Developmentaland ComparativeImmunology,Vol. 14, pp. 151 - 159, 1990 Printed in the USA. All rights reserved.
0145-305X/90 $3.00 + .00 Copyright © 1990 Pergamon Press plc
ONTOGENIC MATURATION OF THE IMMUNE SYSTEM IN REPTILES Somaya Osman El Deeb and Abdel Hakim Mohamed Saad Zoology Department, Facultyof Science, Cairo University,Cairo, Egypt
(Submitted January 1989;Accepted July 1989)
[]Keywords--Reptiles; Ontogeny; Lymphoid organs; T- and B-iymphocytes; Immune responses.
Introduction There has been increased emphasis in recent years on the phylogenetic and ontogenetic aspects of immunity. In contrast to the wealth of information which has been obtained from studying immune reactions in mammals and birds, comparatively little data are available on the structure of lymphoid organs and function of lymphocyte populations in ectothermic vertebrates (fishes, amphibians, and reptiles). There are at least three reasons why reptiles, with a complex "mammal-like" immune system, provide an important research tool for the comparative immunologist interested in the ontogeny of immunity. First, reptiles hold a unique position in the evolut i o n a r y l i n e a g e to b o t h b i r d s and mammals (1). Second, reptiles are the first category of vertebrates to exhibit the change from free-living larvae, as found in fishes and amphibians, to an embryo completely protected by an amnion similar to avian and mammalian embryos (2). Third, reptiles have a prolonged maturation period, making them good candidates for investigating the maturation of immune capacity (3). This brief review focuses on the ontogenetic development of the reptilian imAddress correspondence to Dr. A-H. Saad, Zoology Department, Faculty of Science, Cairo University, Cairo, Egypt.
mune system. We shall review some published data from our laboratory and others showing that a degree of immunological maturation is detectable during reptilian embryonic life. In particular, the lizards Chalcides ocellatus (Scincidae) and Calotes versicolor (Agamidae) have provided a refined experimental model for studying some fundamental aspects concerning ontogeny of reptilian immunity. We hope that this will give the reader some insight into certain evolutionary trends in immunology and lead to a phylogenetic perspective that strengthens this rapidly growing area of research.
Embryonic Development of Lizards Growth and development of reptilian embryos have been studied carefully, and arbitary stages have been established that allow investigators to compare different species or even different populations of the same species. Morphological descriptions of embryonic stages of lizards have dealt principally with representatives of the families Lacertidae, Chamaleonidae, Iguanidae and Scincidae (4). A brief account will be given first on the embryonic development in Calotes versicolor (5) and Chalcides ocellatus (6) which are pertinent to the following discussion. In Calotes, the earliest embryo from normally laid eggs is stage 27. The initial stages (27 to 33) of d e v e l o p m e n t are d i s t i n g u i s h e d by changes in somites, gill clefts, eye and limb-buds. For classifying embryos of 151
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stage 34 to 40, limbs provide the most convenient diagnostic criteria. During the prehatching period (stages 38 to 42), scales and pigmentation on the head and other parts of the embryo are used as distinguishing characters. The interval between two successive stages is approximately 2-3 days during the earlier and 3 - 4 days during the later periods of development (cf. (5)]. The series of developmental stages as reported for Calotes is adopted for classifying Chalcides embryos (6) and is summarized in Table 1.
Embryonic Origin and Migratory Pattern of Precursor Cells Yolk Sac
The developmental pathways of lymphoid cells in embryonic life have been analyzed since the beginning of the century. Several theories have been presented to explain the appearance of lymphocytes in lymphoid organ rudiments (7-12). Despite controversy concerning the source(s) of primary stem cells, there has been a universal agreement that development of lymphoid organs is completely dependent on an inflow of extrinsic stem cells into the epithelial or mesenchymal rudiments. Whatever the origin of the lymphoid stem cells, other questions concerning their differentiation and the nature of the yolk sac in ectotherms remain unanswered. Jordan, in his pioneering studies (13) on hemopoiesis in snapping and box turtles, described a "hemoblast" as an amoeboid cell with a vesicular nucleus, one or two nucleoli, and an envelope of strongly basophilic cytoplasm. This cell was first observed in blood islands of the yolk sac, the earliest hemopoietic tissue in chelonians. Later; these hemoblasts are also found in other foci of hemopoiesis. In this regard, it is incorrect to equate stem cells with " h e m o b l a s t s " which
S.O. El Deeb and A-H. Saad
may or may not be the same cell. Certain labelling studies are necessary in order to demonstrate that they are, indeed, hemopoietic stem cells. Based on observations revealing sequential appearance of hemoblasts in yolk sac blood islands, circulation, mesenchyma and then the thymic primodium, followed by lymphopoiesis in the thymus, Pitchappan and Muthukkaruppan (14) suggested that lymphocyte progenitors in the lizard Calotes versicolor originate from blood-borne stem cells. In accordance, our recent studies on the ontogeny of hemopoietic cell lineages in the lizard Chalcides ocellatus support this hypothesis. In Chalcides, large undifferentiated blasts, characterized by high nucleo-cytoplasmic ratio were first seen in yolk sac at stage 21. These cells were intermingled with primitive erythroblasts and constituted about 57.1% of the total differential count. Gradually, the number of erythroblasts increased together with morphological changes which occur which lead to the appearance of mature forms. Thus, as the embyro ages, mature forms of erythroblasts increase in number accompanied by a gradual disappearance of early erythroid cells. By stage 31, the mature forms of erythrocytes are first detected. It was rather surprising that also the number of undifferentiated blasts increased considerably. In addition, other t y p e s of blood cells were found in varying ratios at different stages, e.g., macrophages, megakaryocytes, myeloid cells, and unidentified blasts (15). By morphological criteria, therefore, the lizard's yolk sac is a hemopoietic site. Since no other hemopoietic organ has been observed to the yolk sacs appearance, it may be coosidered as the first organ which develops hemopoietic cells during ontogeny. These results are in accordance with those observed in case of avian yolk sac hemopoiesis which is more extensive and includes the production of various cell types
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Table 1. Developmental stages of Chalcides ocellatus embryos Important Changes in Development of the Immune System
Stage
Definitive External Characteristics
21-26
The first and second branchial clefts are open, narrow slits. The prospective force- and hind-area are demarcated by mesodermal condensation. The first four branchial clefts appear as open slits. The tail-bud is elongated and curved with unsegmented mesoderm. Fore- and hind-limbs are distinct. Tail elongates but is still unsegmented. Second, third and fourth branchial clefts sunk beneath the surface. Pigmentation begins in the retina. Mandibular process increases in size. The distal part of the limb is broadened (paddle-like) without any demarcation of digits; elbow- and knee-joints are distinct. Beginning of formation of eye-lids. Branchial clefts are no longer visible. Fore- and hind-limbs considerably longer. Digital plate exhibits marked intergrooves delineating the digits. Tongue formation is definite. Digital plate is deeply notched as phalarges elongate. All the five digits are fully demarcated and connected by webs which have curved contours.
