2.3.2 Cells of the monocyte–macrophage lineage
2.3.2.1 Monocytes and macrophages
Macrophages (and their precursors, monocytes) are the main phagocytic cells of the immune system. Tissue macrophages are present throughout the body and contribute to tissue homeostasis and immune surveillance. Upon tissue damage or infection, monocytes are rapidly recruited to the tissue where they differentiate into tissue macrophages that can take up antigens and produce cytokines that modulate the action of other recruited cell types.
Macrophages are remarkably plastic and can change their functional phenotype depending on the environmental cues they receive. Based on these signals, activated macrophages were traditionally divided into classically activated macrophages induced in a Th1 cytokine environment and alternatively activated macrophages induced in a Th2 cytokine environment; however, recent classifications have introduced novel subtypes.
Forlenza et al. (2011) based on the current knowledge on macrophage activation mechanisms in fish, adopted a model with four different phenotypes for activated macrophages. Innate activated macrophages are induced by pathogens (or a microbial stimulus such as pathogen-associated molecular patterns) alone. The pathogens are recognized through toll-like receptors (TLRs) in macrophages and are phagocytosed.
In fish, most of our knowledge regarding innate activation of macrophages comes from stimulating these cells with antigenic molecules like bacterial lipopolysaccharide (LPS), although some studies have also been reported using peptidoglycan, Poly I:C (double stranded RNA), flagellin, or lipoteichoic acid (reviewed in Forlenza et al., 2011).
The effects that these molecules provoke in fish macrophages vary between fish species, but generally lead to the production of oxygen and nitrogen radicals, increased phagocytosis, and production of pro-inflammatory cytokines. It is surprising, however, that fish are much less susceptible to LPS stimulation and septic shock than mammals, and this suggests important differences in the way that LPS is sensed in this group of animals that have to survive aquatic environments with a much higher microbial load.
For example, toll-like receptor 4 (TLR4), responsible for LPS recognition in mammals (see Section 2.5.1), is thought to be absent in salmonids (Rebl et al., 2010), whereas it has been demonstrated that TLR4 does not recognize LPS in some other species such as zebrafish (Sepulcre et al., 2009).
Classically activated macrophages are induced by a combination of IFN-g and a microbial stimulus. IFN-g has been identified in many different teleost species, whereas, in some species, such as zebrafish, catfish, common carp (Cyprinus carpio), and goldfish, two types of IFN-g containing all the conserved IFN-g signature motifs have been identified (see Section 2.4.1.2).
In trout macrophages, recombinant IFN-g has been shown to induce the transcription of IFN-g-inducible protein 10 (gIP-10), MHC class IIa chain, and antiviral genes as well as enhanced respiratory burst activity (Zou et al., 2005). In goldfish, recombinant IFN-g increased the expression of several pro-inflammatory genes and the phagocytic and nitric oxide response of macrophages (Grayfer and Belosevic, 2009).
In carp, however, IFN-g alone produced no effect, whereas in combination with LPS induced a strong synergistic effect on nitric oxide (NO) production, respiratory burst activity, and expression of pro-inflammatory cytokines. In those species that contain a second IFN-g, important differences on their effects on macrophages have been detected between the two proteins, demonstrating that although both genes produce functionally active molecules with effects on macrophages, their role in immunity is not exactly the same (Forlenza et al., 2011).
Alternatively activated macrophages are differentiated in the presence of the Th2 cytokines IL-4 and/or IL-13 (Forlenza et al., 2011). In mammals, alternatively activated macrophages metabolize l-arginine differently from innate/classically activated macrophages (Modolell et al., 1995).
Innate/classically activated macrophages convert l-arginine into l-citrulline and NO, whereas alternatively activated macrophages convert l-arginine into l-ornithine and urea through the activation of arginase, rendering l-arginine unavailable for conversion by iNOS (inducible NO synthase) into NO and, therefore, attenuating the production of NO and acting as an “anti-inflammatory” macrophage (Modolell et al., 1995).
attenuating 약화
Although genes with homology to both IL-4 and IL-13 have been reported in some species (Li et al., 2007; Ohtani et al., 2008), no functional studies have yet been performed with these molecules. Thus, there is no evidence that this activation path is conserved in teleost fish.
Finally, regulatory macrophages are associated with the presence of the regulatory cytokine IL-10 and are, therefore, involved in the down-regulation of inflammation.
They can be generated in response to a combination of TLR ligands and second signals such as immune complexes, prostaglandins, apoptotic cells, glucocorticoids, or G-protein-coupled receptor ligands (Mantovani et al., 2004). Again, even though IL-10 is present in different teleost species (Inoue et al., 2005; Lutfalla et al., 2003; Pinto et al., 2007; Savan et al., 2003; Seppola et al., 2008; Zhang et al., 2005; Zou et al., 2003a), there is no evidence that this macrophage activation pathway functions in a similar fashion to that of mammalian macrophages.
