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The ingestion of microorganisms by phagocytes is not necessarily accompanied by microbial killing (Mazet et a i, 1994). Indeed, phagocytosis may be of only limited value in the defence against intracellular parasites unless it is combined with additional defence reactions. Oxygen-dependent microbial killing by the respiratory burst involves the generation of reactive oxygen species (ROS), (Babior et al., 1973; Badwey & Kai'novsky, 1980). In mammals, the burst occurs if phagocytes ingest particles or bacteria (Badwey & Kamovsky, 1980) or encounter complement factors or various cytokines, often in response to viral infection (Maeda & Akaike, 1991). Membrane-associated NADPH oxidase of stimulated phagocytes produces superoxide anion (0%") (Babior et a l, 1973), which is either converted to hydrogen peroxide (H2O2) spontaneously, is catalyzed by superoxygen dismutase, or

is reduced by metal complexes to form the highly toxic hydroxyl radical ( OH) (Fridovitch, 1978; Nappi et al., 1995). The respiratory burst has been demonstrated in a range of invertebrates, including molluscs (Dikkeboom et a l, 1988; Pipe, 1992), cmstaceans (Bell & Smith, 1993; 1994), echinoderms (Ito e ta l, 1992) and tunicates

General Introduction: Defence Reactions of Crustaceans (Bell & Smith, 1994). However, the strength of the burst varies between phagocytic cells of different species (Bell & Smith, 1994) and the burst is not shown by phagocytes of all groups (Anderson et a l, 1973; Mazet et a l, 1994). Early studies by Anderson et a l (1973) reported that insect phagocytes were unable to exhibit a phagocytic burst. Likewise, Mazet et a l (1994) showed that phagocytes of the moth,

Spodoptera exigua, do not generate ROS upon challenge with different elicitors and are unable to kill fungal cells or bacteria in vitro. In crustaceans, the respiratory burst has been demonstr ated in hyaline cells of C maenas (Bell & Smith, 1993; 1994), but the response is weaker than that recorded for fish or mammals. In mammals, several viral diseases result in the generation of ROS by phagocytic cells (Maeda & Akaike,

1991). During viral infection, phagocytes appear to be activated by interferons, tumor necrosis factor, virus specific antibody or complement factors (Maeda & Akaike, 1991). Human immunodeficiency virus (Kimura et a l, 1993) or influenza-A virus (Kazhdan et a l, 1994) also directly elicit a respiratory burst in human phagocytes in vitro. In the case of influenza-A, the response remains contained within the neutrophil and release of 0%" is not detected (Kazhdan et a l, 1994). It is unknown whether or not viral infection in invertebrates similarly triggers ROS formation.

The ROS generated during viral infection can be dangerous to the host (Maeda & Akaika; 1991). They are the principal factors causing mortality in mice infected with influenza virus, where mortality occured 5-6 days after the virus was cleared and could be considerably reduced by the injection of conjugated antioxidant enzymes (Malda & Akaika, 1991). In C. maenas the antioxidant enzymes superoxide dismutase, catalase and glutathione peroxidase are present within the haemocytes and, to some extent, the plasma (Bell & Smith, 1995), but it is not known whether or not the levels of antioxidant enzymes levels increase during viral infection.

General Introduction: Defence Reactions of Crustaceans

The proPO cascade

The prophenoloxidase (pro-PO) cascade is a cascade of serine proteases, which is activated by exposure to fungal p-1,3 glucans (Unestam & Soderhall, 1977), lipopolysaccharides (LPS) of Gram negative bacteria (Smith & Sôderhâll, 1983; Sôderhâll & Hâll, 1984), or bacterial peptidoglycans (Smith & Sôderhâll, 1983; Sôderhâll et ai, 1986). In crustaceans, components of the proPO cascade mediate a range of defence reactions in vitro (reviewed by Johanssen & Sôderhâll, 1989; Smith, 1991; Smith, 1996). These include phagocytosis (Smith & Sôderhâll, 1983; Sôderhâll et a i, 1986; Thôrnqvist et al., 1994), cell adhesion (Johansson & Sôderhâll, 1988), encapsulation (Smith et a i, 1984; Sôderhâll et al., 1984; Kobayashi etal., 1990) and clotting (Sôderhâll, 1981).

The proPO cascade has been studied in detail in the crayfish, Pacifastacus lenisculus, and is illustrated in Figure 2.1. Peptidoglycans, LPS or p-1,3 glucans trigger degranulation of the granular and semigranular haemocytes and the release of several serine proteases together with proPO and a 76 kDa glycoprotein (Soderhall,

1992) (Figure 1.2.1). The 76 kDa glycoprotein, peroxinecin, is a peroxidase (Johansson et al., 1995) which has multiple functions in the crayfish immune response (Sôderhâll, 1992; Sôderhâll etal. 1994). It causes adhesion of granular and semigranular haemocytes (Johansson & Sôderhâll, 1988), promotes encapsulation of parasites (Kobayashi et al., 1990) and acts as an opsonin for phagocytosis by the hyaline cells (Thôrnqvist et a i, 1994). Il also binds to the granular cells and thus generates a fecdback-loop of cell degranulation (Johansson et al., 1995) (Figure 1.2.1). It rapidly degrades once it has been excreted into the haemolymph (Johansson & Sôderhâll, 1989), ensuring that the maximum response remains concentrated around the site of infection. The proPO response is further modulated by serine

General Introduction: Defence Reactions of Crustaceans

Figure 1.2.1 Activation of the proPO cascade in the crayfish, P. lenisculus

(original drawing). The cascade is activated by LPS, peptidoglycan (not shown) or P-1,3 glucans, which cause degranulation of the granular and semigranular cells (Sôderhâll, 1992). Degranulation of the former involves binding proteins. Activation of the granular cells by P-1,3

glucans is illustrated on the right of the figure. The 100 kDa

P-1,3 glucans binding protein (PGBP) (Duvie & Sôderhâll,

1990), a plasma glycolipoprotein (Cerenius e ta l, 1994; Hall

et a l, 1995), binds to a 340 kDa receptor on the granular haemocytes (Duvic & Sôderhâll, 1992), once it has formed a complex with P-1,3 glucans. The receptor contains a putative recognition site for an RGD (Arg, Gly, Asp), which may indicate that PGBP is similar to the integrin family in vertebrates (Soderhall et a l, 1994). Degranulation leads to the release of several serine proteases together with proPO and a

76 kDa glycoprotein (Sôderhâll, 1992). The proPO of P. lenisculus is an 80 kDa protein (Aspân et a l, 1995), which is cleaved by ProPO activating enzyme (ppA), a 36 kDa serine protease, to yield a 62 kDa phenoloxidase (Aspân et a l, 1990). The 76 kDa glycoprotein, peroxinecin (Johansson et a l, 1995), causes adhesion of granular and semigranular haemocytes (Johansson & Sôderhâll, 1988), promotes encapsulation (Kobayashi e ta l, 1990) and acts as an opsonin (Thôrnqvist et a l, 1995). It also binds to the 340 kDa granulocyte receptor via a KGD (Lys, Gly, Asp) sequence, thus generating a feedback-loop of cell degranulation (Johansson e ta l, 1995).

General Introduction: Defence Reactions of Crustaceans Figure 1.2.1

LPS

/ \

P-l,3-glucan

LPS binding