27
30-31
32
33-34
35 36
37 38 39
40
41
Digits are elongated and their tips are pointed. Webs between the digits are thin and deeply notched. The tympanum is formed. Digits are elongated and thick and appear annulated. Scale primordia are evident. The inner margin of the eye-lid is oval. Very light pigmentation is present on the snout and parietal region. Pigmented scales appear on the tail and dorsal side of the trunk. Claws make their appearance at the extremity of the digits. Scales on the tail, dorso-lateral region of the trunk, and limbs are well pigmented. Slightly pigmented scales begin to appear on the snout and frontal region. The embryo is fully pigmented with a specific pattern. The parietal region is distinctly compressed. Stripes of pigmentation appear on the dorsal side of the head. The embryo at this stage resembles a newborn.
(8,9). On the other hand, these observations are at variance with those described in the mammalian system. In the case of mammals, yolk sac hemopoiesis
Yolk sac exhibit prominent erythropoietic activity. Yolk sac appears to be involved in producing myeloid cells and megakaryocytes together with erythroid cells. Appearance of thymic primordia.
Budding of splenic rudiments.
Detachment of thymic rudiments from the pharyngeal pouches.
Appearance of basophilic cells in the thymus. Onset of haemopoiesis in the spleen. Onset of lymphopoiesis in the thymus. Emergence of Con A responses and MLR reactivity. Spleen is full of erythroblasts and granulocytes. Detection of TA + and Thy-1 ÷ cells in the thymus. Lymphopoiesis begins in the spleen. Detection of surface Ig* and TA + cells in the spleen.
Bone marrow haemopoiesis appears.
Demarcation of thymic cortex and medullary areas as well as splenic white and red pulps. Impaired Con A responses and MLR reactivity.
is limited to the production of a primitive generation of nucleated erythrocytes (7). No sufficient data are available about yolk sac hemopoiesis in fish (16). The
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yolk sac equivalent (ventral blood island) gives rise to erythrocytes, thymocytes and B-lymphocytes in Xenopus laevis (17,18).
Spleen There is a lack of detailed information concerning ontogeny of the spleen in various groups of reptiles. Previous studies available on development of the spleen in snakes and lizards are restricted to the origin and pattern of vascularization of splenic rudiments (19). Kanakambika and Muthukkaruppan analyzed splenic development in Calotes, where the primordium appears at stage 30 as a protuberance of mesenchymal cells from the dorsal mesentary, detected in trunk region serial sections stained routinely with hematoxylin and eosin. In subsequent stages, there is further enlargement of the rudiment due to increase in numbers of mesenchymal cells and blood spaces. At stage 35, the spleen is primarily erythroid in nature. A considerable number of primitive granulocytes are present at stage 36 and mature granulocytes appear in the spleen at stage 38. The initiation of lymphopoiesis occurs at stage 40 (14). Comparable results have also been obtained for Chalcides (15).
Liver and Kidney In Chalcides, no hemopoietic activity is detected at any developmental stage in the liver (in contrast to that found in Calotes, to be discussed later). This absence is similar to birds, but it is in sharp contrast to mammals where hemopoiesis in the liver is extensive and is responsible for the second generation of erythrocytes and of lymphocytes (7-12). Liver hemopoiesis has been demonstrated by Turpen and his colleagues (20) in anuran amphibians revealing the pres-
S.O. El Deeb and A-H. Saad
ence of lymphocytes, plasmatocytes and macrophages at different stages of maturation. If the comparison is valid, no such activity has been recorded in fish. Similarly, no kidney hemopoiesis is detected in lizard embryos at any developmental stages nor in adult Chalcides (15). However, further studies in other reptilian species are warranted before drawing firm conclusions about the existence and/or absence, of liver and kidney hemopoiesis in reptiles.
Bone Marrow Reptilian bone marrow has been studied morphologically in the adult lizards (21,22); it contains mature and transitional forms of various hemopoietic cellular lineages. In this respect, it tends to satisfy the criteria of organbound hemopoiesis as has been described previously for adult marrow of m a m m a l s , b i r d s , and a n u r a n amphibians. No ontogenic study of marrow is yet available, although reptiles are a linking group between lower vertebrates and mammals. Bone marrow in Chalcides becomes hemopoietic at a very late developmental stage (40-41, i.e., just before birth). A few granulocytes and undifferentiated blasts are detected in the bone marrow at stage 41. In considering the evolution of bone marrow, apparently reptiles are the first group of vertebrates in which bone marrow hemopoiesis takes place during embryonic life. From an evolutionary viewpoint, lack of a hemopoietic bone marrow has been repeatedly observed in fish during both embryonic and adult stages (16). Bone marrow without hemopoietic activity has been observed among Urodeles (23,24); it contains mainly fat and fibroblasts. The phylogenetic emergence of a true lymphopoietic, myelopoietic, and erythopoietic bone marrow was first observed in adult anurans (25,26), but
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never encountered during premetamorphic stages (27,28). In an attempt to discuss the relationship between the phylogenetic emergence of bone marrow as a source of hemopoietic cells and the evolution of the terrestial way of life, Cooper et al. (26) suggested that hemopoietic stem cells are protected from irradiation by bones in terrestrial vertebrates, which have substituted for water as a radio-opaque shield. This suggestion would, thus, be most meaningful if Class Reptilia is considered, since it is known to include some species that are the first vertebrate group to become completely free from the aquatic environment. Our study on the ontogeny of hemopoietic tissues in Chalcides leads us to conclude that the first and major hemopoietic organ in the embryo is the yolk sac (15). Later in development, hemopoiesis in the yolk sac is succeeded by blood cell formation in the fetal spleen and fetal bone marrow. Finally, bone marrow becomes the major hemopoietic organ of adult lizards, although the potentiality for hemopoiesis is retained, to some extent, by the spleen (15).
