The Invertebrate Immune System By Eric Borneman and Jonathan Lowrie Aquarium Net April 98
The Immune Response of Corals
Part One: The Invertebrate Immune System
By Eric Borneman and Jonathan Lowrie
To say that invertebrate immunology is in its infancy would be an understatement. To say that almost nothing is known about coral-specific immunity would be an overstatement. In other words, the hypothesis we will present in our next article in the series is being bred in the dark ages of our knowledge. It has been widely recognized, if not only in an anecdotal fashion, that stress has a profound impact on the cnidaria. This concept is certainly not surprising, since the stress response has been shown to play such a role in many organisms, including humans. We also surveyed a host of coral diseases. We are attempting to familiarize the reader with currently proposed models and established data regarding the occurrence and etiology of coral diseases, so that certain points may be illustrated in a soon to be proposed model for rapid tissue necrosis (RTN). We also hope that the study of the cnidarian immune response will be carefully considered in future studies of coral disease with single or multi-causative factors yet to be determined.
Such observations led us to re-examine the current thoughts prevalent among scientists and hobbyists alike regarding certain coral diseases. Jonathan Lowrie’s early work and understanding of invertebrate immunology, coupled with our pragmatic theorizing, some preliminary research trials and some intensive research, have led us to formulate some new ideas toward understanding the cnidarian stress response. While any forthright conclusions, treatment protocols, and indeed comprehensive understanding at a gross and subcellular level may be a long way off, we believe that this paper represents a new direction for all of us to turn our eyes and minds; in the sincere hope of elucidating the etiology of some of the diseases in these most perplexing and yet simple coral animals.
The most basic defintion of invertebrate immunity is stated by Cooper (1996):
"The world is replete with foreign material, antigens of various kinds, some of which threaten the life of invertebrates after they have been exposed to them...The undeniable starting point lies at the basis for the immune response, at least for its initiation. Inherent in this absolute requirement is the need for recognition and the assurance that foreign material, antigens will bind to an appropriate receptor on a potential effector cell or that there is the equivalent of an antigen processing cell that will capture foreign antigens, rendering them palatable to an effector cell. This would serve to set the immune response in motion. At this point we can give some consideration to different forms of immunodefense responses."
Delving further into the depths of specific invertebrate immune systems, we see that the invertebrate immune response consists of three major systems that are fairly universal to all vertebrate and invertebrate life in some form or other. The earliest discovery of immune function in invertebrates was studied by Durham (1888), involving phagocytosis in the starfish (Leclerc 1996). Although less sophisticated than the immune systems of vertebrates, invertebrate immunity is much older in terms of its evolution, likely served as the progenitor to sophisticated vertebrate systems, and also has apparently served these simple animals well enough to keep their lineage alive for the past several hundred million years. The first system is the proPO system, which is measured by phenyloxidase activity, and is involved with host defense and adaptive immunity (Cooper 1996, Johannsen 1996). A similar system utilizing lipopolysccharides (LPS) and glucan-binding proteins may be the analogous sytem in these more primitive cnidarian systems (Smith 1996). Almost all animals, including some fungi and bacteria contain melanin, which is not only the end product of phenyloxidase activity, but also has some cytotoxic abilities (Smith 1996). The second system is the lytic system, which is involved with the cytotoxic response and the lysis of potential invaders. The lytic system is well documented in the cnidaria. The third system is the Immunoglobulin (Ig) superfamily, which is involved with self recogntion and immunity. Coincidentally, the immunoglobulin system is thought to have first evolved in the the nervous system of the Cnidaria(Cooper 1996, Kurosawa, Hashimoto 1996). True immunoglobulins have not been found in cnidarians, although other surface recognition molcules, perhaps limited in their recognition abilities, do exist in these primitive systems (ibid.). Although many aspects of these invertebrate systems have not been studied in corals directly, their homologous nature across phyla and species makes their basic tenets almost assured. Furthermore, animals with extremely long life spans must have some very effective immune systems, even if not complex, in order to assure longevity that is so common to coelenterates like sea anemones and corals.