2.3.2.2 Melanomacrophages
The functions described for melanomacrophage centers present in immune organs, such as the kidney or the spleen, are numerous, including the possibility of being active phagocytic centers for heterogeneous materials such as cell debris, melanin pigments, hemosiderin granules, and lipofuscin residues as well as diverse lipids, protein aggregates, and mucopolysaccharides, acting as metabolic scavengers.
They are also responsible for iron storage, deposition of resistant phases of pathogens, such as bacterial and parasitic spores, and seem to play an active role in antigen processing and presentation. Consequently, they have been proposed as a primitive analog of the germinal centers of mammalian lymph nodes (Agius and Roberts, 2003; Zapata and Amemiya, 2000).
Additionally, the fact that melanin is produced de novo through enzymes belonging to the tyrosinase gene family at the sites of injury or infection in a wide range of species has led authors to speculate that melanin, and/or its quinone precursors, could have antimicrobial properties, as in mammals (Mackintosh, 2001). Although this has to be further explored in fish, the expression of the tyrosinase gene family that occurs in melanomacrophages during chronic inflammation of Atlantic salmon (Salmo salar) (Larsen et al., 2012) and the fact that melanin synthesis is induced in viral disease in this same species (Fagerland et al., 2013) suggests a similar role.
2.3.3 Polymorphonuclear leukocytes
Polymorphonuclear leukocytes (PMNs) are cells of myeloid origin with a distinctive structure based on the polymorphic shape of their nucleus and on the numerous granules present in the cytoplasm. The nomenclature used in mammals for the classification of granulocytes, according to their affinities for acidic or basic dyes, does not always correlate with the characteristics and functions of fish granulocytes; furthermore, the different morphological types of granulocytes are not always present in each fish species.
Neutrophils are the predominant type of granulocytes found in most fish species, but some teleost have been reported to have both acidophilic and/or basophilic granulocytes, in addition to neutrophils (Rowley, 1996).
2.3.3.1 Neutrophils
Neutrophils are one of the first recruited cells at inflammation sites and are known to possess a pivotal antimicrobial activity through their strong phagocytic capacity and, at an extracellular level, by the degranulation of their cytoplasmic granules containing a vast array of soluble mediators, including enzymes, antimicrobial peptides, and potent redox molecules, or by the release of the recently discovered neutrophil extracellular traps (NETs) (Henry et al., 2013; Pijanowski et al., 2013).
degranulation 탈과립
cytoplasmic 세포질의
granules 과립제
Besides, they provide signals for the activation and maturation of other immune cells. Neutrophils (also called heterophils in some species) are present in almost all fish species studied and are critical components of the innate immune defenses. In fish, these cells possess typical neutrophil capacities such as phagocytosis or degranulation.
Their antimicrobial mechanisms also include a strong intracellular (inside the phagosome) and extracellular production of reactive oxygen intermediates (a mechanism called respiratory burst) and nitric oxide metabolites that directly interfere with pathogen survival as well as NET release. Histochemically, neutrophils show strong myeloperoxidase activity, both in elasmobranchs and teleosts (Lieschke et al., 2001).
Recently, thanks to the generation of transgenic zebrafish with neutrophils specifically labeled with fluorescent proteins, many advances have been made to understand neutrophil functionality in fish, including the identification of a tissue gradient of hydrogen peroxide for neutrophil recruitment following tissue injury and direct evidence for reverse migration as a mechanism of inflammation resolution (Henry et al., 2013).
2.3.3.2 Eosinophils and basophils
Eosinophils are PMN leukocytes that retain acid dyes such as eosin. In mammals, these cells are primarily involved in the destruction of internal parasites and in the modulation of allergic inflammatory reactions. Eosinophils have been described in several fish species, including cyprinids. Zebrafish eosinophils have been isolated using a transgene of Gata-2, a transcription factor required for differentiation and maintenance of murine eosinophils, in combination with their light/scatter characteristics (Balla et al., 2010). These zebrafish eosinophils degranulate in vitro in response to an extract of Heligmosomoides polygyrus or in vivo in response to either the same extracts or a live infection with Pseudocapillaria tomentosa; the latter suggests a common response to helminthic infections in all vertebrate eosinophils. In gilthead sea bream (Sparus aurata), cells with the tinctorial properties of eosinophils designated as acidophilic granulocytes show high MHC class II gene expression and phagocytic capacities, suggesting a role for these cells in antigen presentation (Cuesta et al., 2006). The basophils of vertebrates are uncommon granular leukocytes, containing characteristic large basophilic metachromatic granules. Basophils are rarely described in fish, and most of the characterizations are based on their morphology and ultrastructural characteristics (Ellis, 1977).