Early Development of Lymphoid Organs
Thymus In jawed vertebrates there is a separate microenvironment, the thymus, which provides for the differentiation of lymphocytes capable of performing cellmediated immune functions. The thymus is derived from the epithelium which delineates the pharyngeal pouches in different vertebrate groups (29). The epithelial thymus is soon invaded by blood-borne stem cells which differentiate into small lymphocytes before migrating to other lymphoid organs. Similarly, in reptiles, thymic buds arise from outgrowths of the dorsal branchial
155 p o u c h , while the p a r a t h y r o i d s are formed ventrally. The level of origin of thymic primordia varies in reptiles according to s p e c i e s . The t h y m u s of lizards, snakes and turtles develops from d i f f e r e n t pairs of five p h a r y n g e a l pouches. In lizards, anterior and posterior thymic lobes (e.g., Anguis fragilis, Chalcides ocellatus, Lacerta viridis and Lacerta agilus) originate as dorsal outgrowths of the 2nd and 3rd pharyngeal pouches [reviewed in (23)]. In addition, a transient thymic rudiment which is associated with the first (2nd pharyngeal) pouch has been described in L. agilis and L. muralis (30). In contrast, thymic primordia originate from pharyngeal pouches 4 and 5 in snakes, and 3 in turtles (28,29). In Chalcides, histologic, cytologic, and organ culture assays have revealed that prothymocytes arrive in the thymic primordium at stage 35 (15) when hemoblasts also appear first in the thymic rudiment of Calotes (14). Lizards possess two pairs of thymic lobes, one on each side of the neck. Each pair consists of an anterior and posterior lobe situated in close proximity. At stage 36, the number of lizard thymocytes in each lobe varies from 7.5-11.0 × 104. Cell numbers then increase exponentially with time. Lymphoid cell proliferation in the thymus is accompanied by a transition from large to medium and then small lymphocytes. This sequence is in complete accordance with that reported for Agamid lizards (14), fish (16), toads (31), chickens (32), mice and humans (33,34).
Other Lymphoid Organs The spleen begins to develop at stage 31 as a mesenchymal thickening in the dorsal mesogastrium and attains a basic tissue architecture such as red and white pulps at stage 38 (15). The liver in Calotes seems to function also as a secondary lymphoid organ as evidenced by
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the a c c u m u l a t i o n of lymphoid cells (14,19). The importance of the liver in lymphopoiesis will be discussed later. Lymphoid accumulations in the gut and c l o a c a a p p e a r 2 to 6 months after hatching in lizards, snakes and turtles (15,19,35). Observations in reptiles, thus, support the suggestion that the thymus is the first lymphoid organ to develop during vertebrate ontogenesis (36). The origin of prethymic cells is not well established in reptiles.
Ontogeny of T-
and BLymphocyte Subpopulation(s)
Cells of the i m m u n e s y s t e m go through a series of crucial developmental stages before acquiring definitive functional properties. Successive stages in the m o r p h o g e n e s i s of l y m p h o i d organs include interactions of cells of different origins to form progenitors which then migrate to and are processed in appropriate organs, through unknown microenvironmental influences. Because little has been known concerning ontogenesis of cell surface antigens in reptiles, we recently completed an investigation of lymphocyte differentiation antigens and the development of specific cell types in the thymus and spleen of the lizard Chalcides ocellatus (37,38). Marked changes occur in the expression of surface antigens on lizard lymphocytes during emigration from the thymus to the spleen. That seeding occurred was concluded from observations that a n t i - a d u l t t h y m o c y t e s e r u m (AATS), a Chalcides T-cell reagent (21,37), recognized two antigenic entities, one common to intra- and extrathymic T cells and the other present exclusively on intrathymic lymphocytes called TA and Tt, respectively. This suggests that adult thymocytes are TA +, Tt + , whereas splenic T lymphocytes are only TA +. Using AATS and AETS (a second reagent development against
S.O. El Deeb and A-H. Saad
thymocytes of embryos at stages 40 and 41), embryonic thymocytes, like their adult counterparts, carry the TA and Tt differentiation antigens. In addition, our data obtained after absorption of AETS on adult thymocytes (i.e., after totally removing the anti-TA and anti-Tt antibodies), suggest the presence of a third a n t i g e n i c m a r k e r , n a m e l y the TE marker, which is expressed abundantly on embryonic thymocytes and splenocytes, but found on only a minute percentage of adult thymocytes (37). Presence of a third marker suggests that embryonic thymocytes are TA +, Tt + and TE +. Thus, embryonic l y m p h o c y t e s bear a specific membrane determinant not found in adults. Data using AATS revealed that T-cell differentiation antigen bearing lymphocytes appeared as early as stage 37 in the thymus and 39 in the spleen and increased gradually approaching adult values by stage 40 (37). By comparison, the development of T lymphocytes in Xenopus has been recently analyzed using a mouse monoclonal antibody, XT-I, which was produced against surface determinants on thymocytes of J strain toads (39). Subsequent ontogenic analyses reveal that XT-1 + cells were first detected in the thymus of J strain Xenopus (by Nieuwkoop and Faber stage 48, 7 days postfertilization) and then in the spleen, liver and kidney by stage 52 (20 days post-fertilization). XT-1 + cells reached adult levels by stage 52, and those in the spleen, liver, and kidney by stage 56 (40 days post-fertilization). The anti-lizard Ig serum (AGGS) labelled no thymocytes harvested from adult or embryonic lizards (37). AGGS revealed the simultaneous presence of surface and cytoplasmic Ig in a subset of splenic lymphocytes. These data are in accord with those recorded for embryonic birds and fish (10,40), but differ from those in human, mouse, and frogs where the appearance of cytoplasmic Ig precedes surface Ig (34). In contrast to