Since most invertebrates do not have blood vessels, their immunodefensive cells are composed largely of wandering cells that exist extracellularly. Fixed tissue amebocytes are also present in the endothelial cells of corals, which can proliferate and differentiate according to need. Such cells, including those defensive ones involved with phagocytosis and the secretion of antibacterial substances, as well as those involved in immunity, are termed immunocytes (Sawada, Tomonaga 1996). They are not common in the coelenterates, of which corals are a member. However, mesogleal cells and interstitial cells that display glycolytic and other functions have been identified (ibid.). "Scyphozoan and anthozoan mesoglea, while of similar matrix composition, are populated by cells variously called amebocytes, mesogleal cells, or granulocytes." Other cell types may also be capable of ameboid movement (and) are found in the ectoderm and endoderm as well as in the mesoglea and this cell type may sometimes lose and regenerate its granules." (Bigger, Hildemann 1982). Morse (1977) reported a parasitic algae on Gorgonia ventalina (also in Psuedoplexaura flagellosa and Plexaura homomalla, gorgonians with well established antibiotic traits) where "abnormally high numbers of 'mesogleal amoebocytes' and 'abnormal and extensive cellular production'" were found to exist. Amebocytes have been associated with the foreign body responses in jellyfish (Metchnikoff, 1892) and in wound repair. Patterson and Landolt (1979) discovered amebocytes with secondary lysosomes that immediately emigrated to wound repair sites in the anemone A. elegantissima. These cells engulfed necrotic tissue and were responsible for "eliminating it by injection into the surrounding water." The wound response is rapid and complete, with wound cells proliferating and migrating from the mesoglea to digest damaged tissue (Sparks, 1972; Young, 1974). Though not conclusively determined to exist, phagocytes and granular cells are almost universal to invertebrates and vertebrates alike, and are probably present in corals if not in an analogous form. In latest observations, a high level of amobeocycte response was observed to Artemia cysts that become phagocytized by Aurelia medusa during feeding (Lowrie 1998). The inflammation response to a normal food source was observable as the cysts were engulfed and expelled. Furthermore, the cnidocytes, of undifferentiated mesenchymal origin, resemble discharging granulocytes and display a defensive function. Finally, all animals (even single celled ones) have the ability to recognize self from non-self (Tuckova, Bilej 1996, et. al). What may have consequence in coral disease is the markedly reduced ability of invertebrates, especially in coelenterates, to determine between types of non-self.
Lysis is the ability of an organism to destroy foreign or damaged cells. Most invertebrates contain the ability to lyse human blood cells (hemolysis), a relatively standard method for assaying immunodefensive action. Some, such as sea stars and feather worms, do not (Roch 1996). However, the simple supplementation of erythrocytes with calcium induces an antigen response that results in hemolysis. In terms of hemolysis, it is of interest that activity in most invertebrates depends on the presence of molecules of divalent charge, such as calcium and magnesium (ibid.). This would certainly be a likely mechanism for antigen recognition in corals, given the abundant availability of these ions in seawater! Anthopleura elegantissima contains an enzyme capable of lysing the bacterium Micrococcus lysodeiktus, evidence of specifically targeted immunity (Phillips, 1963). Mollusks can even be induced into a lytic response by changes in ionic concentrations, pH, and temperature (ibid). One question that remains is how then do the invertebrates protect themselves from autolysis? This is a very intriguing question in terms of RTN, and will be covered in our next article. Toxins and venoms are also capable of lysis, and the cnidocytes of the cnidarians, previously mentioned in terms of immune function, certainly have this capability in both released substances and in their cnidocytes (ibid.). Celomic fluid and lytic fluids (including the common invertebrate enzyme, lysozyme) in sea stars and other invertebrates have a pronounced bacteriocidal ability that has been shown to kill the pathogen, Vibrio tubiashii (ibid.). There is also the hydrolytic ability of enzymes like peroxidases, lysozymes, toxic factors, estereases, etc. associated with the phagocytic activity of the coelenterates whose role is to "protect the integrity of adjacent colonies" (Leclerc 1996) Some of these enzymes (peroxidase, superoxide dismutase) are already known to exist in corals from previous works discussing oxygen toxicity (Lesser, et. al 1989, 1995).
a2-macroglobulin is an ancient immune system molecule that first occurred in invertebrates some 250 million years before the evolution of the first modern scleractinians in the Pre-Cambrian period (Armstrong 1996). It is almost universal in its occurrence in all vertebrates and invertebrates. Its function is to bind to proteases that can be released either by the host or by invading organisms (ibid.). These enzymes can be very destructive to any surrounding tissue, and are the cause of many degenerative problems from invertebrates to vertebrates. Without functional a2-macroglobulin, or an analogous molecule, coral tissues could be quickly digested through the actions of self-released or antigen-released proteases. This may also have important implications in understanding RTN.
