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Immune mechanisms in fish skin against monogeneans - a model

Kurt Buchmann

Department of Veterinary Microbiology, Section of Fish Diseases, Royal Veterinary and Agricultural University, 13 Bülowsvej,DK-1870 Frederiksberg C, Denmark

Key words: Monogenea, fish immunity, skin immune system, cytokines, complement, humoral factors, cellularresponses

Abstract. Host responses against skin inhabiting monogeneans are commonly observed but the responsible immune mechanismsin the fish skin are insufficiently described. Based on recent knowledge of fish immunity and skin response mechanisms inmammals a model for the skin immunity in fish to monogenean infections is proposed. Important cellular components of themodel are the epithelial cells, the mucous cells and leucocytes. The release of cytokines, e.g. IL-1, following mechanical orchemical injury of the epithelial cells, initiates a series of events leading to decrease of the ectoparasite population. Cytokines(e.g. IL-1, TNF, INF) are suggested to affect secretions from mucous cell and attract neutrophils and macrophages. Leukotrienesare probably involved in the inflammatory reactions. The subsequent production of humoral substances (among otherscomplement factors and peptides) could be responsible for the antiparasitic response in the later stages of infection. Althoughnon-specific factors dominate the response, the involvement of specific antibodies and lymphocytes cannot be excluded.

It has been demonstrated through a number ofinvestigations that teleosts are capable of mountingprotective host responses against infections withectoparasitic monogeneans (Vladimirov 1971, Scott1985, Buchmann and Bresciani 1998). Already sevendecades ago different marine fish species in the NewYork Aquarium exhibited varying responses toinfections with Benedenia (Epibdella) melleni(MacCallum) (Jahn and Kuhn 1932, Nigrelli and Breder1934, Nigrelli 1937). Later, carp (Cyprinus carpio) wasshown to respond effectively against the gill parasiticDactylogyrus vastator Nybelin (Paperna 1964,Vladimirov 1971). Likewise, the European eel Anguillaanguilla was seen to develop partial immunity to thegill monogeneans Pseudodactylogyrus anguillae (Yin etSproston) and P. bini (Kikuchi) (Buchmann 1988,Slotved and Buchmann 1993). The viviparousgyrodactylids are no exception to this rule as a range ofexperiments have documented that sticklebacks (Lesterand Adams 1974), guppies (Scott 1985, Richards andChubb 1996) and salmonids (Cusack 1986, Cone andCusack 1988, Bakke et al. 1991, Bakke and MacKenzie1993, Malmberg 1993, Buchmann 1997, Buchmann andUldal 1997, Buchmann and Bresciani 1998) activaterepelling mechanisms against these monogeneans in thelater stages of infection. Despite this evident response tomonogenean ectoparasites the involved mechanisms arestill insufficiently elucidated. However, recent progressin fish immunology and studies on infections withGyrodactylus derjavini Mikailov on rainbow trout skinhave pointed to both cellular and humoral factors as themain actors in the play. The present work treats theexisting knowledge on interactions between fish and

monogenean, draws parallels to ectoparasitic infectionsof mammals and finally presents a hypothetical modelfor the intricate immunity mechanisms in teleost skin.

THE RAINBOW TROUT EPIDERMIS

The rainbow trout skin is composed of the outerepidermis beneath which the dermis (mainly consistingof connective tissue) is located (Hibiya 1982, Yasutakeand Wales 1983). Various cell types are found in theepidermis. The outermost layer is composed of flattenedepithelial cells tightly bound to each other. Their outercell membranes are folded into fine microridgesproducing fingerprint like patterns on the cell surface(Buchmann and Bresciani 1998). Beneath the epithelialcells mucous (goblet) cells are distributed. They areopening into small pores between the outer epidermalcells. Mucous or goblet cells are numerous, exhibitingcell densities of several hundreds per square mm(Buchmann and Bresciani 1998). Due to their content ofacid mucopolysaccharides they will stain intensely bluewith Alcian blue and the carbohydrate moieties alsoreact with lectins (Fig. 1). The content of the gobletcells are released onto the surface of the fish. Deeper inthe tissue are found chromatophores as melanophoresand xanthophores. Chromatophores have not beenconnected to any function in skin immunity but theyoccur abundantly on the corpus of rainbow trout andless frequently on the fins. In the process ofinflammation different leucocytes as macrophages,neutrophils, eosinophils and other granulocytes arefound distributed between the deeper cells in theepidermis in carp (Cross and Matthews 1993). In