Ontogeny of reptilian immune system
the avian condition (41), the site for Blymphocyte differentiation in reptiles has been less well defined. Studies using the expression of Ig as marker showed that in Calotes, the first B-cells (defined by the presence of surface Ig) are observed at stage 34 in the liver, increase in this organ during stage 36-38 and they subsequently decrease at stage 39-42 in the liver and circulating blood (42). Thus, in analogy with the mammalian system, the embryonic liver in lizards is the "bursal-equivalent" in the sense that it is the most likely organ where B-cell differentiation occurs. In a second comparison, analyses of B-cell ontogeny have been initiated in the anuran amphibians Rana pipiens and Xenopus laevis. In Rana, embryonic urogenital tissues appear to be the earliest site of B-cell generation and B-cells have been identified in embryonic liver at a later d e v e l o p m e n t a l stage (43). In Xenopus, cytoplasmic Ig ÷ surface Igpre-B cells are fist detected in the larval liver (at Nieuwkoop and Faber stage 46), and later in the spleen (at Nieuwkoop and Faber stage 49) (44,45).
Ontogenic Emergence of Cellular Immune Responses Recently, we described for the first time, the emergence of T-cell immune capability in Chalcides throughout embryonic development (stages 36-41) and in newborns (46). The proliferative response of Chalcides thymocytes to Con A is evident as soon as the thymus becomes lymphoid (stage 36), increasing gradually during the successive stages of development to the low level of newly born individuals. Thus, the onset of thymic Con A responsiveness in embryonic lizards occurs at the same time as the overall number of thymocytes progressively increases. These findings correlate well with our previous histological studies on thymic ontogeny in Chalcides
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(15). The fact that newborn lizard thymocytes are low responders to Con A suggests that the responses by Chalcides are dependent on the maturation stages and/or the proportion of Con A-reactive subsets of cultured cells. The difference in Con A responses observed in our lizards might be related to the waves of responsive cells as they enter and multiply within the thymus, events which characterize the ontogeny of reptilian thymus [reviewed in (23)]. If so, the embryonic reptilian thymus may provide a model to study the early events of thymocyte education and differentiation during a broader time frame than is possible in other vertebrates. In many vertebrates, birth and/or hatching are often coincident with full immunological maturation. In reptiles there is general agreement that development of the immune system progresses slowly. According to one in vitro study, maturation of the snapping turtle's immune system occurs several months after hatching (47), so that complete immunological competence may develop quite late (from 4 - 6 months old) (48,49). For example, 4-month-old turtles were unaffected by allogeneic spleen injections, whereas 69% of 1-month-old animals died of graft-versus-host (GVH) disease after undergoing the identical treatment. In studying the immunological competence of newborn Chalcides (46,50), despite having a well-organized thymus and spleen even at birth, no serum antibodies are detectable one month following i.p. injection of 0-, 15and 30-day-old lizards with rat erythrocytes. In addition, newborns readily manifest signs of acute and chronic GVH reactions after administration of spleen cells derived from adult allogeneic donors (50). These results thus indicate that lizards are generally immuno-incompetent at birth. These results vary from those recorded in Calotes, which is able within 24 hours after hatching to mount an immune reaction
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against SRBC in a manner not readily distinguished from adult animals. Furthermore, Calotes hatchlings reject allografts and resist GVH reactions which have been induced by injecting adult cells (51).
Final Remarks Although information is accumulating, the number of species studied is scant, and some fundamental questions
remain to be answered using additional reptilian species. These include: First, whether dual embryonic stem cell compartments exist and what are the migratory patterns of lymphopoietic and hemopoietic stem cell lineages, as well as the mechanism(s) to explain these developmental pathways? Second, what are the embryonic origins of phagocytic leucocytes such as monocytes, macrophages and granulocytes? Availability of newer markers for subpopulations of cells of the reptilian immune system now permits an in-depth analysis.
