Journal of the Royal Society of Medicine Volume 72 September 1979
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Symposium: Antimicrobial defence mechanisms' Evolution of the vertebrate immune system
Margaret J Manning Bsc PhD Department ofZoology, University of Hull, Hull HU6 7RX Introduction The importance of the immune system to the antimicrobial defence mechanisms of man is obvious when one considers, for example, the low resistance to pyogenic infections in patients with Bruton type agammaglobulinaemia, or the susceptibility to certain viral diseases associated with thymic hypoplasia in Di George's syndrome. The vertebrate immune system is superimposed upon more ancient defence mechanisms which rely mainly on phagocytosis and on a variety of antimicrobial humoral substances, but it by no means replaces such mechanisms. On the contrary, it becomes increasingly linked to them with interrelationships at all stages from processing of the antigen by macrophages to its final elimination by phagocytosis or by complement-mediated lysis. In this interrelationship, the recognition molecules of the immune system come to impose a fineness of discrimination upon older defence mechanisms such as phagocytosis which themselves have intrinsically low specificity.
Phylogeny ofimmunoglobulins The immunoglobulin molecule is a vertebrate innovation, both as a secreted molecule and as a specific receptor for antigen recognition on lymphocytes. The evolution of vertebrate immunoglobulins has recently been reviewed in detail (Marchalonis 1974, Litman 1976). The regular occurrence of low molecular weight immunoglobulins whose heavy chains differ from that of IgM seems to have started at the phylogenetic stage when vertebrates were emerging from water to land and may be related to the progressive evolution of high-pressure blood vascular systems (Manning & Turner 1976). Thus changes in the rate of circulation and in the hydrostatic and osmotic pressures of the blood may affect both the migrations of cells from the vessels and the movements of antibody into the extravascular compartments, making it more difficult for high molecular weight antibody to reach invading microorganisms. Low molecular weight (IgG-like) immunoglobulins are less prominent in the amphibian antibody response than in that of mammals, however. Furthermore, the IgA class of immunoglobulin which is found in birds and mammals has not so far been identified in any poikilotherm (Jurd 1977) while IgE, although widespread within the Mammalia, appears to be confined to this advanced vertebrate class.
Transplantation reactions Specific recognition of allogeneic tissue has a long evolutionary history and may well have originated for purposes other than defence. In sponges it occurs in relation to the aggregation of cells of like strain, while in colonial coelenterates and tunicates it helps to maintain the integrity of the colony by preventing fusion with neighbouring colonies of a different kind. The recognition system in corals shows a specific memory component with a high level of discrimination. This must imply an extensive polymorphism of histocompatibility markers (Hildemann et al. 1977) but it is not known whether these markers relate in any way to those on vertebrate cells; nor is it known how they are recognized. Specific second-set memory occurs in both superphyla of coelomate animals, being present in the protostome annelids (Cooper 1976a) as well as in the deuterostome echinoderms (Karp & Hildemann 1976). 1
Papers read to Section of Comparative Medicine, 15 March 1978. Accepted 25 September 1978
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Journal of the Royal Society of Medicine Volume 72 September 1979
Whether the effector mechanisms, antigen recognition systems, and histocompatibility markers in these two divergent coelomate lines are homologous, however, is debatable (see Cooper 1976b). In mammals and birds, histocompatibility markers which elicit strong alloimmune reactions are coded for within a major histocompatibility complex of genes. The phylogeny of this system remains speculative (Klein 1975, Cohen 1976), although there may be an evolutionary link between certain cell surface structures coded for by the major histocompatibility complex and immunoglobulin, since recent evidence suggests an association of major histocompatibility antigens with fl2-microglobulin which itself shows similarities to a domain of the immunoglobulin molecule. Heterogeneity in lymphoid populations Birds and mammals show a clear dichotomy of their lymphocytic populations into T-cells which are responsible for cell-mediated immunity and B-cells which are concerned in antibody synthesis. A similar dichotomy has been demonstrated in anuran amphibians (Manning et al. 1976) but it has yet to be determined whether this distinction is a fundamental one which is present in the immune system of all vertebrates. Mitogens have been used as probes for functional heterogeneity within lymphocytic populations. These experiments indicate differences in the response to T-cell and B-cell mitogens in cells separated on the basis of organ distribution, culture kinetics, or physical properties, in fish (Etlinger et al. 1976, Cuchens et al. 1976), urodeles (Collins & Cohen 1976), anurans (Goldstine, Collins & Cohen 1975) and reptiles (Cuchens et al. 1976), as well as in birds and mammals. Hapten-carrier reactivity involving separable cellular populations also occurs in amphibians and in fishes (Ruben et al. 1973, Yocum et al. 1975), but it is not yet known whether the specific carrier reactive cells are, in fact, a thymus-derived population.
Cell surface immunoglobulins In birds and mammals, the presence of readily demonstrable endogenous immunoglobulin on the surface of lymphocytes is characteristic of B-cells. There is some controversy as to the presence of immunoglobulin on T-cells and this has raised doubts as to whether it is immunoglobulin or some other type of molecule (possibly a product of genes linked within the major histocompatibility complex) which serves as the cell surface receptor for antigen recognition by T-cells (Greaves 1975). In poikilotherms, on the other hand, surface immunoglobulin is readily demonstrable on the thymocytes of elasmobranch fish (Ellis & Parkhouse 1975), teleost fish (Emmrich et al. 1975, Cuchens et al. 1976, Ruben et al. 1977), urodeles (Charlemagne & Tournefier 1975) and anurans (Du Pasquier & Weiss 1973, Jurd & Stevenson 1976), although this immunoglobulin may differ both quantitatively (Du Pasquier & Weiss 1973, Charlemagne & Tournefier 1975) and qualitatively (Fiebig & Ambrosius 1976, Warr et al. 1977) from that on other lymphocytes. In goldfish, there is experimental evidence that the thymocyte membrane immunoglobulin is a functional receptor for antigen (Ruben et al. 1977).
