elasmobranchs and dipnoans (lungfish) which are close to fish ancestors from which tetrapods evolved (Nelson, 1984). Relict groups and species, such as chondrosteans (sturgeons, paddlefishes), and holosteans (bowfin; Amia calva), lead to the teleosts (bony fish) which range from basal groups such as clupeiforms (sprats, anchovies) anguillids (eels) and salmonids (salmon, trout) to advanced groups such as perciforms (Nelson, 1984). II. Granulopoiesis Granulopoiesis, or granulocyte genesis, first occurs in agnathans in isolated blood islands in the prolarva and in the typhlosole and nephric folds of ammocoetes (Percy & Potter, 1976, 1977; Tanaka et al., 1981; F~inge, 1982, 1984; Fujii, 1982; Ardavln & Zapata, 1987). In adult hagfish granulopoiesis occurs in intestinal tissue (Mattisson & F/inge, 1977; Tanaka et al., 1981), which may be equivalent to the spleen (Tavassoli & Yoffey, 1983), and in adult lampreys granulopoiesis occurs in the spleen (Tavassoli & Yoffey, 1983) and supraneural fat column (Kel6nyi & Larsen, 1976; Percy & Potter, 1976, 1977; F~inge, 1982, 1984), but not in the pronephros (Ellis & Youson, 1989). I n elasmobranchs granulopoiesis primarily occurs in the oesophageal Leydig organ, or the epigonal organs (F/inge & Mattisson, 1981; Zapata, 1981; Mattisson & F/inge, 1982; F/inge & Pulsford, 1983; Oguri, 1983; F/inge, 1984; Honma et al., 1984). In the very primitive hexanchiform shark Chlamydoselachus, lymphohaemopoietic tissue occurs in the kidney (F/inge, 1987). In dogfish, the spleen, kidney, the periportal space of the pancreas and intestinal submucosa, may also contain granulopoietic tissue (Tavassoli & Yoffey, 1983; F/inge, 1987). The stingray, Dasyatis, has meningeal granulopoietic tissue, similar to meningeal lymphohaemopoietic masses in holocephalans, chondrosteans, holosteans and some amphibians (see Chiba et al., 1988). In holocephalans, granulopoiesis occurs in lymphomyeloid tissue in depressions of the cranial cartilage around the orbit, the pre-orbital canal, in the basis cranii and in shoulder cartilages (F/inge & Sundell, 1969; F/inge, 1984; Hine & Wain, 1988b; Mattisson et'al., 1990). Lungfish have granulopoietic tissue in the mesonephros, intestine, gonads, liver and pancreas (Jordan & Speidel, 1931; Ward, 1969; Rafn & Wingstrand, 1981), but dividing blasts are common in the blood (Ward, 1969; DeLaney et al., 1976).

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Chondrosteans and holosteans possess granulopoietic tissue between the tubules of the anterior kidney, around and above the anterior spinal cord and medulla oblongata (Robeson, 1932; F~inge, 1984), and to a lesser extent in pericardial tissue (Clawson et al., 1966). Meningeal haemopoietic tissue in ganoids may be homologous to mammalian bone marrow (Tavassoli, 1986) and, as in lampreys (Percy & Potter, 1976, 1977; F~inge, 1982, 1984) and higher vertebrates Tavassoli & Yoffey, 1983), it is closely associated with adipose cells. In teleosts the pronephros (Bayne, 1986; Temminck & Bayne, 1987) or head kidney (Zuasti & Ferrer, 1988; Meseguer et al., 1990) is the usual site of granulopoiesis. III. G r a n u l o c y t e Composition Granulocyte identification, especially among lower fish groups, is currently a confused and difficult area. The inherent weakness of relying on tinctorial properties and the difficulties in resolving cellular detail at the light microscope level, are compounded by the presence of immature cells in the peripheral circulation. Attempts at classification of granulocytes under the light microscope (Kindred, 1971), have not been adopted. Therefore, where possible, identification and determination of homology are here mainly based on ultrastructural studies. AGNATHANS