Address for correspondence: K. Buchmann, Department of Veterinary Microbiology, Section of Fish Diseases, Royal Veterinary and AgriculturalUniversity, 13 Bülowsvej, DK-1870 Frederiksberg C, Denmark. Phone: ++45 35282700; Fax: ++45 35282711; E-mail:kurt.buchmann@vetmi.kvl.dk

FOLIA PARASITOLOGICA 46: 1-9, 1999

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rainbow trout neutrophils and macrophages occurabundantly in inflammatory reactions of the superficialtissue layers (Finn and Nielsen 1971). The mast cell(Reite 1998) is also an important epidermal cell.

Fig. 1. Mucous cells in pectoral fin from rainbow trout. Scalebar = 60 µm. Rainbow trout fins were fixed in 4% neutralformaldehyde for 1 month. Following rinsing in distilledwater mucous cells were visualized by detecting galactosederivatives in the mucopolysaccharides by incubating the fins(blocked for 1 h with 2.5% bovine serum albumin inphosphate buffered saline [PBS]) with biotinylatedconcanavalin A (Sigma C-2272) 10 µg/ ml for 1 h, rinsing inPBS with 0.05% Tween 20 3×10 min, incubating with avidineand biotinylated alkaline phosphatase (DAKO K376).Following a final wash the lectin binding was detected as redcolouring by incubation in Fast Red and Naphthol phosphatein 0.1M Tris-HCl (DAKO K699). After staining specimenswere rinsed in distilled water and mounted in Aquamount.

EPIDERMAL CELLS AND THEIR ROLE INIMMUNITY

Epithelial cellsThe epithelial cells are mechanically injured by the

marginal hooklets of Gyrodactylus derjavini. The largehamuli do not normally penetrate the epithelium butnumerous minute holes are produced by insertion of themarginals (Figs. 2-4). In addition, depressions in theepithelium, presumably caused by the opisthaptor, arefound where these gyrodactylids are fouraging (Fig. 3).Secretions from the parasite are likely to affect theepithelium. A recent study (Buchmann 1998b) showedstrong activities of esterases, aminopeptidases andphosphatases (alkaline and acid) in the intestine of G.derjavini. As monogeneans regurgitate intestinalcontents (Smyth and Halton 1983) these enzymes areprobably released onto the epithelium where theycontribute to injuring or sensitization of the cell layers.Thus, SEM-recordings of G. derjavini show releaseonto the epithelium of material from the mouth region(Fig. 4). A corresponding release of unidentified

substances from Gyrodactylus salaris Malmbergparasitizing the Atlantic salmon was noted by Mo(1994). It is known that keratinocytes from human skinproduce IL-1 in response to infections with Sarcoptesscabiei L. (Arlian et al. 1996). Carp epidermal cells areable to produce an IL-1 like factor (Sigel et al. 1986)and interestingly, it was demonstrated that IL-1 wasproduced in the epidermis of rainbow trout followinginfection with G. derjavini (Buchmann and Bresciani1998). This cytokine has a number of effects on othercells in the epidermis. In fish, IL-1 induce hyperplasia(Balm et al. 1995), macrophages are activated by IL-1(Titus et al. 1991), the response of fish lymphocytes ispotentiated by IL-1 (Hamby et al. 1986, Sigel et al.1986), and it is known that IL-1 induce mucus secretionfrom human goblet cells (Cohan et al. 1991). Thepresent knowledge therefore allows the suggestion thatIL-1 liberated from epithelial cells upon monogeneanattachment elicit a number of reactions in the fish skin.The observed hyperplasia in rainbow trout skin(Buchmann and Bresciani 1998) is probably due to IL-1, the mucins in the goblet cells are released (due to IL-1 activation) onto the skin where the monogenean isentangled in the material (Buchmann 1998b),leucocytes are attracted to the inflammatory site wheremacrophages, granulocytes and lymphocytes will exerttheir effects. It is known from mammalian hosts thatleukotrienes on some occasion will be produced notonly by leucocytes but also by keratinocytes (Thomsen1991). As leukotriene LTB4 acts as a potentchemoattractant for fish leucocytes (Hunt and Rowley1986) it is not excluded that this factor is involved inthe inflammatory reaction in the fish skin followingmonogenean infection. Other cytokines (TNF, IFN) areproduced by human keratinocytes (Bos and Kapsenberg1993) but a corresponding release in fish epithelia isstill to be investigated.Mucous cells