A group of molecules, called inflammatory cytokines, are an important constituent in mediating a cascade of immune functions in the inflammatory reaction. Inflammation is, "the tissue response to a nonlethal injury caused by a chemical, physical, or biological agent" (Habicht, Beck 1996) that results in a rapid (from minutes to hours) change in the host that may include swelling, acute-phase protein synthesis, and an antibody-type response. A small molecule responsible for inflammatory type reactions in starfish was implicated in a specific reaction series of immune hypersensitivity (Bang and Chaet, 1959). In invertebrates, the process includes antigen or damaged-tissue recognition, followed by phagocytes and phagocyte-type cells and molecules appearing at the area to clear it of the source of invasion or damage. Tissue repair is then begun. Cytokines are in charge of all such events, and therefore also mediate phagocytosis (Habicht and Beck 1996). Since phagocytes are present in all vertebrates and invertebrates, their importance in corals consists not only of the transportation and digestion of particulate matter and oxygen transport, but also in the immune response. Phagocytosis is already the primary host defense system in all invertebrates, from single celled animals to highly evolved ones. The main energy source for phagocytosis comes from anaerobic glycolysis (ibid.). Areas of low oxygen tension, such as necrotic tissue and water with a low dissolved oxygen content, would favor increased phagocytosis. This observation may also have important implications in RTN.
One of the most apparent first lines of defense in corals (and many invertebrates) is a physical and chemical barrier. Lacking a hard exoskelton, corals depend primarily on their anti-microbial mucus. First, it is well established now that coral mucus is capable of hosting large numbers of bacteria of varied composition (Ducklow, Mitchell 1979, Sorokin 1973, et. al.). Some (like V. vulnificus, V. alginolyticus and others that are not coincidentally found in marine sediments), are "capable of breaking down not only the mucus, but the intracellular cementing material of the animal as well." (Phillips, 1963). Certainly these bacteria would be capable of producing signs of RTN through endotoxin and protease release. However, marine invertebrates have evolved mechanisms to cope with such bacteria. First, it has been found that 43% of marine mucosal-inhabiting bacteria were unable to grow in the acidic environment of mucus with its pH of 5.9 (Phillips 1963, Bigger and Hildemann 1982). Lowrie found the mucus of healthy Acropora to have a mean value of around 6.5, a value he also correlated with a low colonization rate of Vibrio on nutrient media (Lowrie 1992, 1997). The remaining 57% of mucusal bacteria either consumed or are subjected to an enzyme, like a lysozyme, that can lyse bacteria. This enzyme is common from vertebrate mucus to sea anemone mucus (Phillips 1963), and is therefore likely to be present in coral mucus as well. Gram negative endotoxin reaction also causes the discharge of amebocyte granules to protect against gram negative bacterial invasion. (Bang, 1973, et. al.). Mucus, therefore, not only serves a passive protective role, but is active in the removal, lysis, and consumption of bacterial invaders.
Lectins are molecules that are found in all known taxa, from viruses to vertebrates. They are responsible for surface recognition, and they aid in the attachment of microorganisms and other type antigens to phagocytes and other host defense cells (Olafsen 1996). They act like opsonins (cleverly put into an analogy by an old professor as the molecular "salt" on the surface antigens of tasty bacteria) in phagocytic recognition. Bacteria already have been shown to be a significant part of the diet of corals (Ducklow and Mitchell 1979, Paul et. al 1986, Linley 1986, Schiller et.al. 1989, Sorokin 1973). They colonize readily in corals' mucus and in their coelenteric cavities. In an environment where such prolific microorganisms are found, some invertebrates may even harbor nonindigenous pathogens. In new environments (such as aquaria, environmental changes, or displaced coral colonies), more pathogens, and thus antigens, may be encountered, and the invertebrate must develop ore possess the means to cope with them. Lauckner (Olafsen, 1996) states that "disease may result from massive increase in numbers of otherwise harmless bacteria that proliferate in a compromised invertebrate host or due to changes in environmental factors such as increased temperatures." Among the most common bacteria present are Aeromonas, Psuedomonas, and Vibrio. Of these bacteria, Vibrio were the ones found to increase dramatically under elevated temperature in invertebrate studies. For example, sea urchins could manage normal populations of V. anguillarum unless exposed to increased temperatures or stress (ibid.). Lectins are normally able to agglutinize bacteria, and many invertebrate lectins just happen to show a preference for Vibrios (ibid.)!
