Christopher J. Bayne

The acute phase response and innate immunity of fish.

Tissue trauma or invasion by pathogens or parasites induce changes in the quantities of several macromolecules in animal body fluids. These changes comprise one aspect of the acute phase response (APR), which in toto involves metabolic changes in several organ systems. One clear indication of the response is the increase in synthesis and secretion by the liver of several plasma proteins, with simultaneous decreases in others. These acute phase proteins (APP) function in a variety of defense-related activities such as limiting the dispersal of infectious agents, repair of tissue damage, inactivation of proteases, killing of microbes and other potential pathogens, and restoration of the healthy state. Some APP are directly harmful to microbes, while others modify targets thus marking them for cell responses. Some work alone while others contribute to cascades. Proteins that are APP in mammals, and that have been identified in both teleosts and elasmobranchs include C-reactive protein, serum amyloid P, and several components of the Complement system. Others reported in teleosts include transferrin and thrombin. Of these, only CRP has been reported to increase in acute phase plasma. In trout, a precerebellin-like protein is an APP with unknown functions. A cDNA library enriched in fragments of transcripts that were more abundant in livers from fish undergoing an APR recently yielded sequences resembling 12 additional known APP, and as many others either not known to be APP, or not similar to others yet in public databases. It appears that, as in mammals, hepatocytes are the prime source of APP in fish, and that pro-inflammatory cytokines induce transcription of their genes.

The immediate effects of stress on hormones and plasma lysozyme in rainbow trout.

The fight-or-flight response prepares an animal for coping with alarming situations and their potential consequences, which include injury. The possible involvement of innate components of immunity in the response has received little attention. We determined plasma concentrations of stress hormones and lysozyme activity before and after a 10 min handling stressor. Rainbow trout (Oncorhynchus mykiss) were anesthetized in their home tanks, bled, revived, and then stressed by being held in the air in a net for 30 s and placed in a shallow bucket of water for 10 min. Fish were then captured, concussed (in one of two experiments) and bled again. Control fish were also bled twice, but were kept anesthetized in their holding tanks between bleedings. Following the stressor, plasma cortisol, adrenaline and lysozyme activity were significantly increased. The experiment was repeated 4 months later with a similar outcome. While chronic stress is eventually immunosuppressive, acute stress/trauma may help enhance both cellular and humoral components of innate defenses at times of likely need.

Immune-relevant (including acute phase) genes identified in the livers of rainbow trout, Oncorhynchus mykiss, by means of suppression subtractive hybridization

To develop tools for analysis of the acute phase response, we used suppression subtractive hybridization of cDNAs from the livers of trout in an unchallenged state and in the course of a response to injection with a Vibrio bacterin emulsified in Freunds Incomplete Adjuvant. The resulting cDNA library contains 300-600bp long fragments of 25 or more immune-relevant genes. Fifteen were previously unreported for salmonids, and 12 were not known from any fish species. Known acute phase proteins include serum amyloid A, transferrin and precerebellin-like protein; trout C-polysaccharide-binding protein 1 is probably also an acute phase protein. Components of both the complement system (n=5) and the clotting system (n=3), as well as lectins, various binding proteins, a putative antibacterial peptide, a chemotaxin, an anti-oxidant enzyme, as well as some likely cell-surface receptors and metabolic and lysosomal enzymes are represented in the library. One clone closely resembles a group of Toll-like receptors, including the human IL-1 receptor. Three cDNAs appear to represent complete open reading frames.

The Physiological Ecology of Mytilus californianus Conrad. 1. Metabolism and Energy Balance