Phylogeny of T-cells and B-cells The data shown in Table 1 have led to the suggestion that specialization of lymphocytes into different classes and subsets occurred progressively during the course of vertebrate evolution (see McKinney et al. 1976). Du Pasquier & Wabl (1976) believe that in anurans the ability of the maturing animal to synthesize high affinity antibody of the low molecular weight (IgGlike) class may be correlated with ontogenetic processes that alter the thymus cell populations and result in a lower expression of immunoglobulin on the thymocytes of the adult (see Du Pasquier & Weiss 1973), with ontogeny to some extent recapitulating phylogeny. The question of whether a complete structural and functional dichotomy of T-cells and B-cells first occurs at around the level of adult anurans, or whether it is present throughout the vertebrates is, however, still debatable.
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Table 1. The T-cell: B-cell dichotomy in vertebrates. Some characteristics ofthe immune system ofpoikilotherms and mammals Fish
Demonstration of both thymus-dependent and thymus-independent responses to mitogens and to antigens Separable populations of hapten and carrier reactive cells Heterogeneity in mitogen responses of lymphocytic populations Readilydetectableendogenous immunoglobulin on thymocytes Presence of low-molecular-weight class of immunoglobulin
Urodeles
+
+
+
++
+
+
-
-
Anurans
Reptiles
Mammals
+
+
+
+ +
+
+
-
-
+
+
+
A blank space indicates that sufficient information is not yet available.
Lymphoid organs Thymus: The definitive thymus may be derived from different pharyngeal pouches in different groups of jawed vertebrates (see Brachet 1921). Its histology is essentially similar throughout these groups although in teleosts and urodeles there is little distinction between cortex and medulla. The thymic epithelium, once evolved, provides an important micro-environment for T-cell differentiation, possibly an essential one from the outset, although this is not certain. Nor is it clear to what extent B-cells as well as T-cells may be spawned within the thymus of poikilotherms (see Turpen et al. 1975). Bone marrow: Bone marrow does not occur with any regularity until the evolution of anuran amphibians, reptiles, birds and mammals; it probably represents a rehousing of stem-cell populations previously situated elsewhere in the body.
Kidney: The kidneys of fish and of anuran amphibians contain simple accumulations of lymphocytes in the intertubular regions. These are situated in close proximity to the blood sinuses at sites where cells can cluster and respond to the presence of antigen. Antigen may reach the kidney via the circulation, or in mobile macrophages, or by phagocytosis by cells of the renal tubules (Turner 1973). In the metanephric kidneys of amniotes the role of the kidney as a lymphoid organ wanes and correspondingly greater emphasis is placed on other secondary lymphoid organs.
Lymph nodes: Anurans are the first vertebrates to possess simple lymph nodes (Table 2), but it is doubtful whether these represent anything more than additional organs with sinusoidal blood flow which, like the kidney, provide sites where lymphoid cells accumulate in response to antigen. Scattered lymphoid nodules are found in certain reptiles but, unlike 'true' lymph nodes of homoiotherms, these are not associated with distinct afferent and efferent lymphatic channels. Gut-associated lymphoid tissue: Gut-associated lymphoid nodules first appear in anuran amphibians (Table 2). These are similar to a single nodule of a mammalian Peyer's patch but in poikilotherms they lack germinal centres (Goldstine, Manickavel & Cohen 1975).
Spleen: The spleen of poikilotherms already displays some of the complex architecture found in the spleen of mammals. In the clawed toad Xenopus laevis a distinct boundary layer separates white pulp from red pulp (Sterba 1951) and arteries entering the spleen terminate as arterioles in the marginal zone in a manner resembling that described by Mitchell (1973) in rats and mice. Furthermore, in the spleen of anuran amphibians antigen is trapped on the
686
Journal ofthe Royal Society of Medicine Volume 72 September 1979
Table 2. Summary of the major lymphoid tissues ofpoikilothermic vertebrates Gut-associated
Thymus
Bone marrow
lymphoid nodules Majorsecondarylymphoid organs
Agnathans (lampreys + hagfish) Fish Urodeles
No true thymus
Lacking
Lacking
Scattered foci (including kidney in hagfish) Spleen, head kidney, kidney
Present Present
Usually lacking Usually lacking
Lacking Lacking
Anurans
Present
Present
Present
Spleen (plus perihepatic plasmocytes) Spleen, kidney, primitive lymph
Reptiles
Present
Present
Present
nodes Spleen, primitive lymph nodes
processes ofdendritic cells in a pattern similar to that seen in the germinal centres of birds and mammals (Diener & Marchalonis 1970, Horton & Manning 1974). In amphibians (Turner & Manning 1973) and in reptiles (Borysenko 1975), antigenic stimulation results in blast cell proliferation in the white pulp of the spleen but there is no formation of distinct germinal centres. Discussion The cell-mediated responses of vertebrates may well have arisen from pre-existing capabilities already present in the deuterostome invertebrates and perhaps of even earlier origin, since graft rejection phenomena look remarkably alike in sea-stars (Karp & Hildemann 1976), seasquirts (Reddy & Bryan 1974) and vertebrates. How this T-cell-like activity relates to the emergence of cells capable of secreting immunoglobulin remains problematical. Both capabilities are present in agnathans which lack a hierarchy of discrete lymphoid organs. The fishes have taken a step forward by isolating the primary organ (the thymus) from secondary lymphoid organs where antigen is encountered (see Table 2). Indeed, the general trend in vertebrates is clearly one from relatively scattered lymphoid