Hagfishes have a fine granulocyte that has pleomorphic, but usually small dense rod-like granules and a band nucleus (0stberg et al., 1976; Mattisson & F~inge, 1977; Tanaka et al., 1981; Chiba & Honma, 1986). Lampreys also have a neutrophilic granulocyte with granules of similar shape that contain an electron dense (Page & Rowley, 1983: fig. 6) or electron transparent [Keldnyi & Larsen, 1976: fig. 7(d),(f)] crystalline rod. Like immature mammalian neutrophils, agnathan neutrophilic granulocytes have a band nucleus and may contain DShle bodies (Fujii, 1981). Lampreys also have eosinophilic granulocytes with large spherical or ovoid uniformly electron dense granules (Rowley & Page, 1985), here called homogeneous granule eosinophils (HGE). DIPNOI A N D E L A S M O B R A N C H S

The Australian lungfish has four granulocyte types (Hine et al., 1990a), eosinophils with granules containing an axial crystalline rod (here called axial rod eosinophils [ARE]), heterophils with ovoid or rod-like eosinophilic granules around the centrosome, neutrophils with small dense azurophil granules, and basophils with a central nucleus and layer of large dense spherical amorphous granules. The classification of equivalent elasmobranch granulocytes is shown in Table 1. Dogfishes have HGE, and heterophils, but lack ARE and basophils. They also have a neutrophilic (Hine & Wain, 1987b) or modified heterophilic (Mattisson & F/inge, 1982) granu'locyte with small granules containing dense cores. These dense cores may lose their contents leaving a central hole. A similar granulocyte in holocephalans (Mattisson & F~inge, 1986; Hine & Wain, 1988a; Mattisson et al., 1990) appears to be a progranulocytic stage of HGE

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(Mattisson et al., 1990). The concurrent presence of lucent core granules and HGE type granules in these cells in Etmopterus spinax, reported by Mattison & F~inge [1982: fig. 3(d)], suggests that may also be a progranulocytic stage of HGE i n dogfish, the type A heterophilic granulocyte of Mattisson & F~inge (1982: fig. 5) being the true heterophil. The granulocytes of the Shark Scyliorhinus described by Morrow & Pulsford (1980), F~inge & Pulsford (1983) and Mainwaring & Rowley (1985a,b) are similar to those of the lungfish, but Scyliorhinus has HGE, not ARE. However, Parish et al. (1986a) regarded cells with large (la) or smaller (lb) granules as sub-types of one granulocyte, and distinguished degranulated HGE (type 3 granulocytes) as separate from un-degranulated (la) cells. They also distinguished two forms of ' thrombocyte ', type a, which appear to be true thrombocytes, and type b, corresponding to the G4 granulocytes of Mainwaring & Rowley (1985a,b). These type 4 granulocytes are ultrastructurally similar t o t h e basophil[mast cell lineage as they contain a layer of granules around a central nucleus, as in the basophils of sturgeons (Hine & Wain, 1988d), lungfish (Hine et al., 1990a) and many vertebrates up to humans (Zucker-Franklin, 1980). Two enzymes, acetyl-Ltyrosine-a-naphthyl esterase and tosyl-L-lysine-a-naphthyl esterase, selectivity stain basophils in sturgeons (Hine & Wain, 1988d), axolotls (Ambystoma mexicanum), the tuatara (Sphenodon punctatus) a primitive reptile, chickens and the SD granulocytes from the blood of a shark, Galeorhinus sp. (Hine, ounpubl, data), which, like those of Mustelus and Apristurus (Table 1; Hine & Wain, 1987d), morphologically resemble basophils. Thrombocytes of Scyliorhinus are phagocytic (Hunt & Rowley, 1986a) and phagocytosed material may be confused with granules. Dogfishes and rays also have thrombocytes, but type 4 granulocytes have never been ultrastructurally reported in them. Other studies on sharks (F~inge & Mattison, 1981; Morillas, 1981a,b; Hyder et al., 1983; Hyder Smith et al., 1989), dogfishes (Hine & Wain, 1987b) and rays (Hine & Wain, 1987c), including enzyme cytochemical studies (Mainwaring & Rowley, 1985b; Hine & Wain, 1987a) suggest a close inter-relationship between HGE, ARE and heterophils. All are eosinophilic, and morphologically their granules show varying combinations from eosinoph!l (HGE) granules that may be amorphous [Hine & Wain, 1987c: fig. 5(b)] or contain fibrils (F~inge & Mattisson, 1981: fig. 13; Hyder et al., 1983: fig. 7) or an obscured axial rod [Mainwaring & Rowley, 1985b: fig. 11; Hine & Wain, 1987c: fig. 5(c)], to classical ARE with an axial rod [Hine & Wain, 1987c: fig. 4(b)]. Lungfish heterophils have an eccentric nucleus with granules distributed around the centrosome (Hine et al., 1990b: fig. 7). Similar distributions can be seen in dogfish (Etmopterus baxteri) cells with ovoid eosinophilic DA granules containing fibrillar inclusions (Hine & Wain, 1987b: fig. 2), in Halaelurus heterophils (Morillas, 1981a: fig. 7, 1981b, figs 2 and 6), in rays [Hine & Wain, 1987c: figs 2(a) and 3] and in Scyliorhinus canicula [F~inge & Pulsford, 1983: fig. 5(d); F~inge, 1984: fig. 7(b); Mainwaring & Rowley, 1985a: fig. 2]. However, the heterophil granules are very variable in appearance [Kel~nyi, 1972: fig. 1; F~inge & Mattisson, 1981; Morillas, 1981b; F~inge & Pulsford, 1983; Hyder et al., 1983; Mainwaring & Rowley, 1985a,b: fig. 7: Hine & Wain, 1987b, d: fig. 3(c)], and there is no point at which elongated heterophil granules can be distinguished from ARE granules (Hine & Wain, 1987c).