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brane determinants in the lizard embryo. Dev. Comp. Immunol. 10:353-364; 1986. El Deeb, S.; Saad, A-H; Zapata, A. G. Detection of Thy-1 + cells in the developing thymus of the lizard, Chalcides ocellatus. Thymus 12:3-9; 1988. Nagata, S. Development of T lymphocytes in Xenopus laevis: Appearance of the antigen recognized by an anti-thymocyte mouse monoclonal antibody. Dev. Biol. 114:389-394; 1986. Grossi, C. E.; Lydard, P. M.; Cooper, M. D. Changing patterns of cytoplasmic IgM expression and of modulation requirements of surface IgM by anti-M antibodies. J. Immunol. 119:749-755; 1977. Le Douarin, N. M.; Dieterlen-Lievre, E ; Oliver, P. D. Ontogeny of primary lymphoid organs and lymphoid stem cells. Am. J. Anat. 170:261-299; 1981. Natarajan, K.; Muthukkaruppan, V. R. Distribution and ontogeny of B cells in the garden lizard, Calotes versicolor. Dev. Comp. Immunol. 9:301-310; 1985. Zettergren, L. D. Ontogeny and distribution of cells in B lineage in the American leopard frog, (Rana pipiens). Dev. Comp. Immunol. 6:311320; 1982. Hadji-Azimi, I.; Schwager, J.; Thiebaud, C. B lymphocyte differentiation in Xenopus laevis larvae. Dev. Biol. 90:253-258; 1982. Tochinai, S.; Nagata, S. Distribution of thymus-homing lymphoid precursor cells in larval and adult Xenopus hemopoietic organs. Dev. Growth Differ. 27:509-517; 1985. El Deeb, S.; Saad, A-H. Ontogeny of Con A responsiveness and mixed leucocyte reactivity in the lizard, Chalcides ocellatus. Dev. Comp. Immunol. 11:595-604; 1987. Sidky, Y. A.; Auerbach, R. Tissue culture analysis of immunological capacity of snapping turtle, Chelydra serpentina. J. Exp. Zool. 170:359-364; 1969. Borysenko, M. The maturation of the capacity to reject skin allografts and xenografts in the snapping turtle, Chelydra serpentina. J. Exp. Zool. 170:359-364;1969. Borysenko, M.; Tulipan, P. The graft-versushost reaction in the snapping turtle, Chelydra serpentina. Transplantation 16:496-504; 1973. Saad, A-H.; El Ridi, R. Mixed leucocyte reaction, graft-versus-host reaction, and skin allograft rejection in the lizard, Chalcides ocellatus. Immunobiology 166:484-493; 1984. Kanakambika, P.; Muthukkaruppan, V. R. Immunological competence in the newly hatched lizard, Calotes versicolor. Proc. Soc. Exp. Biol. Med. 140:21-23; 1972,
0145-305X/90 $3.00 + .00 Copyright © 1990 Pergamon Press plc
ONTOGENIC MATURATION OF THE IMMUNE SYSTEM IN REPTILES Somaya Osman El Deeb and Abdel Hakim Mohamed Saad Zoology Department, Facultyof Science, Cairo University,Cairo, Egypt
(Submitted January 1989;Accepted July 1989)
[]Keywords--Reptiles; Ontogeny; Lymphoid organs; T- and B-iymphocytes; Immune responses.
Introduction There has been increased emphasis in recent years on the phylogenetic and ontogenetic aspects of immunity. In contrast to the wealth of information which has been obtained from studying immune reactions in mammals and birds, comparatively little data are available on the structure of lymphoid organs and function of lymphocyte populations in ectothermic vertebrates (fishes, amphibians, and reptiles). There are at least three reasons why reptiles, with a complex "mammal-like" immune system, provide an important research tool for the comparative immunologist interested in the ontogeny of immunity. First, reptiles hold a unique position in the evolut i o n a r y l i n e a g e to b o t h b i r d s and mammals (1). Second, reptiles are the first category of vertebrates to exhibit the change from free-living larvae, as found in fishes and amphibians, to an embryo completely protected by an amnion similar to avian and mammalian embryos (2). Third, reptiles have a prolonged maturation period, making them good candidates for investigating the maturation of immune capacity (3). This brief review focuses on the ontogenetic development of the reptilian imAddress correspondence to Dr. A-H. Saad, Zoology Department, Faculty of Science, Cairo University, Cairo, Egypt.
mune system. We shall review some published data from our laboratory and others showing that a degree of immunological maturation is detectable during reptilian embryonic life. In particular, the lizards Chalcides ocellatus (Scincidae) and Calotes versicolor (Agamidae) have provided a refined experimental model for studying some fundamental aspects concerning ontogeny of reptilian immunity. We hope that this will give the reader some insight into certain evolutionary trends in immunology and lead to a phylogenetic perspective that strengthens this rapidly growing area of research.
Embryonic Development of Lizards Growth and development of reptilian embryos have been studied carefully, and arbitary stages have been established that allow investigators to compare different species or even different populations of the same species. Morphological descriptions of embryonic stages of lizards have dealt principally with representatives of the families Lacertidae, Chamaleonidae, Iguanidae and Scincidae (4). A brief account will be given first on the embryonic development in Calotes versicolor (5) and Chalcides ocellatus (6) which are pertinent to the following discussion. In Calotes, the earliest embryo from normally laid eggs is stage 27. The initial stages (27 to 33) of d e v e l o p m e n t are d i s t i n g u i s h e d by changes in somites, gill clefts, eye and limb-buds. For classifying embryos of 151
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stage 34 to 40, limbs provide the most convenient diagnostic criteria. During the prehatching period (stages 38 to 42), scales and pigmentation on the head and other parts of the embryo are used as distinguishing characters. The interval between two successive stages is approximately 2-3 days during the earlier and 3 - 4 days during the later periods of development (cf. (5)]. The series of developmental stages as reported for Calotes is adopted for classifying Chalcides embryos (6) and is summarized in Table 1.