2.3.3.3 Mast cells/eosinophilic granule cells
Cells with structural and functional properties resembling those of mammalian mast cells (MCs) have been described in the digestive tract, gills, and other tissues of most teleosts.
These cells present a characteristic basic staining of the cytoplasmic granules with alcoholic thionin and toluidine blue, or Alcian blue. After water fixation, the granules present eosinophilic characteristics and, for this reason, these cells have been denominated “eosinophilic granule cells” (EGCs) in some studies (Reite and Evensen, 2006).
The granules contain phospholipids and acidic mucopolysaccharides and present acid and alkaline phosphatase, arylsulfatase, and 5N-nucleotidase activities. Several studies made in teleosts suggest that, as in mammals, mast cell precursors leave the hematopoietic system and enter the tissues via blood circulation in order to undergo the maturation process (Bergeron and Woodward, 1983).
The acute responses of MCs/EGCs observed in fish after intraperitoneal or intestinal administration of bacteria or after natural infection consists of a typical degranulation, followed by an inflammatory reaction and vasodilatation (Dezfuli and Giari, 2008; Reite and Evensen, 2006). The recruitment and accumulation of MCs/EGCs observed in different species after parasitic infections or dermal lesions indicates that this recruitment is a general response in persistent inflammatory reactions in teleosts.
intraperitoneal 복강내의
2.3.3.4 Rodlet cells
In teleost, the rodlet cells are secretory cells located in the endothelium of the cardiovascular system, the intestinal epithelia, and other epithelia of many fish species that release their products at epithelial, mesothelial, or endothelial surfaces.
These cells have a characteristic structure with cytoplasmic inclusions that possess a crystalline inner core. The recruitment and accumulation of rodlet cells in tissues has been correlated with the presence of different parasites, including cestodes, trematodes, encysted larvae of helminths, myxosporodian, or copepods, usually parallel to the accumulation of MCs/EGCs (Bielek, 2002; Reite and Evensen, 2006; Yokoya and Ebina, 1981).
Additionally, rodlet cells seem to respond to the presence of parasites on epithelial surfaces by degranulation, secreting substances that dampen the infections and contribute to the elimination of parasites (Leino, 1996; Manera and Dezfulli, 2004).
2.3.4 Dendritic cells
Dendritic cells (DCs) in mammals constitute the most important antigen-presenting cell with the broadest range of antigen presentation capacities. Although the presence of a well-defined DC population has not been reported in fish, some recent studies point to subpopulations of leukocytes with characteristics or functions similar to those of DCs.
Fish cells positive to human antibodies characteristic of DCs or with DC-like morphology have been reported in salmonids (Haugland et al., 2012; Lovy et al., 2006, 2009). In zebrafish, a cell population with high affinity for the lectin peanut agglutinin (PNA) and the classical morphological features of mammalian DCs has been described and is capable of phagocytizing bacteria and activating T lymphocytes in an antigen-dependent manner (Lugo-Villarino et al., 2010).
In rainbow trout, a protocol for the enrichment of head kidney and spleen cultures for DCs was adapted from mammals (Bassity and Clark, 2012). These cells were phagocytic, exhibited a DC-like morphology and were activated by TLR ligands. However, due to the fact that B lymphocytes and phagocytes can also be considered professional antigen-presenting cells capable of phagocytizing antigens and displaying them in the context of MHC class II and having evidence that, in fish, some of these mammalian DC-signature markers do not seem exclusive of DCs (Johansson et al., 2012), the real nature of these cell populations remains unclear until specific fish markers are developed for their in-depth study.
2.3.5 Thrombocytes
Thrombocytes are the equivalent of mammalian platelets and are involved in blood clotting. On the contrary to mammalian platelets, teleost thrombocytes have a nucleus and thus some additional functions have been described for them. Thrombocytes from carp (Stosik et al., 2002), rainbow trout (Hill and Rowley, 1998), and sea bass (Dicentrarchus labrax) (Meseguer et al., 1992) showed phagocytic capacity and thus a possible role in antigen presentation has been speculated for them (Kollner et al., 2004).
blood clotting 혈액응고
Furthermore, trout thrombocytes express components of the MHC class I pathway, IL-1b, TNF-a, transforming growth factor b (TGF-b), the IL receptor common g chain, as well as CXC and CC chemokines; however, their exact role in immunity has still not been clearly elucidated (Kollner et al., 2004).