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Heterophils, HGE and ARE have a similar enzyme content (Mainwaring & Rowley, 1985b; Hine & Wain, 1987a), and all stain positively with luxol fast blue, as do the eosinophils of higher vertebrates (Shoham et al., 1974), and the heterophils of reptiles and birds (Hine, unpubl, data). Heterophils, HGE and ARE are therefore regarded as different expressions of the eosinophil lineage. HOLOCEPHALANS

Holocephalan granulocytes are difficult to identify and granulocyte composition differs between families. Most simple are those of Rhinochimaera which only has a fine granulocyte with granules containing whorls, vesicles, plate-like or rod-like inclusions. Harriotta, another rhinochimaerid, has similar fine granulocytes, with granules apparently lacking complex sub-structures, and has HGE in orbital tissue (Hine & .Wain, 1988a). Hydrolagus sp. and Chimaera monstrosa, both chimaerids, have fine granulocytes with lucent cores (Hine & Wain, 1988a; Mattisson et al., 1990) that may expel an ovoid core surrounding the lucent space, to develop into cells with HGE granules (Mattisson et al., 1990). In addition they also have fine granulocytes without lucent cores (Mattisson & F~inge, 1986; Hine & Wain, 1988a), similar to those in Rhinochimaera. The elephant fish, Callorhynchus millii, also has granulocytes with lucent cores, designated LVGI and LVGII by Hine & Wain (1988a: figs 10-14), that may develop to HGE. However, expelled ovoid cores similar to those observed by Mattisson & Fiinge (1982: fig. 4) and Hine & Wain (1988a: fig. 7) in HGE of Chimaera and Hydrolagus, were not observed in CallorhynchusHGE, and their origin from lucent core progranulocytes remains uncertain. The lighter cortex and darker medulla of HGE granules in Chimaera and Callorhynchus may be due to partial degranulation or fixation (Mattisson & F~inge, 1986). CttoNDRoSTEANS AND HOLOSTEANS

Chondrosteans have HGE (Hine & Wain, 1988d) that may contain an unorientated fibrillar element, which Clawson et al. (1966) compared with the axial crystalloids of mammalian ARE. Chondrosteans also have neutrophils (Clawson et al., 1966; Keldnyi, 1972) with the characteristic fusiform granules containing longitudinal fibrils, called ' n u c l e a t e d ' granules in mammalian neutrophils (Brederoo et al., 1983). Sturgeons (Acipenser brevirostrum) have neutrophils with fibrillar granules and large elongated granules that resemble the granules of reptilian and avian heterophils, but which are not eosinophilic (Hine & Wain, 1988d). The holostean, Amia calva, has HGE, heterophils ('eosinophils with bacillary granules') and possibly neutrophils ('special granulocytes '), at the light microscope level (Robeson, 1932). TELEOSTS

The granulocyte composition of teleosts is very variable with some species apparently lacking neutrophils or eosinophils, and identifiable basophils being rarely seen (Hine et al., 1987). Some species and groups have granulocytes that are readily recognisable at the EM level. Neutrophils with granules containing an axial fibrous sub-structure, closely resembling the ' nucleated' granules of mammalian neutrophils (Brederoo et al., 1983), have been reported from plaice

