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Alternatively activated macrophages are generated in the presence of the T_H2 cytokines IL-4 and/or IL-13 (Gordon and Martinez, 2010). A feature typical of alternatively activated macrophages is that they metabolize L-arginine differently from the way innate/

classically activated macrophages do (Modolell et al., 1995). Where the enzyme iNOS is omnipresent in innate/classically activated macrophages and converts L-arginine into L-citrulline and NO, alternatively activated macrophages compete for the same substrate by up-regulating arginase enzymatic activity, and convert L-arginine into L-ornithine and urea (Figure 2). Thus, alternatively activated macrophages, through the activation of arginase, rendering L-arginine unavailable for conversion by iNOS into NO, may act as

‘anti-inflammatory’ macrophages by attenuating the production of NO (Modolell et al., 1995).

Figure 2. L-Arginine metabolism of macrophages. The enzymes iNOS and arginase use a common substrate, L-arginine, to initiate different pathways that negatively regulate each other as indicated by the dashed lines. The pathway initiated by iNOS leads to production of NO and L-citrulline and a phenotype associated with antimicrobial activity and inflammation, hallmarks of (innate and) classically activated macrophages. The pathway initiated by arginase leads to the formation of urea, and metabolites (L-proline and polyamines) linked to extracellular matrix production, cell proliferation and tissue repair and is associated with the development of alternatively activated macrophages.

In fish, two genes with homology to both IL-4 and IL-13 have been reported (Li et al., 2007; Ohtani et al., 2008). However, unlike IL-4 and IL-13 of mammalian vertebrates, the two fish genes are not located in one T_H2 locus on a single chromosome. At present, it is unknown whether the two molecules represent duplicated genes that have acquired distinct functions, similar to what has been described for the IFN-γ isoforms, or whether they display functional redundancy. The two homologous genes are tentatively referred to as IL-4-like (IL-4L) and IL-4-related (IL-4rel) and it seems that only functional studies can conclusively classify these genes as IL-4 or IL-13 orthologues. At this moment, no functional studies on the 4L protein have been reported. However, a recombinant IL-4rel protein has recently been produced and functional studies in zebrafish have shown that injection of IL-4rel leads to enhanced B-cell proliferation (Hu et al., 2010), which is in accordance with the functional properties of mammalian vertebrate IL-4 (Callard, 1989; Clark et al., 1989). In another study, the same recombinant IL-4rel protein

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increased DC-SIGN/CD209 expression on leukocyte cell surfaces (Lin et al., 2009). The latter observation especially, is in agreement with the functional effects of IL-4/IL-13 on alternatively activated macrophages in mammalian vertebrates. These effects include increased expression of DC-SIGN along with increased endocytosis of mannosylated ligands, down-regulation of latex bead phagocytosis, increased antigen presentation to T_H2 cells and decreased microbicidal functions (Martinez et al., 2009). In this context it could be rewarding to study the presence of IL-4rel-specific receptors on fish macrophages.

In mammalian vertebrates, the heterodimeric receptor complex for IL-4 and IL-13 consists of one receptor subunit in common for both cytokines (IL-4Rα), which is in fact the molecular basis for their overlapping biological functions, and one receptor subunit specific for each cytokine (Mueller et al., 2002). Although similar sequences for these T_H2 cytokine receptors are present in the zebrafish database (Acc. No. NP 001013300), no studies have been performed to link the presence of IL-4/IL-13 cytokine receptors to macrophage polarization. No doubt, functional studies on the effects of 4-like and IL-4-rel proteins on (purified) macrophage cell populations should be helpful to study the link between these presumed T_H2 cytokines with the generation of alternatively activated macrophages in fish.

Arginase activity as a marker for alternatively activated macrophages

Arginase is a manganese metallo-enzyme that catalyses the hydrolysis of L-arginine to L-ornithine and urea and is the main enzyme responsible for the cyclic nature of the urea cycle (Wu and Morris, 1998). In mammalian vertebrates, arginase activity can be indicative of either of two distinct isoforms encoded by two separate genes (Jenkinson et al., 1996). Arginase-1 takes part in the ornithine-urea cycle aimed at ammonia detoxification, is located in the cell cytosol and is mainly expressed in the liver, whereas arginase-2 is located in the mitochondria. Arginase is necessary for the production of proline and glutamate, important for cell proliferation and collagen production during extracellular matrix regeneration (Albina et al., 1990; Vincendeau et al., 2003). In mice it is the expression of arginase-1 in particular (Munder et al., 1999), that is associated with the presence of IL-4/IL-13 induced alternatively activated macrophages, whereas LPS+IL-10-induced regulatory macrophages are characterized by enhanced expression of arginase-2 (Lang et al., 2002).