tissues to discrete and highly structured organs, and from in situ reactions to infection to specialized regional organization (typified by the mammalian system of lymph nodes which provide for discrete and wellcontrolled local responses while, at the same time, being part of a highly integrated total immune system). The immune apparatus possessed by fishes is adequate for animals with a single-circuit circulatory system (cf. the double circulation of higher vertebrates). No doubt it is supplemented by more ancient mechanisms involving nonspecific phagocytosis and nonimmunoglobulin humoral factors. In regions where blood flow is relatively sluggish, cells can cluster and respond to invading microorganisms utilizing reactions based on high molecular weight antibody and aided by complement. (The full complement system is first present in jawed vertebrates although some of its components, particularly those of the alternative pathway for activation, are phylogenetically much older (Day 1974).) The necessity for low molecular weight antibodies and elaborate lymphocytic migratory pathways perhaps relates to the advanced circulatory systems which evolved when the vertebrates emerged onto land. At this stage, architectural modifications within the lymphoid organs may be required to ensure that cells can move in and out of the circulation at appropriate sites. Apart from the urodeles and apodans (which remain fish-like in many ways), amphibians seem to have achieved at least incipient development of many of the advanced characters of higher vertebrates. They have low molecular weight antibody, surface immunoglobulins are decreased on adult thymocytes, bone marrow and gut-associated lymphoid nodules are present and primitive lymph nodes appear in the higher anurans, dendritic antigen-trapping occurs in distinct areas and the immune system is clearly delineated functionally into thymus-dependent (T-cell) and thymus-independent (B-cell) components. These features of the immune system therefore date back in phylogeny at least some 300 million years.
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Amphibians as a class have not, however, lost the free-living larval stage in their life history. Like other vertebrates, amphibians are subject to the induction of specific immunological tolerance both to soluble antigens (Marchalonis & Germain 1971) and to allografts (Volpe 1970, Houillon 1972, Clark & Newth 1972). Free-living larvae, however, are vulnerable to infection from the environment at a stage when their lymphoid system is still very immature and this may necessitate a rapid maturation of pathways which lead to positive immune responses, rather than to tolerance, perhaps at the expense of more advanced differentiation. It has been demonstrated experimentally that early exposure of larvae to antigenic stimulation does not necessarily preclude the possibility of an immune response in the more mature animal (Horton 1969). In amniotes (reptiles, birds and mammals), protection of the embryo may make the need to produce mature immunocompetent cells less urgent and perhaps there can be a build-up of cellular populations before functional commitments need be made. This, together with homoiothermy, must have provided a strong evolutionary incentive for the increased diversity and efficiency of the immune system as seen in birds and mammals.
References Brachet A (1921) Traite d'Embryologie de Vertebres. Masson, Paris Borysenko M (1975) Advances in Experimental Medicine and Biology 64, 277-291 Charlemagne J & Tournefier A (1975) Advances in Experimental Medicine and Biology 64, 251-255 Clark J C & Newth D R (1972) Experientia 28,951-953 Cohen N (1976) In: Phylogeny of Thymus and Bone Marrow - Bursa Cells. Ed. R K Wright and E L Cooper. Elsevier/North Holland Biomedical Press, Amsterdam; p 169 Collins N H & Cohen N (1976) In: Phylogeny of Thymus and Bone Marrow - Bursa Cells. Ed. R K Wright and E L Cooper. Elsevier/North Holland Biomedical Press, Amsterdam; p 143 Cooper E L (1976a). In: Phylogeny of Thymus and Bone Marrow - Bursa Cells. Ed. R K Wright and E L Cooper. Elsevier/North Holland Biomedical Press, Amsterdam; p 9 Cooper E L (1976b) Comparative Immunology. Prentice-Hall, Inc., Englewood Cliffs, New Jersey Cuchens M, McLean E & Clem L W (1976) In: Phylogeny of Thymus and Bone Marrow - Bursa Cells. Ed. R K Wright & E L Cooper. Elsevier/North Holland Biomedical Press, Amsterdam; p 205 Day E (1974) Progress in Immunology 2, 287-291 Diener E & Marchalon J J (1970) Immunology 18, 279-293 Du Pasquier L & WabI M R (1976) Cold Spring Harbor Symposia on Quantitative Biology 41, 771-779 Du Pasquier L & Weiss N (1973) European Journal of Immunology 3, 773-777 Ellis A E & Parkhouse R M E (1975) European Journal of Immunology 5, 726-728 Emmnrich F, Richter R F & Ambrosius H (1975) European Journal of Immunology 5, 76-78 Etliager H M, Hodgins HO & Chiller J M (1976) Journal of Immunology 116, 1547-1553 Fiebig H & Ambrosius H (1976) In: Phylogeny of Thymus and Bone Marrow - Bursa Cells. Ed. R K Wright and E L Cooper. Elsevier/North Holland Biomedical Press, Amsterdam; p 195 Godstine S N, Collins N H & Cohen N (1975) Advances in Experimental Medicine and Biology 64, 343-351 Goldstine S N, Manickavel V & Cohen N (1975) American Zoologist 15, 107-118 Greaves M F (1975) Cellular Recognition. Chapman and Hall, London Hfldemau W H, Raison R L, Cheung G, Hull C J, Akaka L & Okaamoto J (1977) Nature (London) 270, 219-223 Horton J D (1969) Journal of Experimental Zoology 170, 449-466 Horton J D & Manning M J (1974) Immunology 26, 797-807 Houlllo C (1972) In: L'etude phylogenique et ontogenique de la reponse immunitaire et son apport a la theorie immunologique. Societe Fran9aise d'Immunologie, Paris; p 97 Jurd R D (1977) In: Developmental Immunobiology. Ed. J B Solomon and J D Horton. Elsevier/North Holland Biomedical Press, Amsterdam; p 307 Jurd R D & Stevenson G T (1976) Comparative Biochemistry and Physiology 53A, 381-387 Karp R D & Hiblemann W H (1976) Transplantation 22, 434-439 Klein J (1975) Advances in Experimental Biology and Medicine'64, 467-478 Litman G W (1976) In: Comparative Immunology. Ed. J J Marchalonis. Blackwell Scientific Publications, Oxford; p 239 Manning M J, Donnely N & Cohen N (1976) In: Phylogeny of Thymus and Bone Marrow - Bursa Cells. Ed. R K Wright and E L Cooper. Elsevier/North Holland Biomedical Press, Amsterdam; p 123 Manning M J & Turer R J (1976) Comparative Immunobiology. Blackie and Son Limited, Glasgow and London Marchaloi J J (1974) Progress in Immunology 2, 249-259 Maalois J J & Genmain R N (1971) Nature (London) 231, 321-322