The presence of a dense layer of mucous cellsbeneath the epithelial cells in the rainbow trout hasprompted suggestions that this cell type could beinvolved in fish immunity to ectoparasitic infections(Pickering 1974, Wells and Cone 1990, Buchmann andBresciani 1998). This hypothesis is related to the factthat mucus and goblet cells from mammalian mucosalsurfaces are known to participate in the host responseagainst intestinal nematodes (Castro and Harari 1982,Miller 1987, Rabel et al. 1994, Ishikawa et al. 1994).The presence in mucus scrapings from fish of severalbiologically active substances as immunoglobulin (St.Louis-Cormier et al. 1984, Rombout et al. 1993,Buchmann and Bresciani 1998), complement factors(Vladimirov 1971, Harris et al. 1997, Buchmann andBresciani 1998), peptides (Buchmann and Bresciani1998), lysozyme and proteases (Fletcher 1982,

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Figs. 2-4. Scanning electron micrographs of Gyrodactylus derjavini. Fig. 2. Marginal hooklets penetrating epithelial cells on arainbow trout fin (pectoral fin). Fig. 3. Depression in the epithelium of a rainbow trout tail fin parasitized by G. derjavini. Fig. 4.G. derjavini on a rainbow trout tail fin. Note the release of materials from the mouth region.. Scanning electron microscopy wasperformed on specimens of G. derjavini parasitizing fins of rainbow trout by fixing in 2.5% cacodylate buffered glutaraldehydefor 24 h, post-fixing in osmium tetroxide, whereafter specimens were critical point dried, sputtered with gold and studied in aJeol scanning electron microscope. Scale bars: Fig. 2 = 10 µm; Figs. 3, 4 = 100 µm.

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Yano 1996), varying carbohydrates (Buchmann andBresciani 1998) and lectins (Yano 1996) add further tothe suggestion that mucus affects survival of invadingmonogeneans. The release of the goblet cell contentsdue to IL-1 production by injured epithelial cells couldconnect these two cell types with monogenean invasion.

Cells in the inflammatory reactionsFollowing injuries or parasitic invasion of the fish

epidermis inflammatory reactions occur with infiltrationof various cell types including neutrophils andmacrophages in rainbow trout (Finn and Nielsen 1971)and, in addition, eosinophils and basophils in carp(Cross and Matthews 1993). This corresponds well tomammalian reactions to Sarcoptes scabiei infections(Arlian et al. 1994b). Activation of leucocytes elicit theproduction of leukotrienes as LTB4, a substance knownto induce migration in fish neutrophils and eosinophils(Hunt and Rowley 1986). Both macrophages andneutrophils from fish are known to secrete IL-1(Verburg-van Kemenade et al. 1995) which again willaffect other macrophages and fish lymphocytes (Hambyet al. 1986, Secombes and Fletcher 1992). TNF and INFhave been shown to regulate mucus production ingoblet cells from the human colon (Jarry et al. 1992).As TNF-like molecules are present in rainbow trout(Ahne 1993) and related molecules are produced bycells in the inflammatory reaction (Alexander andIngram 1992, Verburg-van Kemenade et al. 1996) it ispossible that they participate in the increased mucusproduction already involving IL-1. These cytokines allaffect the different leucocytes which probably haveimportant functions in the reactions to monogeneans.Thus, Hardie et al. (1994) found that TNF in interactionwith macrophage activating factor (MAF) positivelyaffects rainbow trout macrophage respiratory burstactivity. Such events are highly antiparasitic in vitro(Whyte et al. 1989) and it cannot be excluded that thereactive oxygen metabolites released in these processeswill affect the monogeneans. The demonstration of adelayed type hypersensitivity in fish against theprotozoan parasites Cryptobia salmositica (Thomas andWoo 1990) and Ichthyophthirius multifiliis (Sin et al.1996) suggests that a similar response could beactivated in rainbow trout against monogeneans. Such areaction would involve release of macrophage migrationinhibition factor (MIF) from sensitized lymphocytes atthe site of infection. Thereby the reactive cells would beconcentrated near the invading pathogen. However,such a reaction in rainbow trout against monogeneanshas not yet been demonstrated.