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(Ferguson, 1976; MacArthur et al., 1984), trout ( Bielek, 1980, 1981; Sharp et al., 1991), cyprinids (Bielek, 1981; Cenini, 1984; Temminck & Bayne, 1987; Imagawa et al., 1989; Rombout et al., 1989; Fujimaki & Isoda, 1990), catfish (Cannon et al., 1980a,b; Breazile et al., 1982), icefish (Barber et al., 1981), striped bass (Bodammer, 1986), tilapia (Suzuki K., 1986; Doggett & Harris, 1989, 1991), eels (Hine et al., 1986a,b), sea bass (Meseguer et al., 1990) and probably pike (Savage, 1983) and African bream (Boomker, 1981: fig. 11). In catfish the axial fibrous sub-structure may be reduced to a rod-like inclusion (Lester & Desser, 1975), resembling the plate in granules of agnathan fine granulocytes (Page & Rowley, 1983). The granular fibrils in cyprinid neutrophils are closely packed to resemble the axial rod of ARE, leading to the mis-identification of neutrophils as eosinophils (Weinreb, 1963; Hendrick et al., 1986). Conversely, HGE or heterophils in African catfish have been mis-identified as neutrophils (Boomker, 1981: fig. 9). The blood granulocytes of sparids, described'as heterophils (Zuasti & Ferrer, 1988) and neutrophils (Roubal, 1986), are eosinophilic (Roual, 1986; Hine et al., 1987) and do not ultrastructurally resemble neutrophils. They more closely resemble HGE (Roubal, 1986: figs 10 and 11; Zuasti & Ferrer, 1988: figs 4-8), although some granulocytes illustrated by Roubal (1986: figs 16 and 20) have elongated granules similar to heterophils of lungfish (Hine et al., 1990b), reptiles (Kel~nyi & N~meth, 1969) and birds (Enbergs, 1975), and may best be regarded as heterophils until ontogeny is established. Some cells identified as granulocytes (Ferri & Hernandez Blazquez, 1987; Imagawa et al., 1989: fig. 8) resemble mammalian natural killer cells (Kang et al., 1987). All teleost eosinophils appear to be HGE, although a granule with a l i g h t axial band has been reported from eels (Hine & Wain, 1989: fig. 5) and sea bass [Meseguer et al., 1990: fig. 3(e)]. The electron density of the granules may obscure sub-structures such as rod-like crystalloids (Fujimaki & Isoda, 1990: figs 5 and 6) or fibrils (Cross & Matthews, 1991). When extravascular, eosinophils are described as eosinophilic granule cells (EGC) (Ezeasor & Stokoe, 1980; Ellis, 1985; Hine & Wain, 1989; Vallejo & Ellis, 1989; Powell et al., 1990; Lamas et al., 1991), to distinguish them from blood eosinophils. HGE have been reported from many species (Boomker, 1981; Bodammer, 1986; Hine et al., 1986b; Suzuki, K. 1986; Zuasti & Ferrer, 1988; Doggett & Harris, 1989, 1991; Meseguer et al., 1990), but in some species, particularly cyprinids, it is not possible to distinguish eosinophils from basophils with certainty (Cenini, 1984; Temminck & Bayne, 1987; Imagawa et al., 1989; Rombout et al., 1989; Fujimaki & Isoda, 1990). Many teleosts have a peripheral blood granulocyte with large spherical granules which are amylase-resistant periodic acid-Schiff (PAS) positive, nominated PAS-GL, and thought to be the piscine forerunner of the basophil/mast cell (Barber & Mills Westermann, 1975, 1978a). It was postulated that PAS-GL became basophils by sulphation of granule polysaccharide to give heparin, and by storage of histamine (Barber & Mills Westermann, 1978b). Eosinophil]basophil granulocytes of cyprinids lose granule contents to leave large lucent cores (Cenini, 1984; Suzuki, K., 1986; Temminck & Bayne, 1987; Imagawa et al., 1989; Rombout et al., 1989), that in some cases (Bielek, 1981: fig. 3) resemble the lucent core granulocytes of holocephalans that develop to HGE (Mattisson et al., 1990). Some HGE have dark cores and a light cortex (Lester & Desser, 1975; Roubal, 1986; Rombout et al., .1989), that may represent early