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McKinney E C, Ortiz G, Lee J C, Sigel M M, Lopez D M, Epstein R S & McLeod T F (1976) In: Phylogeny of Thymus and Bone Marrow - Bursa Cells. Ed. R K Wright and E L Cooper. Elsevier/North Holland Biomedical Press, Amsterdam; p 73 Mitchell J (1973) Immunology 24, 93-107 Reddy A L and Bryan B (1974) Progress in Immunology 2, 292-296 Ruben L N, Van der Hoven A & Dutton R W (1973) Cellular Immunology 6, 300-314 Ruben L N, Waff G W, Decker J M & Marchalonis J J (1977) Cellular Immunology 31, 266-283 Sterba G (1951) Morphologisches Jahrbuch 90, 221-248 Turmer R J (1973) Journal ofExperimental Zoology 183, 35-45 Turmer R J & Manning M J (1973) Journal of Experimental Zoology 183, 21-33 Turpen J B, Volpe E P & Cohen N (1975) American Zoologist 15, 51-61 Volpe E P (1970) Transplantation Proceedings 2, 287-292 Waff G W, DeLuca D, Decker J M, Marcalonis J J & Ruben L N (1977) In: Developmental Immunobiology. Ed. J B Solomon and J D Horton. Elsevier/North Holland Biomedical Press, Amsterdam; p 241 Yocum D, Cuchens M & Clem L W (1975) Journal of Immunology 114, 925-927
Defence of invertebrates against bacterial infection Professor R P Dales PhD Bedford College, University of London Regents Park, London NWJ 4NS Dr Cameron of University College Hospital Medical School injected earthworms with enough diphtheria toxin to kill 300 guinea-pigs and found that most worms survived. He also found that huge doses of bacteria such as Staphylococcus aureus, pneumococcus, paratyphoid bacilli, Vibrio cholerae, Bacillus pestis, and Shigella dysenteriae, could all be injected into common earthworms (Lumbricus terrestris) without obvious result (Cameron 1932). We should not be surprised by this, for insect vectors can convey bacteria pathological to man, yet are themselves immune, but we would be wrong to think that invertebrates were not open to bacterial attack. Vertebrates maintaining body fluids at a high and constant temperature present an ideal medium for bacterial growth but invertebrates present growth media for other kinds of bacteria and therefore require a defensive system. It is wrong to think of invertebrates as simply producing large numbers of offspring so that those which become infected are considered expendable, or that invertebrates do not live long enough to justify an immune system. How septic are invertebrate body fluids? Cameron (1932) found that he could culture the same bacteria from earthworm body fluid that occurred in the soil in which they were found, on the skin or from the gut. More recently Marks & Cooper (1977) have found Aeromonas hydrophila consistently present in the Lumbricus terrestris kept in culture. Other invertebrates when carefully sampled may be aseptic or show only minor contamination, often with characteristic bacteria. Careful studies of animals such as oysters show that they have a characteristic commensal flora but can accumulate gram-positive bacteria (Colwell & Liston 1960). Blue crabs and other crustaceans can harbour Vibrio parahaemolyticus which are common in the sea (Kaneko & Colwell 1973) especially at some times of year (Tubiash et al. 1975), Colwell et al. (1975) concluding that the haemolymph of blue crabs was probably never sterile. Accidental wounds in all animals present a means of entry for bacteria, and those mishandled are often infected. Commercially important shellfish may often be spoiled in this way (Johnson 1976). The balance between health and disease is a delicate one and crabs are vulnerable organisms in any but ideal conditions. In some invertebrates agglutination of cells or clotting proteins can serve to close a wound. In crustaceans the cells caught up in a clot may release bactericidal substances which will minimize bacterial entry and at the same time stimulate phagocytosis. The gelling of limulus fluid which helps to close wounds (Shirodkar et al. 1960) is cell-derived and is gelled by endotoxin which causes clumping of cells and their
0141-0768/79/090688--09/$01.00/0
%:v,.', 1979 The Royal Society of Medicine
683
Symposium: Antimicrobial defence mechanisms' Evolution of the vertebrate immune system
Margaret J Manning Bsc PhD Department ofZoology, University of Hull, Hull HU6 7RX Introduction The importance of the immune system to the antimicrobial defence mechanisms of man is obvious when one considers, for example, the low resistance to pyogenic infections in patients with Bruton type agammaglobulinaemia, or the susceptibility to certain viral diseases associated with thymic hypoplasia in Di George's syndrome. The vertebrate immune system is superimposed upon more ancient defence mechanisms which rely mainly on phagocytosis and on a variety of antimicrobial humoral substances, but it by no means replaces such mechanisms. On the contrary, it becomes increasingly linked to them with interrelationships at all stages from processing of the antigen by macrophages to its final elimination by phagocytosis or by complement-mediated lysis. In this interrelationship, the recognition molecules of the immune system come to impose a fineness of discrimination upon older defence mechanisms such as phagocytosis which themselves have intrinsically low specificity.