Also in fish, arginase-1 and arginase-2 are present as distinct isoforms that cluster with their respective mammalian counterparts, suggesting a conservation of structural differences. Polyploid fish have undergone further duplications of the arginase genes, sometimes resulting in multiple copies of both arginase isoforms (Wright et al., 2004;

Joerink et al., 2006c). Both fish arginases contain a mitochondrial targeting sequence and the absence of a cytosolic form of arginase in fish may be related to the fact that most

fish are ammoniotelic animals, excreting their excess nitrogenous wastes as ammonia directly into the water. In carp, arginase-1 gene expression was found mainly in the mid kidney, whereas arginase-2 gene expression was detected in all organs, with the highest expression in liver (Joerink et al., 2006c). In rainbow trout, although arginase-1 gene expression is highest in liver, fasting animals regulate arginase-2 gene expression (Wright et al., 2004). In addition, up-regulation of either arginase gene leads to increased arginase enzyme activity and may thus be associated with the presence of alternatively activated macrophages in fish. It may be clear that, until the exact role of the two arginase isoforms in fish has been resolved, it is best to determine the gene expression of both genes.

There are a number of studies in fish that have measured arginase gene expression as part of an immune response to infection. In carp, infection with the protozoan parasite Trypanosoma carassii induced an up-regulation of arginase-1, but not arginase-2 gene expression in head kidney (Joerink et al., 2006a); mechanical skin injury induced arginase-2, but not arginase-1 gene expression during the first few hours after skin damage (Gonzalez et al., 2007) and injection of zymosan in the peritoneum resulted in an influx of phagocytes with increased gene expression of, among others, arginase-2 (arginase-1 not measured; (Chadzinska et al., 2008)), that could be enhanced by co-injection of morphine (Chadzinska et al., 2009). In Atlantic salmon, infection with ectoparasitic caligid crustaceans induced up-regulation of arginase-1 gene expression in intact skin of infected fish (arginase-2 not measured) (Skugor et al., 2008); infection with the bacterium Aeromonas salmonicida showed induced gene expression for both arginase-1 and -2 (Fast et al., 2009) and injection with oil-adjuvanted vaccines against this bacterium induced granulomatous reactions associated with increased arginase-1 gene expression in head kidney (Mutoloki et al., 2010). Although it is evident that gene expression studies can only be suggestive of the presence in vivo of alternative macrophage activation, the use of an iNOS/arginase gene expression index (Fast et al., 2009) may be a useful way to quantify macrophage polarization during immune responses to pathogens in vivo.

A lack of arginase mRNA expression does not always imply a lack of arginase activity, or vice versa and total arginase enzymatic activity can be indicative of the activation of arginase-1, arginase-2 or both (Laberge et al., 2009). Thus, the most reliable marker for the presence of alternatively activated macrophages in fish is arginase enzymatic activity.

Studies in mice have shown that arginase activity can be induced via the increase of intracellular cAMP and tyrosine kinase phosphorylation as well as by the administration of exogenous cAMP (Munder et al., 1999). Similarly, studies in carp using head kidney-derived macrophages have shown that administration of exogenous cAMP induced arginase-2, but not arginase-1 gene expression and clearly induced arginase activity but not nitric oxide production in these macrophages (Joerink et al., 2006c). In carp, as in mice,

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arginase enzymatic activity could be specifically inhibited by N^G-hydroxy-L-arginine (Figure 2) which, as intermediate in the conversion of L-arginine into L-citrulline and NO, inhibits arginase by specifically interacting with the manganese-cluster of the active site of the arginase enzyme. Altogether, these in vitro results provide the best evidence to date that arginase enzymatic activity could be a useful marker for the presence of alternatively activated macrophages in fish.