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degranulation (Roubal, 1986; Rombout et al., 1989), especially in inflammatory tissues (Lester & Desser, 1975). Basophils (Ferri, 1984; Zuasti & Ferrer, 1988; Meseguer et al., 1990) and mast cells (Weiss, 1979) have been reported from teleosts, but too little is known to ultrastructurally identify them as such at present. Studies on cyprinids suggest that cells with features of eosinophils and basophils are common in peripheral blood. OVERVIEWOFFISHGRANULOCYTEULTRASTRUCTURE All the granu]ocyte types seen in higher vertebrates are present in fish. Granu]ocytes closelyresemblingmammalianneutrophils in the possession of fusiform fibril]ar or azurophil granules occur in agnathans, lungfish, chondrosteans, teleosts, amphibians (Curtis et al., 1979), reptiles (Frye, 1981) and mammals(Brederooet al., 1983). Thefine granulocytesin holocephalansand the G2 granulocytesof Scyliorhinus may also be regarded as neutrophils, but their affinitiesare uncertain. Heterophils, HGE, ARE, and HGE developing from granules with lucent cores are regarded as different expressions of the eosinophil lineage. Heterophils occur in e]asmobranchs,possibly some teleosts, lungfish, reptiles (Frye, 1981) and birds (Enbergs, 1975). HGE occur in lampreys,ho]ocepha]ans,e]asmobranchs, chodrosteans, teleosts, amphibians and reptiles (Kel6yi & N6meth, 1969; Frye, 1981), some birds (Enbergs, 1975) and some mamma]s (Sonoda & Kobayashi, 1970). In some amphibians the dense granule matrix may obscure pleomorphic crystalloidsub-structure(Curtis et al., 1979; Mesegderet al., 1985). HGE developing from granules with lucent cores are on]yknown from dogfishes and holocephalans. ARE occur in elasmobranchs;lungfish, some birds, particularly anseriforms(Maxwell, 1978) and mammals(Scott & Horn, 1970). Basophi]s are known from elasmobranchs, chondrosteans, some te]eosts, lungfish, amphibians, in some of which the granules contain an axial rod-like core (Curtis et al., 1979), are commonin reptiles (Frye, 1981) and birds (Enbergs, 1975), but less commonin mamma]s(Dvoraket al., 1982).

IV. F u n c t i o n a n d C y t o c h e m i s t r y AGNATHANS

Both neutrophils and eosinophils are phagocytic in larval lampreys, but neutrophils are more phagocytic than eosinophils (Rowley & Page, 1985). However, neither granulocyte contains peroxidase (Kel~nyi & Larsen, 1976; Page & Rowley, 1983; Hine et al., 1987), a key enzyme in the killing of phagocytosed micro-organisms. Agnathans granulocytes contain alkaline and acid phosphatases (Hine et al., 1987). DIPNOI, ELASMOBRANCHSAND HOLOCEPHALANS

An increase in peripheral blood neutrophils has been reported in infected lungfish (DeLaney et al., 1976), only eosinophils are known to be phagocytic (Fishman et al., 1979), and peroxidase only occurs in eosinophils and macrophages (Hine et al., 1990b).

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Very little is known about granulocyte function in the inflammatory response in elasmobranchs. Studies on natural skin lesions (Woodhead, 1982) and on turpentine-induced inflammation (Reznikoff & Reznikoff, 1934), report lymphocytes and macrophages in lesions, but lesions in experimental vibriosis contained macrophages and neutrophilic granulocytes (Grimes et al., 1985). Chemotaxis assays indicate the G1 (HGE) and G3 (heterophil) granulocytes (Mainwaring & Rowley, 1985a,b) of Scyliorhinus are chemokinetic (Hunt & Rowley, 1986b), and G1 chemokinesis is enhanced by leukotriene B4 (LTB4) (Hunt & Rowley, 1986c). However, Scyliorhinus granulocytes do not respond in bipolar shape assays to N-formyl-methionyl-phenylalanine (Hunt & Rowley, 1986b), and higher levels of LTB4 are needed to enhance chemokinesis than in mammals (Hunt & Rowley, 1986c). Phagocytic assays on Scyliorhinus granulocytes conflictingly report only monocytes and thrombocytes (Hunt & Rowley, 1986a), or G2 (neutrophil) cells, as well as monocytes and thrombocytes, are phagocytic (Parish et al.,r1985, 1986b,c). However, all granulocyte types have similar weak or rare peroxidase, very rare alkaline phosphatase and flglucuronidase, and variable but often strong acid phosphatase, esterase and PAS content (Mainwaring & Rowley, 1985b; Hine & Wain, 1987a; Hine et al., 1987). Holocephalan peripheral blood granulocytes lack peroxidase, have weak acid phosphatase and moderate esterases, but, except in Harriotta and Rhinochimaera, stain strongly for alkaline phosphatase (Hine & Wain, 1988a). Tissue resident eosinophils may contain peroxidase. CHONDROSTEANS AND HOLOSTEANS