Phylogeny ofimmunoglobulins The immunoglobulin molecule is a vertebrate innovation, both as a secreted molecule and as a specific receptor for antigen recognition on lymphocytes. The evolution of vertebrate immunoglobulins has recently been reviewed in detail (Marchalonis 1974, Litman 1976). The regular occurrence of low molecular weight immunoglobulins whose heavy chains differ from that of IgM seems to have started at the phylogenetic stage when vertebrates were emerging from water to land and may be related to the progressive evolution of high-pressure blood vascular systems (Manning & Turner 1976). Thus changes in the rate of circulation and in the hydrostatic and osmotic pressures of the blood may affect both the migrations of cells from the vessels and the movements of antibody into the extravascular compartments, making it more difficult for high molecular weight antibody to reach invading microorganisms. Low molecular weight (IgG-like) immunoglobulins are less prominent in the amphibian antibody response than in that of mammals, however. Furthermore, the IgA class of immunoglobulin which is found in birds and mammals has not so far been identified in any poikilotherm (Jurd 1977) while IgE, although widespread within the Mammalia, appears to be confined to this advanced vertebrate class.
Transplantation reactions Specific recognition of allogeneic tissue has a long evolutionary history and may well have originated for purposes other than defence. In sponges it occurs in relation to the aggregation of cells of like strain, while in colonial coelenterates and tunicates it helps to maintain the integrity of the colony by preventing fusion with neighbouring colonies of a different kind. The recognition system in corals shows a specific memory component with a high level of discrimination. This must imply an extensive polymorphism of histocompatibility markers (Hildemann et al. 1977) but it is not known whether these markers relate in any way to those on vertebrate cells; nor is it known how they are recognized. Specific second-set memory occurs in both superphyla of coelomate animals, being present in the protostome annelids (Cooper 1976a) as well as in the deuterostome echinoderms (Karp & Hildemann 1976). 1
Papers read to Section of Comparative Medicine, 15 March 1978. Accepted 25 September 1978
0141-0768/79/090683-.06/$01.00/0
wC, 1979 The Royal Society of Medicine
684
Journal of the Royal Society of Medicine Volume 72 September 1979
Whether the effector mechanisms, antigen recognition systems, and histocompatibility markers in these two divergent coelomate lines are homologous, however, is debatable (see Cooper 1976b). In mammals and birds, histocompatibility markers which elicit strong alloimmune reactions are coded for within a major histocompatibility complex of genes. The phylogeny of this system remains speculative (Klein 1975, Cohen 1976), although there may be an evolutionary link between certain cell surface structures coded for by the major histocompatibility complex and immunoglobulin, since recent evidence suggests an association of major histocompatibility antigens with fl2-microglobulin which itself shows similarities to a domain of the immunoglobulin molecule. Heterogeneity in lymphoid populations Birds and mammals show a clear dichotomy of their lymphocytic populations into T-cells which are responsible for cell-mediated immunity and B-cells which are concerned in antibody synthesis. A similar dichotomy has been demonstrated in anuran amphibians (Manning et al. 1976) but it has yet to be determined whether this distinction is a fundamental one which is present in the immune system of all vertebrates. Mitogens have been used as probes for functional heterogeneity within lymphocytic populations. These experiments indicate differences in the response to T-cell and B-cell mitogens in cells separated on the basis of organ distribution, culture kinetics, or physical properties, in fish (Etlinger et al. 1976, Cuchens et al. 1976), urodeles (Collins & Cohen 1976), anurans (Goldstine, Collins & Cohen 1975) and reptiles (Cuchens et al. 1976), as well as in birds and mammals. Hapten-carrier reactivity involving separable cellular populations also occurs in amphibians and in fishes (Ruben et al. 1973, Yocum et al. 1975), but it is not yet known whether the specific carrier reactive cells are, in fact, a thymus-derived population.
Cell surface immunoglobulins In birds and mammals, the presence of readily demonstrable endogenous immunoglobulin on the surface of lymphocytes is characteristic of B-cells. There is some controversy as to the presence of immunoglobulin on T-cells and this has raised doubts as to whether it is immunoglobulin or some other type of molecule (possibly a product of genes linked within the major histocompatibility complex) which serves as the cell surface receptor for antigen recognition by T-cells (Greaves 1975). In poikilotherms, on the other hand, surface immunoglobulin is readily demonstrable on the thymocytes of elasmobranch fish (Ellis & Parkhouse 1975), teleost fish (Emmrich et al. 1975, Cuchens et al. 1976, Ruben et al. 1977), urodeles (Charlemagne & Tournefier 1975) and anurans (Du Pasquier & Weiss 1973, Jurd & Stevenson 1976), although this immunoglobulin may differ both quantitatively (Du Pasquier & Weiss 1973, Charlemagne & Tournefier 1975) and qualitatively (Fiebig & Ambrosius 1976, Warr et al. 1977) from that on other lymphocytes. In goldfish, there is experimental evidence that the thymocyte membrane immunoglobulin is a functional receptor for antigen (Ruben et al. 1977).