Embryonic Origin and Migratory Pattern of Precursor Cells Yolk Sac
The developmental pathways of lymphoid cells in embryonic life have been analyzed since the beginning of the century. Several theories have been presented to explain the appearance of lymphocytes in lymphoid organ rudiments (7-12). Despite controversy concerning the source(s) of primary stem cells, there has been a universal agreement that development of lymphoid organs is completely dependent on an inflow of extrinsic stem cells into the epithelial or mesenchymal rudiments. Whatever the origin of the lymphoid stem cells, other questions concerning their differentiation and the nature of the yolk sac in ectotherms remain unanswered. Jordan, in his pioneering studies (13) on hemopoiesis in snapping and box turtles, described a "hemoblast" as an amoeboid cell with a vesicular nucleus, one or two nucleoli, and an envelope of strongly basophilic cytoplasm. This cell was first observed in blood islands of the yolk sac, the earliest hemopoietic tissue in chelonians. Later; these hemoblasts are also found in other foci of hemopoiesis. In this regard, it is incorrect to equate stem cells with " h e m o b l a s t s " which
S.O. El Deeb and A-H. Saad
may or may not be the same cell. Certain labelling studies are necessary in order to demonstrate that they are, indeed, hemopoietic stem cells. Based on observations revealing sequential appearance of hemoblasts in yolk sac blood islands, circulation, mesenchyma and then the thymic primodium, followed by lymphopoiesis in the thymus, Pitchappan and Muthukkaruppan (14) suggested that lymphocyte progenitors in the lizard Calotes versicolor originate from blood-borne stem cells. In accordance, our recent studies on the ontogeny of hemopoietic cell lineages in the lizard Chalcides ocellatus support this hypothesis. In Chalcides, large undifferentiated blasts, characterized by high nucleo-cytoplasmic ratio were first seen in yolk sac at stage 21. These cells were intermingled with primitive erythroblasts and constituted about 57.1% of the total differential count. Gradually, the number of erythroblasts increased together with morphological changes which occur which lead to the appearance of mature forms. Thus, as the embyro ages, mature forms of erythroblasts increase in number accompanied by a gradual disappearance of early erythroid cells. By stage 31, the mature forms of erythrocytes are first detected. It was rather surprising that also the number of undifferentiated blasts increased considerably. In addition, other t y p e s of blood cells were found in varying ratios at different stages, e.g., macrophages, megakaryocytes, myeloid cells, and unidentified blasts (15). By morphological criteria, therefore, the lizard's yolk sac is a hemopoietic site. Since no other hemopoietic organ has been observed to the yolk sacs appearance, it may be coosidered as the first organ which develops hemopoietic cells during ontogeny. These results are in accordance with those observed in case of avian yolk sac hemopoiesis which is more extensive and includes the production of various cell types
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Table 1. Developmental stages of Chalcides ocellatus embryos Important Changes in Development of the Immune System
Stage
Definitive External Characteristics
21-26
The first and second branchial clefts are open, narrow slits. The prospective force- and hind-area are demarcated by mesodermal condensation. The first four branchial clefts appear as open slits. The tail-bud is elongated and curved with unsegmented mesoderm. Fore- and hind-limbs are distinct. Tail elongates but is still unsegmented. Second, third and fourth branchial clefts sunk beneath the surface. Pigmentation begins in the retina. Mandibular process increases in size. The distal part of the limb is broadened (paddle-like) without any demarcation of digits; elbow- and knee-joints are distinct. Beginning of formation of eye-lids. Branchial clefts are no longer visible. Fore- and hind-limbs considerably longer. Digital plate exhibits marked intergrooves delineating the digits. Tongue formation is definite. Digital plate is deeply notched as phalarges elongate. All the five digits are fully demarcated and connected by webs which have curved contours.
27
30-31
32
33-34
35 36
37 38 39
40
41
Digits are elongated and their tips are pointed. Webs between the digits are thin and deeply notched. The tympanum is formed. Digits are elongated and thick and appear annulated. Scale primordia are evident. The inner margin of the eye-lid is oval. Very light pigmentation is present on the snout and parietal region. Pigmented scales appear on the tail and dorsal side of the trunk. Claws make their appearance at the extremity of the digits. Scales on the tail, dorso-lateral region of the trunk, and limbs are well pigmented. Slightly pigmented scales begin to appear on the snout and frontal region. The embryo is fully pigmented with a specific pattern. The parietal region is distinctly compressed. Stripes of pigmentation appear on the dorsal side of the head. The embryo at this stage resembles a newborn.
(8,9). On the other hand, these observations are at variance with those described in the mammalian system. In the case of mammals, yolk sac hemopoiesis
Yolk sac exhibit prominent erythropoietic activity. Yolk sac appears to be involved in producing myeloid cells and megakaryocytes together with erythroid cells. Appearance of thymic primordia.
Budding of splenic rudiments.
Detachment of thymic rudiments from the pharyngeal pouches.
Appearance of basophilic cells in the thymus. Onset of haemopoiesis in the spleen. Onset of lymphopoiesis in the thymus. Emergence of Con A responses and MLR reactivity. Spleen is full of erythroblasts and granulocytes. Detection of TA + and Thy-1 ÷ cells in the thymus. Lymphopoiesis begins in the spleen. Detection of surface Ig* and TA + cells in the spleen.
Bone marrow haemopoiesis appears.
Demarcation of thymic cortex and medullary areas as well as splenic white and red pulps. Impaired Con A responses and MLR reactivity.
is limited to the production of a primitive generation of nucleated erythrocytes (7). No sufficient data are available about yolk sac hemopoiesis in fish (16). The
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yolk sac equivalent (ventral blood island) gives rise to erythrocytes, thymocytes and B-lymphocytes in Xenopus laevis (17,18).
Spleen There is a lack of detailed information concerning ontogeny of the spleen in various groups of reptiles. Previous studies available on development of the spleen in snakes and lizards are restricted to the origin and pattern of vascularization of splenic rudiments (19). Kanakambika and Muthukkaruppan analyzed splenic development in Calotes, where the primordium appears at stage 30 as a protuberance of mesenchymal cells from the dorsal mesentary, detected in trunk region serial sections stained routinely with hematoxylin and eosin. In subsequent stages, there is further enlargement of the rudiment due to increase in numbers of mesenchymal cells and blood spaces. At stage 35, the spleen is primarily erythroid in nature. A considerable number of primitive granulocytes are present at stage 36 and mature granulocytes appear in the spleen at stage 38. The initiation of lymphopoiesis occurs at stage 40 (14). Comparable results have also been obtained for Chalcides (15).
Liver and Kidney In Chalcides, no hemopoietic activity is detected at any developmental stage in the liver (in contrast to that found in Calotes, to be discussed later). This absence is similar to birds, but it is in sharp contrast to mammals where hemopoiesis in the liver is extensive and is responsible for the second generation of erythrocytes and of lymphocytes (7-12). Liver hemopoiesis has been demonstrated by Turpen and his colleagues (20) in anuran amphibians revealing the pres-
S.O. El Deeb and A-H. Saad
ence of lymphocytes, plasmatocytes and macrophages at different stages of maturation. If the comparison is valid, no such activity has been recorded in fish. Similarly, no kidney hemopoiesis is detected in lizard embryos at any developmental stages nor in adult Chalcides (15). However, further studies in other reptilian species are warranted before drawing firm conclusions about the existence and/or absence, of liver and kidney hemopoiesis in reptiles.