SummaryThe rates of oxygen consumption, filtration and ammonia excretion by Mytilus californianus have been related to body size and to ration. The rate of oxygen consumption (VO2) by individuals while immersed, measured on the shore, resembled rates recorded for mussels starved in the laboratory. VO2 by M. californianus was relatively independent of change in temperature, with a Q10 (13–22° C) of 1.20. In contrast, the frequency of heart beat was more completely temperature dependent [Q10 (13–22° C)=2.10]. Filtration rate showed intermediate dependence on temperature change [Q10 (13–22° C)=1.49] up to 22° C, but declined at 26° C. Both VO2 and filtration rate declined during starvation. The utilisation efficiency for oxygen was low (approx. 4%) between 13 and 22° C, but increased to 10% at 26° C. Three components of the “routine” rate of oxygen consumption are recognised and estimated; viz. a basal rate (0.136 ml O2 h-1 for a mussel of 1 g dry flesh weight), a “physiological cost” of feeding (which represented about 6% of the calories in the ingested ration), and a “mechanical cost” of feeding which was three times higher than the physiological cost. The ratio oxygen consumed to ammonia-nitrogen excreted was low, and it declined during starvation. These data are compared with previously published measurements on Mytilus edulis, and the two species of mussel are shown to be similar in some of their physiological characteristics, though possibly differing in their capacities to compensate for change in temperature. For M. californianus, the scope for growth was highest at 17–22° C and declined at 26° C; it is suggested that exposure to temperatures in excess of 22° C, as for example during low tides in the summer, might result in a cumulative stress on these populations of mussels by imposing a metabolic deficit which must be recovered at each subsequent high tide. The high “mechanical cost” of feeding imposes a more general constraint on the scope for activity of the species.

Schistosoma mansoni: cytotoxicity of hemocytes from susceptible snail hosts for sporocysts in plasma from resistant Biomphalaria glabrata.

Abstract Sporocysts of Schistosoma mansoni (PR1 strain) survive and grow in Biomphalaria glabrata PR albino strain snails, whereas they are encapsulated and die in B. glabrata 10R2 strain snails. These processes also occur in an in vitro system in which the only living cells are those of sporocysts and snail hemolymph. Hemocytes of the susceptible snail are normally not effective in damaging sporocysts. However, when the encounter occurred in the presence of cell-free plasma from resistant snails, previously impotent hemocytes severely damaged sporocysts in 24 hr. The cytotoxic capacity of resistant strain hemocytes was not altered by plasma from susceptible snails. Furthermore, it was retained even when plasma was replaced by culture medium free of snail components. The nature of the plasma factor(s) which facilitated damage by otherwise impotent hemocytes is discussed, and evidence is evaluated for the hypothesis that snail resistance is dependent upon the specificity of cytophilic factors present both in the plasma and on the hemocyte plasma membranes.

Mechanisms of molluscan host resistance and of parasite strategies for survival.

In parallel with massive research efforts in human schistosomiasis over the past 30 years, persistent efforts have been made to understand the basis for compatibility and incompatibility in molluscan schistosomiasis. Snail plasma contains molecules that are toxic to trematodes, but these seem to kill only species that never parasitize the mollusc used as the source of plasma. A sporocyst will be killed actively by haemocytes alone if they are from a snail that is resistant to the trematode. Oxygen-dependent killing mechanisms play a major role. Enzymes such as NADPH oxidase, superoxide dismutase, myeloperoxidase and nitric oxide synthase are critical components of the putative killing pathways. Metabolic intermediates such as hydrogen peroxide and nitric oxide appear to be more important against trematodes than the shorter-lived intermediates that are more important in anti-microbial defences. Products secreted by trematode larvae influence the physiology of snail haemocytes, implying active counter-defences mounted by the parasite, but these remain largely unexplored. A possible molecular basis for the susceptibility/resistance dichotomy in molluscan schistosomiasis is suggested to be deficient forms of enzymes in the respiratory burst pathway, and a selective disadvantage for schistosome resistance is an integral component of this model.

KILLING OF SCHISTOSOMA MANSONI SPOROCYSTS BY HEMOCYTES FROM RESISTANT BIOMPHALARIA GLABRATA: ROLE OF REACTIVE OXYGEN SPECIES

The fate of Schistosoma mansoni (Trematoda) sporocysts in its molluscan host Biomphalaria glabrata (Gastropoda) is determined by circulating phagocytes (hemocytes). When the parasite invades a resistant snail, it is attacked and destroyed by hemocytes, whereas in a susceptible host it remains unaffected. We used 3 inbred strains of B. glabrata: 13-16-R1 and 10-R2, which are resistant to the PR-1 strain of S. mansoni, and M-line Oregon (MO), which is susceptible to PR-1. In an in vitro killing assay using plasma-free hemocytes from these strains, the rate of parasite killing corresponded closely to the rate by which S. mansoni sporocysts are killed in vivo. Hemocytes from resistant snails killed more than 80% of S. mansoni sporocysts within 48 hr, whereas sporocyst mortality in the presence of hemocytes from susceptible snails was <10%. Using this in vitro assay, we assessed the involvement of reactive oxygen species (ROS) produced by resistant hemocytes, during killing of S. mansoni sporocysts. Inhibition of NADPH oxidase significantly reduced sporocyst killing by 13-16-R1 hemocytes, indicating that ROS play an important role in normal killing. Reduction of hydrogen peroxide (H2O2) by including catalase in the killing assay increased parasite viability. Reduction of superoxide (O2−), however, by addition of superoxide dismutase or scavenging of hydroxyl radicals (·OH) and hypochlorous acid (HOCl) by addition of hypotaurine did not alter the rate of sporocyst killing by resistant hemocytes. We conclude that H2O2 is the ROS mainly responsible for killing.