SPECIFIC ANTIBODIES

In mammalians infected with Sarcoptes scabieiserum antibody titers increase significantly after infec-

tion although this factor is considered of minor impor-tance (Arlian et al. 1994a). Production in fish of specificserum immunoglobulin against gill monogeneans hasbeen seen (Vladimirov 1971, Buchmann 1993).However, gill tissue is far more fragile and blood filledthan fish skin whereby gill parasite antigens are morelikely to come in direct contact with host blood. Inaddition, recent immunocytochemical experiments withwhole worms have shown that rainbow troutimmunoglobulins from infected fish do not bind to thetegument of G. derjavini (Buchmann 1998a) cor-responding to the lack of antibody binding of sciaenidfishes to monogenean antigens (Thoney and Burreson1988). Some studies (e.g. Scott 1985) found that theacquired protection of fish against gyrodactylids waslimited to few weeks. This supports that the protectionmainly is based on non-specific reactions. However,this does not exclude that antibodies could bind tomolecules in the parasite intestine or other internalstructures. In addition, it is known from studies on aparasitic ciliate (Ichthyophthirius multifiliis) infectingthe epidermis of a range of freshwater fishes that serumantibody titres are raised following infection (Clark etal. 1988, Clark and Dickerson 1997). In that system thespecific humoral response is considered protective andshows that specific antibodies indeed are produced inresponse to parasites invading the epidermis.

COMPLEMENT AND NON-SPECIFICHUMORAL FACTORS

Recently complement from rainbow trout was seen tobind to and kill G. derjavini (Buchmann 1998a), whichcorresponds to the lethal effect of salmon complementto G. salaris (Harris et al. 1997). As complement factorsare produced not only by host liver cells but also bymacrophages (Lappin and Whaley 1993, Dalmo et al.1997) this system is probably an important factor in theresponse to ectoparasites. The glycocalyx of G. der-javini is composed of various carbohydrates consistingof galactose derivatives, lactose derivatives and promi-nent mannose derivatives in the cephalic duct openings.They are easily binding lectins and activate thealternative complement pathway (Buchmann 1998a).This corresponds partly to the response of rainbow troutto Cryptobia salmositica. In that system both theclassical antibody dependent and the alternativepathway are involved in the protective immunity (Woo1996). The presence of carbohydrates in the glycocalyxof monogeneans (Smyth and Halton 1983, Buchmann1998a) leads to the suggestion that lectins in fish mucuscould contribute to the host response againstmonogeneans. Fish mucus is a rich source of lectins(Yano 1996). Their function is elusive (Arason 1996)but could take part in the non-specific defence againstexternal pathogens. The observed binding of mucus to

Buchmann: Fish skin immunity and monogeneans

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Fig. 5. Schematic illustration of the hypothetical network of cellular and humoral interactions involved in the response of the fishepithelium against infections with monogeneans. Abbreviations: Mo – monogenean parasite, E – epithelial cell, M – mucouscell, Leu – leucocyte, T – T-lymphocyte, B – B-lymphocyte.

the parasite surface (Buchmann 1998b) could be due tolectins. No immediate injury of the parasite tegument iscaused by mucus binding. However, incubation of G.derjavini in mucus scrapings from rainbow trout islethal within few hours but that effect could be causedby the complement content.

A number of other non-specific molecules in fishepidermis could play a role in the response to mono-geneans. Lysozyme is present in high amount in fishepidermis (Fletcher 1982, Lie et al. 1989, Alexanderand Ingram 1992) but does not seem to correlate withresistance to monogenean infection (Buchmann andBresciani 1998). Proteases are present in fish epithelia(Hjelmeland et al. 1983, Braun et al. 1990) but theireffects on monogeneans are unknown.

Peptides as somatostatin and ACTH have beendetected in mucus scrapings. Their possible antiparasiticrole remains undetermined but as monogeneans areknown to posses peptidergic innervations of theirnervous systems (Reuter 1987, Maule et al. 1990,Halton et al. 1993, Cable et al. 1996) an effect on

parasite behaviour is not to be excluded. It is knownfrom amphibians that skin secretions contain a vastamount of bioactive peptides and defensins (Lazarusand Attila 1993) and it is possible that monogeneans areaffected by secretions of fish skin. Thus, cestodesrespond actively to somatostatin (Sukhdeo et al. 1984).