Bone Marrow Reptilian bone marrow has been studied morphologically in the adult lizards (21,22); it contains mature and transitional forms of various hemopoietic cellular lineages. In this respect, it tends to satisfy the criteria of organbound hemopoiesis as has been described previously for adult marrow of m a m m a l s , b i r d s , and a n u r a n amphibians. No ontogenic study of marrow is yet available, although reptiles are a linking group between lower vertebrates and mammals. Bone marrow in Chalcides becomes hemopoietic at a very late developmental stage (40-41, i.e., just before birth). A few granulocytes and undifferentiated blasts are detected in the bone marrow at stage 41. In considering the evolution of bone marrow, apparently reptiles are the first group of vertebrates in which bone marrow hemopoiesis takes place during embryonic life. From an evolutionary viewpoint, lack of a hemopoietic bone marrow has been repeatedly observed in fish during both embryonic and adult stages (16). Bone marrow without hemopoietic activity has been observed among Urodeles (23,24); it contains mainly fat and fibroblasts. The phylogenetic emergence of a true lymphopoietic, myelopoietic, and erythopoietic bone marrow was first observed in adult anurans (25,26), but
Ontogeny of reptilian immune system
never encountered during premetamorphic stages (27,28). In an attempt to discuss the relationship between the phylogenetic emergence of bone marrow as a source of hemopoietic cells and the evolution of the terrestial way of life, Cooper et al. (26) suggested that hemopoietic stem cells are protected from irradiation by bones in terrestrial vertebrates, which have substituted for water as a radio-opaque shield. This suggestion would, thus, be most meaningful if Class Reptilia is considered, since it is known to include some species that are the first vertebrate group to become completely free from the aquatic environment. Our study on the ontogeny of hemopoietic tissues in Chalcides leads us to conclude that the first and major hemopoietic organ in the embryo is the yolk sac (15). Later in development, hemopoiesis in the yolk sac is succeeded by blood cell formation in the fetal spleen and fetal bone marrow. Finally, bone marrow becomes the major hemopoietic organ of adult lizards, although the potentiality for hemopoiesis is retained, to some extent, by the spleen (15).
Early Development of Lymphoid Organs
Thymus In jawed vertebrates there is a separate microenvironment, the thymus, which provides for the differentiation of lymphocytes capable of performing cellmediated immune functions. The thymus is derived from the epithelium which delineates the pharyngeal pouches in different vertebrate groups (29). The epithelial thymus is soon invaded by blood-borne stem cells which differentiate into small lymphocytes before migrating to other lymphoid organs. Similarly, in reptiles, thymic buds arise from outgrowths of the dorsal branchial
155 p o u c h , while the p a r a t h y r o i d s are formed ventrally. The level of origin of thymic primordia varies in reptiles according to s p e c i e s . The t h y m u s of lizards, snakes and turtles develops from d i f f e r e n t pairs of five p h a r y n g e a l pouches. In lizards, anterior and posterior thymic lobes (e.g., Anguis fragilis, Chalcides ocellatus, Lacerta viridis and Lacerta agilus) originate as dorsal outgrowths of the 2nd and 3rd pharyngeal pouches [reviewed in (23)]. In addition, a transient thymic rudiment which is associated with the first (2nd pharyngeal) pouch has been described in L. agilis and L. muralis (30). In contrast, thymic primordia originate from pharyngeal pouches 4 and 5 in snakes, and 3 in turtles (28,29). In Chalcides, histologic, cytologic, and organ culture assays have revealed that prothymocytes arrive in the thymic primordium at stage 35 (15) when hemoblasts also appear first in the thymic rudiment of Calotes (14). Lizards possess two pairs of thymic lobes, one on each side of the neck. Each pair consists of an anterior and posterior lobe situated in close proximity. At stage 36, the number of lizard thymocytes in each lobe varies from 7.5-11.0 × 104. Cell numbers then increase exponentially with time. Lymphoid cell proliferation in the thymus is accompanied by a transition from large to medium and then small lymphocytes. This sequence is in complete accordance with that reported for Agamid lizards (14), fish (16), toads (31), chickens (32), mice and humans (33,34).
Other Lymphoid Organs The spleen begins to develop at stage 31 as a mesenchymal thickening in the dorsal mesogastrium and attains a basic tissue architecture such as red and white pulps at stage 38 (15). The liver in Calotes seems to function also as a secondary lymphoid organ as evidenced by
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the a c c u m u l a t i o n of lymphoid cells (14,19). The importance of the liver in lymphopoiesis will be discussed later. Lymphoid accumulations in the gut and c l o a c a a p p e a r 2 to 6 months after hatching in lizards, snakes and turtles (15,19,35). Observations in reptiles, thus, support the suggestion that the thymus is the first lymphoid organ to develop during vertebrate ontogenesis (36). The origin of prethymic cells is not well established in reptiles.