Sturgeon eosinophils (Hine & Wain, 1988d), and the monocytes and macrophages, but not the neutrophils, of a holostean, Lepisosteus, are phagocytic (McKinney et al., 1977). Sturgeon eosinophils, but not neutrophils, contain peroxidase, but neutrophils, not eosinophils, contain PAS positive substance (Hine & Wain, 1988d). TELEOSTS

Neutrophils As in mammals (Montali, 1988; Benestad & Laerum, 1989), the neutrophils of teleosts mediate the acute inflammatory response (Finn, 1970; Finn & Nielsen, 1971a,b; Nagamura & Wakabayashi, 1983; MacArthur et al., 1984; Suzuki, K., 1986; Suzuki & Hibiya, 1988; Ventura & Grizzle, 1988). After vasodilation, marginated neutrophils enter the peripheral circulation by diapedesis (Bly et al., 1990), and the marginated pool and pronephric neutrophils become depleted (Park & Wakabayashi, 1989). Bacterial endotoxin (LPS) may cause an elevated left shift (MacArthur et al., 1984) resulting in' toxic' neutrophils (Hine & Wain, 1988c). LPS may also cause a febrile response, with infected fish seeking elevated water temperatures (Reynolds et al., 1978). Neutrophils move to the site of inflammation by chemokinesis (Liewes & van Dam, 1982; Nash et al., 1986), but the kinetics of the acute inflamrhatory response are slower in fish than in mammals (Finn & Nielsen, 1971b; MacArthur et al., 1984; Yasuda et al., 1984; MacArthur et al., 1985), possibly because low levels of endogenous chemoattractants (MacArthur et al., 1985).

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In fish, a peripheral blood neutrophilia may indicate the early phase of acute inflammation resulting from infection (Bruno & Munro, 1986; Lowe-Jinde, 1986; Clifton-Hadley et al., 1987; Steinhagen et al., 1990). However, neutrophilia is also a non-specific response to stressors (Ellis, 1981a; Peters et al., 1991) such as transport and handling (Ellsaesser & Clem, 1986), trauma (Majahan & Dheer, 1982), exposure to some chemicals (Hlavek & Bulkley, 1980), and starvation (Mahajan & Dheer, 1983). Temperature may also affect neutrophil abundance (Dunn et al., 1989), causing neutrophilia (Bennett & Neville, 1975) or neutropenia (Dheer, 1988). The degree to which neutrophils phagocytose pathogens/particles/phlogistons is variable between fish species, and individuals (Moritomo et al., 1988). The neutrophils of most species are phagocytic in vivo (MacArthur & Fletcher, 1985; Suzuki, K., 1986; Siwicki & Studnicka, 1987) and in vitro (Suzuki, 1984; Hine et al., 1986a; Finco-Kent & Thune, 1987; Thuvander et al., 1987; Moritomo et al., 1988; Ainsworth & Dexiang, 1990). During inflammation, neutrophil subpopulations may differ in their phagocytic capability (SSv~nyi & Kusuda, 1987). Although phagocytosis by plaice (Pleuronectes platessa) neutrophils has been clearly demonstrated (MacArthur et al., 1984; MacArthur & Fletcher, 1985), this was not observed by Ellis (1976), who later suggested neutrophils may be bactericidal extracellularly (Ellis, 1981b). Extracellular bactericidal activity may occur (Waterstrat et al., 1991), but as in mammals, the way in which neutrophils of one fish species combat pathogens may vary with the virulence and species of pathogen (Ainsworth & Dexiang, 1990). However, studies on the effects of complement and opsonisation on enhancement of phagocyte chemiluminescence (Sakai, 1984; Iida & Wakabayashi, 1988; Moritomo et al., 1988i Yoshida & Kitao, 1991), and superoxide anion production (Plytycz et al., 1989), suggest similar bactericidal and respiratory burst processes in neutro'phils of fish, as in mammals. Phagocytosis in fish is temperature dependent (O'Neill, 1985), which may explain their preference for warm water as a febrile response to bacterial infection (Reynolds et al., 1978). The enzyme cytochemistry of teleost leucocytes has been extensively reviewed by Hine et al. (1987). Granulocyte peroxidase content varies between (Hine & Wain, 1988e) and within (Hine et al., 1986b), groups. In general, occurrence of eosinophil peroxidase decreases and neutrophil peroxidase increases from basal groups up to advanced groups, with some exceptions, such as in pleurinectiforms (Hine & Wain, 1988e). HGE cannot easily internalise particles because of their large granules, and they are effectors by degranulation, causing release of enzymes such as peroxidase, that may damage nearby cells. The small granules of neutrophils, and phagocytosis, overcome these problems and may account for the change in granulocyte peroxidase distribution. During inflammation, granulocyte enzyme content may change as different sub-populations enter the peripheral blood (Yasuda et al., 1984; Sakai et al., 1987; Hine & Wain, 1988c; Waago et al., 1988). Eosinophils and basophils Peripheral blood eosinophils are uncommon in many teleost species (Hine et al., 1987), and therefore researchers have had to study EGC at extravascular sites (Ezeasor & Stokoe, 1980; Ellis, 1985; Suzuki, K., 1986; Reimschuessel et al.,