Phylogeny of T-cells and B-cells The data shown in Table 1 have led to the suggestion that specialization of lymphocytes into different classes and subsets occurred progressively during the course of vertebrate evolution (see McKinney et al. 1976). Du Pasquier & Wabl (1976) believe that in anurans the ability of the maturing animal to synthesize high affinity antibody of the low molecular weight (IgGlike) class may be correlated with ontogenetic processes that alter the thymus cell populations and result in a lower expression of immunoglobulin on the thymocytes of the adult (see Du Pasquier & Weiss 1973), with ontogeny to some extent recapitulating phylogeny. The question of whether a complete structural and functional dichotomy of T-cells and B-cells first occurs at around the level of adult anurans, or whether it is present throughout the vertebrates is, however, still debatable.
Journal of the Royal Society ofMedicine Volume 72 September 1979
685
Table 1. The T-cell: B-cell dichotomy in vertebrates. Some characteristics ofthe immune system ofpoikilotherms and mammals Fish
Demonstration of both thymus-dependent and thymus-independent responses to mitogens and to antigens Separable populations of hapten and carrier reactive cells Heterogeneity in mitogen responses of lymphocytic populations Readilydetectableendogenous immunoglobulin on thymocytes Presence of low-molecular-weight class of immunoglobulin
Urodeles
+
+
+
++
+
+
-
-
Anurans
Reptiles
Mammals
+
+
+
+ +
+
+
-
-
+
+
+
A blank space indicates that sufficient information is not yet available.
Lymphoid organs Thymus: The definitive thymus may be derived from different pharyngeal pouches in different groups of jawed vertebrates (see Brachet 1921). Its histology is essentially similar throughout these groups although in teleosts and urodeles there is little distinction between cortex and medulla. The thymic epithelium, once evolved, provides an important micro-environment for T-cell differentiation, possibly an essential one from the outset, although this is not certain. Nor is it clear to what extent B-cells as well as T-cells may be spawned within the thymus of poikilotherms (see Turpen et al. 1975). Bone marrow: Bone marrow does not occur with any regularity until the evolution of anuran amphibians, reptiles, birds and mammals; it probably represents a rehousing of stem-cell populations previously situated elsewhere in the body.
Kidney: The kidneys of fish and of anuran amphibians contain simple accumulations of lymphocytes in the intertubular regions. These are situated in close proximity to the blood sinuses at sites where cells can cluster and respond to the presence of antigen. Antigen may reach the kidney via the circulation, or in mobile macrophages, or by phagocytosis by cells of the renal tubules (Turner 1973). In the metanephric kidneys of amniotes the role of the kidney as a lymphoid organ wanes and correspondingly greater emphasis is placed on other secondary lymphoid organs.
Lymph nodes: Anurans are the first vertebrates to possess simple lymph nodes (Table 2), but it is doubtful whether these represent anything more than additional organs with sinusoidal blood flow which, like the kidney, provide sites where lymphoid cells accumulate in response to antigen. Scattered lymphoid nodules are found in certain reptiles but, unlike 'true' lymph nodes of homoiotherms, these are not associated with distinct afferent and efferent lymphatic channels. Gut-associated lymphoid tissue: Gut-associated lymphoid nodules first appear in anuran amphibians (Table 2). These are similar to a single nodule of a mammalian Peyer's patch but in poikilotherms they lack germinal centres (Goldstine, Manickavel & Cohen 1975).
Spleen: The spleen of poikilotherms already displays some of the complex architecture found in the spleen of mammals. In the clawed toad Xenopus laevis a distinct boundary layer separates white pulp from red pulp (Sterba 1951) and arteries entering the spleen terminate as arterioles in the marginal zone in a manner resembling that described by Mitchell (1973) in rats and mice. Furthermore, in the spleen of anuran amphibians antigen is trapped on the
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Journal ofthe Royal Society of Medicine Volume 72 September 1979
Table 2. Summary of the major lymphoid tissues ofpoikilothermic vertebrates Gut-associated
Thymus
Bone marrow
lymphoid nodules Majorsecondarylymphoid organs
Agnathans (lampreys + hagfish) Fish Urodeles
No true thymus
Lacking
Lacking
Scattered foci (including kidney in hagfish) Spleen, head kidney, kidney
Present Present
Usually lacking Usually lacking
Lacking Lacking
Anurans
Present
Present
Present
Spleen (plus perihepatic plasmocytes) Spleen, kidney, primitive lymph
Reptiles
Present
Present
Present
nodes Spleen, primitive lymph nodes