Ontogeny of T-
and BLymphocyte Subpopulation(s)
Cells of the i m m u n e s y s t e m go through a series of crucial developmental stages before acquiring definitive functional properties. Successive stages in the m o r p h o g e n e s i s of l y m p h o i d organs include interactions of cells of different origins to form progenitors which then migrate to and are processed in appropriate organs, through unknown microenvironmental influences. Because little has been known concerning ontogenesis of cell surface antigens in reptiles, we recently completed an investigation of lymphocyte differentiation antigens and the development of specific cell types in the thymus and spleen of the lizard Chalcides ocellatus (37,38). Marked changes occur in the expression of surface antigens on lizard lymphocytes during emigration from the thymus to the spleen. That seeding occurred was concluded from observations that a n t i - a d u l t t h y m o c y t e s e r u m (AATS), a Chalcides T-cell reagent (21,37), recognized two antigenic entities, one common to intra- and extrathymic T cells and the other present exclusively on intrathymic lymphocytes called TA and Tt, respectively. This suggests that adult thymocytes are TA +, Tt + , whereas splenic T lymphocytes are only TA +. Using AATS and AETS (a second reagent development against
S.O. El Deeb and A-H. Saad
thymocytes of embryos at stages 40 and 41), embryonic thymocytes, like their adult counterparts, carry the TA and Tt differentiation antigens. In addition, our data obtained after absorption of AETS on adult thymocytes (i.e., after totally removing the anti-TA and anti-Tt antibodies), suggest the presence of a third a n t i g e n i c m a r k e r , n a m e l y the TE marker, which is expressed abundantly on embryonic thymocytes and splenocytes, but found on only a minute percentage of adult thymocytes (37). Presence of a third marker suggests that embryonic thymocytes are TA +, Tt + and TE +. Thus, embryonic l y m p h o c y t e s bear a specific membrane determinant not found in adults. Data using AATS revealed that T-cell differentiation antigen bearing lymphocytes appeared as early as stage 37 in the thymus and 39 in the spleen and increased gradually approaching adult values by stage 40 (37). By comparison, the development of T lymphocytes in Xenopus has been recently analyzed using a mouse monoclonal antibody, XT-I, which was produced against surface determinants on thymocytes of J strain toads (39). Subsequent ontogenic analyses reveal that XT-1 + cells were first detected in the thymus of J strain Xenopus (by Nieuwkoop and Faber stage 48, 7 days postfertilization) and then in the spleen, liver and kidney by stage 52 (20 days post-fertilization). XT-1 + cells reached adult levels by stage 52, and those in the spleen, liver, and kidney by stage 56 (40 days post-fertilization). The anti-lizard Ig serum (AGGS) labelled no thymocytes harvested from adult or embryonic lizards (37). AGGS revealed the simultaneous presence of surface and cytoplasmic Ig in a subset of splenic lymphocytes. These data are in accord with those recorded for embryonic birds and fish (10,40), but differ from those in human, mouse, and frogs where the appearance of cytoplasmic Ig precedes surface Ig (34). In contrast to
Ontogeny of reptilian immune system
the avian condition (41), the site for Blymphocyte differentiation in reptiles has been less well defined. Studies using the expression of Ig as marker showed that in Calotes, the first B-cells (defined by the presence of surface Ig) are observed at stage 34 in the liver, increase in this organ during stage 36-38 and they subsequently decrease at stage 39-42 in the liver and circulating blood (42). Thus, in analogy with the mammalian system, the embryonic liver in lizards is the "bursal-equivalent" in the sense that it is the most likely organ where B-cell differentiation occurs. In a second comparison, analyses of B-cell ontogeny have been initiated in the anuran amphibians Rana pipiens and Xenopus laevis. In Rana, embryonic urogenital tissues appear to be the earliest site of B-cell generation and B-cells have been identified in embryonic liver at a later d e v e l o p m e n t a l stage (43). In Xenopus, cytoplasmic Ig ÷ surface Igpre-B cells are fist detected in the larval liver (at Nieuwkoop and Faber stage 46), and later in the spleen (at Nieuwkoop and Faber stage 49) (44,45).
Ontogenic Emergence of Cellular Immune Responses Recently, we described for the first time, the emergence of T-cell immune capability in Chalcides throughout embryonic development (stages 36-41) and in newborns (46). The proliferative response of Chalcides thymocytes to Con A is evident as soon as the thymus becomes lymphoid (stage 36), increasing gradually during the successive stages of development to the low level of newly born individuals. Thus, the onset of thymic Con A responsiveness in embryonic lizards occurs at the same time as the overall number of thymocytes progressively increases. These findings correlate well with our previous histological studies on thymic ontogeny in Chalcides
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(15). The fact that newborn lizard thymocytes are low responders to Con A suggests that the responses by Chalcides are dependent on the maturation stages and/or the proportion of Con A-reactive subsets of cultured cells. The difference in Con A responses observed in our lizards might be related to the waves of responsive cells as they enter and multiply within the thymus, events which characterize the ontogeny of reptilian thymus [reviewed in (23)]. If so, the embryonic reptilian thymus may provide a model to study the early events of thymocyte education and differentiation during a broader time frame than is possible in other vertebrates. In many vertebrates, birth and/or hatching are often coincident with full immunological maturation. In reptiles there is general agreement that development of the immune system progresses slowly. According to one in vitro study, maturation of the snapping turtle's immune system occurs several months after hatching (47), so that complete immunological competence may develop quite late (from 4 - 6 months old) (48,49). For example, 4-month-old turtles were unaffected by allogeneic spleen injections, whereas 69% of 1-month-old animals died of graft-versus-host (GVH) disease after undergoing the identical treatment. In studying the immunological competence of newborn Chalcides (46,50), despite having a well-organized thymus and spleen even at birth, no serum antibodies are detectable one month following i.p. injection of 0-, 15and 30-day-old lizards with rat erythrocytes. In addition, newborns readily manifest signs of acute and chronic GVH reactions after administration of spleen cells derived from adult allogeneic donors (50). These results thus indicate that lizards are generally immuno-incompetent at birth. These results vary from those recorded in Calotes, which is able within 24 hours after hatching to mount an immune reaction
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against SRBC in a manner not readily distinguished from adult animals. Furthermore, Calotes hatchlings reject allografts and resist GVH reactions which have been induced by injecting adult cells (51).
Final Remarks Although information is accumulating, the number of species studied is scant, and some fundamental questions
remain to be answered using additional reptilian species. These include: First, whether dual embryonic stem cell compartments exist and what are the migratory patterns of lymphopoietic and hemopoietic stem cell lineages, as well as the mechanism(s) to explain these developmental pathways? Second, what are the embryonic origins of phagocytic leucocytes such as monocytes, macrophages and granulocytes? Availability of newer markers for subpopulations of cells of the reptilian immune system now permits an in-depth analysis.
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