Besides being of general importance these non-specific reactions are suggested to play a role in thevarying susceptibility of different salmonids togyrodactylids which has been documented during thelast years (Bakke et al. 1990, Bakke and MacKenzie1993, Buchmann and Uldal 1997).

LYMPHOCYTES

Generally infiltration with B-lymphocytes is weak inthe inflammatory reactions to ectoparasites in mam-malian skin. However T-lymphocytes are importantfactors involved in the reaction to these infections(Arlian et al. 1997) and do generally play an importantrole in skin immunity (Bos and Kapsenberg 1993). In

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fish B-cells are recognized as immunoglobulin positivelymphocytes. Due to the lack of T-cell markers fish T-cells are often referred to as immunoglobulin-negativelymphocytes. However, recently molecular evidencewas presented for the existence of a T-cell receptor inrainbow trout (Partula et al. 1995) and the occurrence infish of B- and T-cell equivalents are now accepted. Therole of lymphocytes in the response of fishes tomonogenean invasion is still unknown. It is acceptedthat Langerhans cells (LC) of the mammalian epidermistake up antigen from the external pertubator and migratethrough the dermal lymphatics to the draining lymphnodes. In this region the antigen/MHC complex on theLC surface is presented to T-cells (Bos and Kapsenberg1993, Stingl et al. 1993). Future work should establishif similar events occur in fish skin. It was suggested byGraves et al (1985) that non-specific cytotoxic cells areresponsible for the protective response of fish to skin-invading ciliates. Whether a corresponding cell type haseffects on monogeneans should be elucidated.

CONCLUSIONS

The demonstration of strong anti-monogeneanfactors in the activated fish skin has prompted ahypothetical model for the skin immune system in fish(Fig. 5). This is based on the present knowledge of theanatomy of the fish skin, the findings of inflammatorycells in the skin, the demonstration of cytokines,complement, immuno-globulin and peptides in the skinand mucus. Research in mammalian systems hasestablished links between several factors in the skin and

some parallels seem to exist in fish. The activation ofthe fish skin epidermis by monogeneans (mechanicallyand/or chemically) causes production of cytokines (IL-1and possibly TNF and INF ) from the epithelial cells(and leucocytes). The IL-1 (and TNF and INF) affectsthe mucous cells and causes mucus secretion. Theleucocytes (and possibly epithelial cells) subsequentlyproduce leukotrienes, IL-1, INF and TNF with a rangeof effects on granulocytes and macrophages. Cytokinesdirect the leucocytes to the site of inflammation andnoxious substances including reactive oxygenmetabolites are released by the leucocytes affecting themonogenean. The secreted mucus and cellularliberations include complement factors reacting with theglycocalyx of the monogenean. In addition, releasedpeptides could affect worm behaviour. Antibodies mayor may not bind to the worm but it is entangled inmucus containing complement, lectins and C-reactiveproteins. Involvement of specific B and T cells ispossible but even without this specificity the non-specific responses in the epidermis would create amicroenvironment hostile to the parasites. This wouldlead to either a killing of the monogeneans or an escapeto less hostile surfaces (uninfected naive fish, fish tankbottom or less reactive sites on the host).

Acknowledgements. This study was supported by the DanishAgricultural and Veterinary Research Council, the DanishMinistry of Food, Agriculture and Fisheries and the Elisabethand Knud Petersen Foundation. The author is indebted to Dr.José Bresciani, Section of Zoology, Royal Veterinary andAgricultural University, for assistance with the scanningelectron microscopic recordings.

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Received 23 December 1997 Accepted 28 July 1998

May 3 – 5, 1999

Course:Evolution of Parasites and of Host-parasite Relationships

(Évolution des parasites et des relations hôtes-parasites)Paris, France

Contact:Dr. Marie-Claude Durette-Desset or Prof. Jean-Lou Justine

Laboratoire de Biologie Parasitaire, Helminthologie, ProtistologieMuséum National d´Histoire Naturelle,

61 rue Buffon,75231 Paris cedex 05, France

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e-mail: mcdd@mnhn.fr or justine@mnhn.frProgramme: http://www.mnhn.fr/ens/ed/07-justi.htm