processes ofdendritic cells in a pattern similar to that seen in the germinal centres of birds and mammals (Diener & Marchalonis 1970, Horton & Manning 1974). In amphibians (Turner & Manning 1973) and in reptiles (Borysenko 1975), antigenic stimulation results in blast cell proliferation in the white pulp of the spleen but there is no formation of distinct germinal centres. Discussion The cell-mediated responses of vertebrates may well have arisen from pre-existing capabilities already present in the deuterostome invertebrates and perhaps of even earlier origin, since graft rejection phenomena look remarkably alike in sea-stars (Karp & Hildemann 1976), seasquirts (Reddy & Bryan 1974) and vertebrates. How this T-cell-like activity relates to the emergence of cells capable of secreting immunoglobulin remains problematical. Both capabilities are present in agnathans which lack a hierarchy of discrete lymphoid organs. The fishes have taken a step forward by isolating the primary organ (the thymus) from secondary lymphoid organs where antigen is encountered (see Table 2). Indeed, the general trend in vertebrates is clearly one from relatively scattered lymphoid tissues to discrete and highly structured organs, and from in situ reactions to infection to specialized regional organization (typified by the mammalian system of lymph nodes which provide for discrete and wellcontrolled local responses while, at the same time, being part of a highly integrated total immune system). The immune apparatus possessed by fishes is adequate for animals with a single-circuit circulatory system (cf. the double circulation of higher vertebrates). No doubt it is supplemented by more ancient mechanisms involving nonspecific phagocytosis and nonimmunoglobulin humoral factors. In regions where blood flow is relatively sluggish, cells can cluster and respond to invading microorganisms utilizing reactions based on high molecular weight antibody and aided by complement. (The full complement system is first present in jawed vertebrates although some of its components, particularly those of the alternative pathway for activation, are phylogenetically much older (Day 1974).) The necessity for low molecular weight antibodies and elaborate lymphocytic migratory pathways perhaps relates to the advanced circulatory systems which evolved when the vertebrates emerged onto land. At this stage, architectural modifications within the lymphoid organs may be required to ensure that cells can move in and out of the circulation at appropriate sites. Apart from the urodeles and apodans (which remain fish-like in many ways), amphibians seem to have achieved at least incipient development of many of the advanced characters of higher vertebrates. They have low molecular weight antibody, surface immunoglobulins are decreased on adult thymocytes, bone marrow and gut-associated lymphoid nodules are present and primitive lymph nodes appear in the higher anurans, dendritic antigen-trapping occurs in distinct areas and the immune system is clearly delineated functionally into thymus-dependent (T-cell) and thymus-independent (B-cell) components. These features of the immune system therefore date back in phylogeny at least some 300 million years.
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Amphibians as a class have not, however, lost the free-living larval stage in their life history. Like other vertebrates, amphibians are subject to the induction of specific immunological tolerance both to soluble antigens (Marchalonis & Germain 1971) and to allografts (Volpe 1970, Houillon 1972, Clark & Newth 1972). Free-living larvae, however, are vulnerable to infection from the environment at a stage when their lymphoid system is still very immature and this may necessitate a rapid maturation of pathways which lead to positive immune responses, rather than to tolerance, perhaps at the expense of more advanced differentiation. It has been demonstrated experimentally that early exposure of larvae to antigenic stimulation does not necessarily preclude the possibility of an immune response in the more mature animal (Horton 1969). In amniotes (reptiles, birds and mammals), protection of the embryo may make the need to produce mature immunocompetent cells less urgent and perhaps there can be a build-up of cellular populations before functional commitments need be made. This, together with homoiothermy, must have provided a strong evolutionary incentive for the increased diversity and efficiency of the immune system as seen in birds and mammals.
References Brachet A (1921) Traite d'Embryologie de Vertebres. Masson, Paris Borysenko M (1975) Advances in Experimental Medicine and Biology 64, 277-291 Charlemagne J & Tournefier A (1975) Advances in Experimental Medicine and Biology 64, 251-255 Clark J C & Newth D R (1972) Experientia 28,951-953 Cohen N (1976) In: Phylogeny of Thymus and Bone Marrow - Bursa Cells. Ed. R K Wright and E L Cooper. Elsevier/North Holland Biomedical Press, Amsterdam; p 169 Collins N H & Cohen N (1976) In: Phylogeny of Thymus and Bone Marrow - Bursa Cells. Ed. R K Wright and E L Cooper. Elsevier/North Holland Biomedical Press, Amsterdam; p 143 Cooper E L (1976a). In: Phylogeny of Thymus and Bone Marrow - Bursa Cells. Ed. R K Wright and E L Cooper. Elsevier/North Holland Biomedical Press, Amsterdam; p 9 Cooper E L (1976b) Comparative Immunology. Prentice-Hall, Inc., Englewood Cliffs, New Jersey Cuchens M, McLean E & Clem L W (1976) In: Phylogeny of Thymus and Bone Marrow - Bursa Cells. Ed. R K Wright & E L Cooper. Elsevier/North Holland Biomedical Press, Amsterdam; p 205 Day E (1974) Progress in Immunology 2, 287-291 Diener E & Marchalon J J (1970) Immunology 18, 279-293 Du Pasquier L & WabI M R (1976) Cold Spring Harbor Symposia on Quantitative Biology 41, 771-779 Du Pasquier L & Weiss N (1973) European Journal of Immunology 3, 773-777 Ellis A E & Parkhouse R M E (1975) European Journal of Immunology 5, 726-728 Emmnrich F, Richter R F & Ambrosius H (1975) European Journal of Immunology 5, 76-78 Etliager H M, Hodgins HO & Chiller J M (1976) Journal of Immunology 116, 1547-1553 Fiebig H & Ambrosius H (1976) In: Phylogeny of Thymus and Bone Marrow - Bursa Cells. Ed. R K Wright and E L Cooper. Elsevier/North Holland Biomedical Press, Amsterdam; p 