MACROPHAGELIKE HEMOCYTES OF RESISTANT BIOMPHALARIA GLABRATA ARE CYTOTOXIC FOR SPOROCYSTS OF SCHISTOSOMA MANSONI IN VITRO

When Schistosoma mansoni miracidia penetrate resistant individuals of the intermediate host snail Biomphalaria glabrata, the sporocyst is encapsulated by hemocytes (macrophagelike cells of the snail circulation) and killed. In our in vitro model the same fate requires only sporocysts and snail hemolymph. However, when cultured in plasma alone (cell-free hemolymph), sporocysts remain viable for more than 3 days, regardless of whether the plasma is from susceptible or resistant snails. When hemolymph is used from susceptible snails, the sporocysts retain a normal healthy appearance. Furthermore, the parasite appears to express an offensive response to the hemocytes of susceptible snails. Ultrastructural study reveals that resistant-strain hemocytes destroy the parasite tegument; within 24 hr the sporocyst is damaged severely throughout. This cell-mediated, internal, defensive response of an invertebrate host closely resembles antibody-dependent, cell-mediated damage to schistosomula in mammalian hosts.

Modulation of the oxidative burst in trout myeloid cells by adrenocorticotropic hormone and catecholamines: mechanisms of action.

The oxidative burst of rainbow trout (Oncorhynchus mykiss) phagocytes was previously found to be differentially modulated by adrenocorticotropic hormone (ACTH) and the catecholamine receptor agonists phenylephrine and isoproterenol. From data obtained using both luminol‐enhanced chemiluminescence (LECL) and ferricytochrome C (cyt C) reduction to measure oxidative burst kinetics, we postulated that the observed modulation was mediated by affects on enzymes responsible for the production and metabolism of superoxide anion. Using exogenous superoxide dismutase (SOD) and catalase as scavengers, nitroprusside to poison endogenous SOD, and an assay for hydrogen peroxide, we have tested our postulates by exploiting the differences with which various reactive oxygen intermediates influence LECL and cyt C reduction. The ability of ACTH to potentiate both assays of the oxidative burst appears due to its enhancing influence on the production of superoxide. Phenylephrine, an α‐adrenergic receptor agonist, appears to enhance the activity of endogenous SOD, whereas isoproterenol, a β‐adrenergic receptor agonist, may suppress SOD activity. This work reveals how components of the natural immune system may be regulated by products of the neuroendocrine system. Also, lymphocyte‐derived ACTH may provide a novel pathway for lymphoid regulation of inflammation.

Ultrastructure of encapsulation of Schistosoma mansoni mother sporocysts by hemocytes of juveniles of the 10-R2 strain of Biomphalaria glabrata.

Dynamic aspects of the cellular responses of juvenile (2-3 mm shell diameter) 10-R2 strain Biomphalaria glabrata to newly penetrated Schistosoma mansoni (NIH-Sm-PR-1 strain) were studied at the ultrastructural level. As early as 3 hr postexposure (PE), host hemocytes had contacted the parasites surface and by 7.5 hr, had phagocytosed sporocyst microvilli and small pieces of underlying tegument. Most sporocysts observed at 24 hr PE lacked tegumental cytoplasm, and germinal cells and other internal structures showed extensive pathological changes. By 48 hr, capsules were filled with hemocytes containing numerous, large phagosomes, and only scattered remnants of sporocyst material remained. Capsules were difficult to find at 4 days PE, suggesting that hemocytes participating in the encapsulation response had dispersed. Hemocytes responsible for the rapid and consistent destruction of S. mansoni sporocysts in the head-foot region typically formed extensive pseudopodia and contained large numbers of lysosomelike bodies, characteristics associated with granulocytes of B. glabrata. No evidence was found to suggest that hemocytes were lysed, or formed multinuclear syncytia, during the encapsulation response.