Other molecules which may serve analogous functions to antibody recognition have been identified in the cnidarians (Kurosawa, Hashimoto 1996). These molecules serve in surface recognition and protein interaction functions. Opsonizing agents, perhaps akin to antibodies, have been found in sea anemone tissue. When exposed to an antigen in low levels, the anemone antibody-like substance reacts in an immune type response. Perhaps more notable is that in high concentration to an antigen, the effect is inhibitory (Phillips, 1963). There is also data to suggest that this immunity is passively transferrable to adjacent anemones (Bigger and Hildemann 1982) amd that passive transfer of immune memory exists in corals (ibid., Bigger 1988). This is an important observation, in that it may be theorized that healthy corals could transfer a degree of immunity to each other for antigens already present in the confines of a closed system. It is also proof of a memory induced response, a key feature of true immune function. While these findings have not been studied in corals, much less RTN-affected corals, it is not unreasonable to assume that a similar mechanism exists. Thus, if corals were exposed to an unreasonably high level of antigen, the normal immune response could be suppressed. As an example, if a coral were exposed to a normal population of bacteria or chemical antigen, opsonizing agents could be released for immunorecognition, triggering phagocytosis. Under more adverse conditions, such as high sedimentation, overpopulation of potential pathogens in the coral mucus, or other antigen overexposure, the immune response of opsonizing agents would be inhibited. Such decreased level of immune function would result in impairment of the coral's ability to manage antigen exposure. The final aspect of coral immunity covered in this section is histocompatability. The zooxanthellae/coral symbiosis may already be thought of as "evolutionary forerunners of the vertebrate histocompatability reactions," (Olafsen 1996) in terms of the ability of a coral to recognize and accept the algae as "self." Thirty five years ago, Prazdnikov and Mikhailova (1962) inserted a thread into jellyfish mesoglea. Within three hours, the ectoderm sloughed and an immune response began; early proof of the ability of invertebrates to act and react to "non-self." Corals have evolved highly effective wound-repair abilities and multiple means to determine self from non-self (Bigger and Hildemann 1982, et. al.). They have numerous defensive and aggressive structures that participate in this recognition. Allo- and xeno-recognition is reported in nearly every cnidarian group, including all stony and soft corals (Hildemann 1975, et. al.). Five effector responses have been identified; mesenterial filament attack, contact avoidance, barrier formation/autolysis, nematocyst-based responses, and stolon overgrowth (Raftos 1996). These responses are well known to both aquarists and scientists alike. First, avoidance reactions are based on the fact that glycoprotein histocompatability antigens (likely to be involved with corals' recognition of "self") have been isolated in coral mucus (ibid.) Second, recognition signals can trigger autolysis of cells in the "war zone" between proximate corals (ibid.). Enzymes released from lysed cells cause local tissue degeneration of both species. Thus corals can and do sacrifice their own tissues and cells through autolysis. Third, nematocysts can discharge neurotoxic, myotoxic, hemolytic and necrotic factors to damage the tissue of the "non-self" entities (ibid.). While able to recognize "self" in the use of nematocysts, the chemical factors released by the nematocysts are not specific, and they are just as degenerative to "self” as "non-self". The normal acrorhagial response is stronger to allogeneics than xenogeneics, implicating again the potential of self attack (Francis, 1973; Bigger, 1976; Benacerraf and Burakoff, 1978). Fourth, in stolon overgrowth, hydroids have been shown to produce offspring with autoreactive qualities. Such colonies used their own defense systems to actively destroy their own tissues. Thus, the reduced ability of cnidarians to determine “self” from “non-self” in the above examples, conclusively shows that corals are capable and can engage in autolytic behavior (ibid, Bigger and Hildemann 1982.). Van-Praet and Doumenc (1974) found unique macrophages in the anemone Actinia equina. These cells were found in endodermal and mesogleal tissue and phagocytized nematocysts and epithelio-muscle cells. Metchnikoff (1892), (in Bigger, Hildemann 1982), found anthozoan mesodermic phagocytes that "responded to foreign bodies or their own cells by phagocytosing them." Tokin and Yericheva (1961) found an invertebrate non-specific response to foreign material where phagocytosis occurred throughout the whole animal and not just in localized areas. Bigger (1979) showed non-fusion reactions and acute necrotic responses to foreign tissue in gorgonians. Finally, Acropora have been shown to possess transitive histocompatability where, if species A is compatable with species B, and species B is compatable with species C, then Species A is compatable with species C. Corals are therefore capable of recognizing degrees of "self," and their reactions to "non-self" are determined on degrees of genetic disparity (Raftos 1996).