195 Godstine S N, Collins N H & Cohen N (1975) Advances in Experimental Medicine and Biology 64, 343-351 Goldstine S N, Manickavel V & Cohen N (1975) American Zoologist 15, 107-118 Greaves M F (1975) Cellular Recognition. Chapman and Hall, London Hfldemau W H, Raison R L, Cheung G, Hull C J, Akaka L & Okaamoto J (1977) Nature (London) 270, 219-223 Horton J D (1969) Journal of Experimental Zoology 170, 449-466 Horton J D & Manning M J (1974) Immunology 26, 797-807 Houlllo C (1972) In: L'etude phylogenique et ontogenique de la reponse immunitaire et son apport a la theorie immunologique. Societe Fran9aise d'Immunologie, Paris; p 97 Jurd R D (1977) In: Developmental Immunobiology. Ed. J B Solomon and J D Horton. Elsevier/North Holland Biomedical Press, Amsterdam; p 307 Jurd R D & Stevenson G T (1976) Comparative Biochemistry and Physiology 53A, 381-387 Karp R D & Hiblemann W H (1976) Transplantation 22, 434-439 Klein J (1975) Advances in Experimental Biology and Medicine'64, 467-478 Litman G W (1976) In: Comparative Immunology. Ed. J J Marchalonis. Blackwell Scientific Publications, Oxford; p 239 Manning M J, Donnely N & Cohen N (1976) In: Phylogeny of Thymus and Bone Marrow - Bursa Cells. Ed. R K Wright and E L Cooper. Elsevier/North Holland Biomedical Press, Amsterdam; p 123 Manning M J & Turer R J (1976) Comparative Immunobiology. Blackie and Son Limited, Glasgow and London Marchaloi J J (1974) Progress in Immunology 2, 249-259 Maalois J J & Genmain R N (1971) Nature (London) 231, 321-322
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McKinney E C, Ortiz G, Lee J C, Sigel M M, Lopez D M, Epstein R S & McLeod T F (1976) In: Phylogeny of Thymus and Bone Marrow - Bursa Cells. Ed. R K Wright and E L Cooper. Elsevier/North Holland Biomedical Press, Amsterdam; p 73 Mitchell J (1973) Immunology 24, 93-107 Reddy A L and Bryan B (1974) Progress in Immunology 2, 292-296 Ruben L N, Van der Hoven A & Dutton R W (1973) Cellular Immunology 6, 300-314 Ruben L N, Waff G W, Decker J M & Marchalonis J J (1977) Cellular Immunology 31, 266-283 Sterba G (1951) Morphologisches Jahrbuch 90, 221-248 Turmer R J (1973) Journal ofExperimental Zoology 183, 35-45 Turmer R J & Manning M J (1973) Journal of Experimental Zoology 183, 21-33 Turpen J B, Volpe E P & Cohen N (1975) American Zoologist 15, 51-61 Volpe E P (1970) Transplantation Proceedings 2, 287-292 Waff G W, DeLuca D, Decker J M, Marcalonis J J & Ruben L N (1977) In: Developmental Immunobiology. Ed. J B Solomon and J D Horton. Elsevier/North Holland Biomedical Press, Amsterdam; p 241 Yocum D, Cuchens M & Clem L W (1975) Journal of Immunology 114, 925-927
Defence of invertebrates against bacterial infection Professor R P Dales PhD Bedford College, University of London Regents Park, London NWJ 4NS Dr Cameron of University College Hospital Medical School injected earthworms with enough diphtheria toxin to kill 300 guinea-pigs and found that most worms survived. He also found that huge doses of bacteria such as Staphylococcus aureus, pneumococcus, paratyphoid bacilli, Vibrio cholerae, Bacillus pestis, and Shigella dysenteriae, could all be injected into common earthworms (Lumbricus terrestris) without obvious result (Cameron 1932). We should not be surprised by this, for insect vectors can convey bacteria pathological to man, yet are themselves immune, but we would be wrong to think that invertebrates were not open to bacterial attack. Vertebrates maintaining body fluids at a high and constant temperature present an ideal medium for bacterial growth but invertebrates present growth media for other kinds of bacteria and therefore require a defensive system. It is wrong to think of invertebrates as simply producing large numbers of offspring so that those which become infected are considered expendable, or that invertebrates do not live long enough to justify an immune system. How septic are invertebrate body fluids? Cameron (1932) found that he could culture the same bacteria from earthworm body fluid that occurred in the soil in which they were found, on the skin or from the gut. More recently Marks & Cooper (1977) have found Aeromonas hydrophila consistently present in the Lumbricus terrestris kept in culture. Other invertebrates when carefully sampled may be aseptic or show only minor contamination, often with characteristic bacteria. Careful studies of animals such as oysters show that they have a characteristic commensal flora but can accumulate gram-positive bacteria (Colwell & Liston 1960). Blue crabs and other crustaceans can harbour Vibrio parahaemolyticus which are common in the sea (Kaneko & Colwell 1973) especially at some times of year (Tubiash et al. 1975), Colwell et al. (1975) concluding that the haemolymph of blue crabs was probably never sterile. Accidental wounds in all animals present a means of entry for bacteria, and those mishandled are often infected. Commercially important shellfish may often be spoiled in this way (Johnson 1976). The balance between health and disease is a delicate one and crabs are vulnerable organisms in any but ideal conditions. In some invertebrates agglutination of cells or clotting proteins can serve to close a wound. In crustaceans the cells caught up in a clot may release bactericidal substances which will minimize bacterial entry and at the same time stimulate phagocytosis. The gelling of limulus fluid which helps to close wounds (Shirodkar et al. 1960) is cell-derived and is gelled by endotoxin which causes clumping of cells and their
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