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1987; Hine & Wain, 1989; Vallejo & Ellis, 1989; Powell et al., 1990; Cross & Matthews, 1991; Lamas et al., 1991). Although eosinophils may accumulate in parasitic infections (Lester & Daniels, 1976) and inflammation (Roubal, 1986), there is not good evidence of antiparasitic function similar to that seen in mammals (Butterworth et al., 1980). In fish lacking eosinophils, or in which eosinophils are scarce, neutrophils may respond to helminth infections (Elarifi, 1982; Sharp et al., 1991). The confusion between basophils and eosinophils at the EM level in cyprinids, also occurs between EGC, PAS-GL and basophils cytochemically. Basophils do not show the same pattern of metachromasia (Suzuki, Y., 1986) as in mammals, and whereas mammalian histamine derives from mast cells, there is some evidence that in fish histamine comes from PAS-GL (Barber & Mills Westermann, 1987b), but stronger evidence that it derives from EGC (Ellis, 1982, 1985; Vallejo & Ellis, 1989). EGC histamine may be released in such concentrations as to cause an anaphylactic-like response, but details of granule release differ from those in mammals (Ellis, 1985; Vallejo & Ellis, 1989). Interestingly, degranulated cells regenerate granules (Vallejo & Ellis, 1989), which in mammals is characteristic of basophils (Galli et al., 1984). Mammalian basophils and eosinophils derive from a common progenitor (Denburg et al., 1985).

V. S u m m a r y Fish show great, unpredictable variability in granulocyte composition "and function, even within families. Four granulocyte types may be distinguished; neutrophils, heterophils, eosinophils and basophils. Knowledge of mammalian granulocytes can be applied only loosely to those of fish, the closest similarity being to the neutrophils of some teleosts. However, as pointed out by Ellis (1981b), teleost and mammalian neutrophils may not have similar activities and functions in inflammation and pathology. Granulocyte morphology and peroxidase distribution suggest that eosinophils are important in bactericidal mechanisms in lower groups (elasmobranchs, holocephalans, chondrosteans), but the neutrophil developed as a more efficient phagocyte. Neutrophils and monocytes/macrophages are the primary phagocytes in many vertebrates, both usually contain peroxidase and in reptiles cannot be readily differentiated. Eosinophils and basophils/mast cells are distinctive end cells in mammals, but are poorly differentiated in teleosts. The eosinophils/basophils of teleosts, including EGC and PAS-GL, may therefore be developmentally immature compared with those of mammals. Ellis (1977) suggested classification of granulocytes of fish be based on ontogeny, morphology and function, rather than tinctorial properties. Ontogenetically granulocytes derive from common progenitor cells, and therefore ontogeny is of limited value in classification. Morphological studies do not readily differentiate eosinophils and basophils, and enzyme cytochemistry may not reliably indicate function as there is growing evidence in mammals that leucocytes may utilise each others products (Leung & Goren, 1989). Where possible, fish granulocytes should be nominated in mammalian terms, where this is not possible, terminology based on function should be considered.

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