Whether or not specific factors are involved, or whether simple dissimilarity is involved, either scenario can be applied to the reaction of corals with RTN. Histocompatability in cnidarians requires either direct or close contact with incompatable organisms. Hildemann suggests that histocompatability molecules functioning as antigen recognition molecules are founded on cell surface immunorecognition (Hildemann 1975). Because of spatial competition, the degree of specificity and inter-individual interactions of histocompatability will be close to the smallest possible (Stoddart, 1988). Coral mucus is a likely place for histocompatability markers to occur, and Ertman and Davenport (1981) showed that mucus from the cnidarian, A. elegantissima, will cause the nematocysts of A. xanthogrammica to discharge. The reverse was not true. Thus, it appears that biochemical markers, and not necessarily the recognition of histocompatability antigens, may be involved (Raftos 1996). Davenport further theorized that inhibitory agents prevent discharge of an animal’s own nematocysts in response to mucus effectors. Normally seen, though perhaps immunally incorrect, cross-specific compatability could therefore occur because of cross- or con-specific inhibitory substances. Other experiments have shown that proteins, perhaps glycoproteins, are acting as histocompatability antigens in other defensive responses (ibid.). Therefore, the chemical mechanisms for eliciting potential autoimmune responses exist in and between various species.
A study of "major immunological importance" occured when "M. verrucosa allograft rejection fulfilled all three criteria of (a true) immunological response." These three criteria are a cytotoxic response, specificity, and inducible memory (Bigger 1998). Hildemann (1974, 1975) showed that coral genera (Montipora verrucosa, an Acroporid) of different species could, upon contact, induce necrosis of one or both animals. Montipora also show a delayed cytotoxic response. Further observations show that memory is involved, implying that all-or-none type reactions may be averted by standard immunocompetence measures such as slow acclimation and gradual introduction to normal immune response compatable levels of antigenic material. Burkholder (1973) used incubated colonies of stony and soft corals in a study on antimicrobial activity to common marine bacteria. He found that some corals had a pronounced wide spectrum antimicrobial ability, some had specific antimicrobial activity, and some had little to no response. Certain corals (Parazoanthus spp.) had a lytic response that was delayed by a week to inital exposure. Gorgonians had very strong antimicrobial responses. As seen, corals have all of the following aspects of coelenterate (cnidarian) cellular defense: antimicrobial activity, non-specific cellular response to foreign materials, allogeneic incompatability reactions, viable allograft/xenografts persisting indefinitely, immunological responsiveness and cancers. This is the greatest number of immune responses known presently in the cnidaria.
This has been a comparatively basic review of some of the typical immune responses known to exist in the cnidarians. While it would have been possible to supplementally explore the more unique cellular and subcellular physiologic and biochemical aspects of invertebrate immune response, we feel that this background is sufficient to allow for some understanding of how we will arrive at the hypothesis stated in our next article. There are many described experiments and references in the literature listed below that will allow any interested readers to further their studies in this area. Our hopes for this paper is to bring to light a topic not yet covered in the aquarium literature, and to hopefully present some information that will, when coupled with our future corroboratory evidence for our hypothesis, allow a more complete understanding of the potential physiologic, biochemical and pathoimmunologic response to corals under stress. In conclusion, we leave the reader with the following quotation:
"...the long standing, converse notion that all invertebrates lack specific immunological capability must now be rendered untenable." (Hildemann 1975)
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