EVOLUTION OF THE INNATE AND ADAPTIVE IMMUNE SYSTEMS:... : Transplantation (original) (raw)
Since 1907, when Wilson (1) described the species-specific reaggregation of cells from marine sponges (Porifera), they have become a classical model system for the study of self/self- and self/non-self-recognition of metazoan cells. Almost simultaneously, the same phenomenon was described for freshwater sponges as well (2). The ability of sponges to fuse with autografts and to reject allografts was discovered by Paris (3) and Moscona (4). Based on the occurrence of an accelerated rate of rejection of second-set grafts, a primordial immune memory was postulated for the sponge Callyspongia diffusa(5). After the identification of the first cell-cell adhesion molecules in sponges, the intercellular aggregation factor (AF*) and the cell surface-associated aggregation receptor (AR) to which the AF binds, studies about molecules potentially involved in immune response became possible on cellular level (6). The AFs, have been found simultaneously in two marine sponges, Microciona prolifera(7, 8) and Geodia cydonium(9).
Porifera are the phylogenetically oldest extant metazoan phylum. Paleontological evidence suggests that unicellular eukaryotes evolved from prokaryotes approximately 2,000 million years ago (MYA) (10), while multicellular Metazoa have been traced back to 800-1,000 MYA (11). The taxon Geodiidae represents the oldest living group (550 MYA) within the Demospongiae. Hence, it can be deduced that the ancestor of the present-day _Geodia cydonium_-one of the model species used in our present studies- already lived before the onset of the "Cambrian explosion," the time of main divergence of metazoan phyla.
Immune systems are universally present in every kingdom of life. In Metazoa in particular, immunity is an integral part of the homeostatic system. The immune systems protect animals against attacking organisms and also hinder malformation during morphogenetic growth and aging; in addition, they prevent parasitism and fusion with genetically unrelated organisms that would result in chimerism and loss of individuality (reviewed in 12). The extent of commonality and evolutionary origin of the metazoan immune system remained poorly understood until recently (reviewed in 13). This status changed after the first nucleotide (nt) sequences, coding for immune and immune-related molecules, were isolated from sponges. The rationale to screen for those molecules in sponges is based upon the recent conclusion that all metazoan animals are of monophyletic origin (14-16). Studies concentrating on those molecules that are considered to be characteristic for metazoan cells revealed that protein sequences, deduced from cDNAs obtained from the lowest metazoan phylum, the Porifera (sponges), are highly similar to those found in other metazoan phyla (reviewed in 17). These molecules include primarily G-protein-linked transmembrane receptors and transmembrane adhesion molecules, which were isolated mainly from the marine species G. cydonium and S. domuncula. In addition, the cell culture system became recently available from sponges (18, 19), and has successfully been applied for functional studies of adhesion molecules (20).
This review summarizes recent progress in the cloning of molecules, potentially involved in self/self- and self/non-self-recognition from sponges, and discusses their relationship to similar molecules of the defense/immune systems in mammals. Two types of immunity are distinguished: the natural (innate) pattern and the adaptive system (21). Recent studies on sponges suggest that invertebrates too are capable of both types of immunity.
AUTOGRAFT FUSION AND ALLOGRAFT REJECTION IN SPONGES
Sponges possess remarkable regeneration capabilities (1). The capacity of regeneration in combination with inflammatory responses to injury (22) is an essential component of their ability to survive. In addition, sponges have developed mechanisms to distinguish between self and non-self. Little is known about natural challenges to self-integrity in sponges (23, 24); most of it is available from experimental transplantation studies. Smith and Hildemann in their extensive review (25) have grouped sponge alloimmune responses in experimental transplantations into two major rejection processes. Some species may form barriers to separate from non-self tissue, e.g., the marine sponge Axinella verrucosa(26) or the freshwater sponge Ephydatia muelleri(27), while others may react by cytotoxic factors that destroy the transplant, e.g., the marine sponges Callyspongia diffusa(5) or G. cydonium(28).
Progress in understanding allorecognition in sponges has been made on the cellular level. In grafted branches from two individuals of M. prolifera, a specific cell type, the gray cells, accumulate in the contact areas (29). Furthermore, some combinations of dissociated cells originating from unrelated sponges react in vitro by immediate cytotoxic reactions (30). Based upon these data, the authors suggested that the gray cells may be the functional immunocytes of sponges.
In our approach, we searched for putative structures of invertebrate immune systems by cloning genes in sponges. Those gathered to date display surprisingly high sequence homologies to the receptors involved in self/non-self-recognition in vertebrates. The marine sponges G. cydonium(Fig. 1A) and S. domuncula(Fig. 1I) served as model systems (6, 31).
Tissue and cell recognition in sponges. (A-D) Recognition of autografts and allografts of the sponge G. cydonium using the parabiotic attachment technique. (A) One animal of 45 cm in diameter in a depth of 25 m on muddy sand bottom (original magnification, ×0.05). (B) Autograft after a 2 days transplantation period (original magnification, ×0.3). Fusion of an autograft (C) and formation of a boundary zone in one allograft at the recognition zone (D); (original magnification for both, ×0.5). The fusion zone (fz) and rejection zone (rz) are marked. (E-H) Application of the insertion technique for grafting experiments with G. cydonium. (E) Tissue pieces were removed with a cork drill from one specimen and inserted into holes in the recipients, which had a slightly narrower diameter. (F) Autograft fused after 5 days. Allografts initially fused together but underwent necrotic degeneration after 3 days (G), a process that ultimately resulted in resorption (H); (original magnification, ×0.5). (I-L) Autograft fusion and allograft rejection of transplants from S. domuncula. (I) Two specimens in red and blue are shown (original magnification, ×0.5). (J) Two grafts are tied together with a fiber (original magnification, ×1). (K) Autograft 4 days (cross-section) after transplantation (original magnification, ×1). (L) 4-day-old allograft (cross-section) from a red and a whitish specimen, which reject each other (original magnification, ×1). (M-P) Cell recognition in sponges. Formation of primmorphs from cells of S. domuncula. (M) Dissociated single cell suspension and small cell clumps (original magnification, ×100); (N) primmorph (original magnification, ×10); (O) immunocytochemical detection of proliferating (BrdU-labeled) cells from primmorphs. After incubation of the primmorphs with BrdU, the cells were dissociated and subjected to staining with anti-BrdU monoclonal antibody. The dark brownish stained nuclei are those that have incorporated BrdU. The arrow marks a BrdU-positive cell; (original magnification, ×200). (P) Species-specific reaggregation. Cells from Thoosa istriaca (lilac-purple) and Thoosa mollis (white-yellow) were mixed, and the two AFs from both species were added. After 5 hr, the species-specific reaggregates are formed (original magnification, ×10).
Using the parabiotic attachment technique, slices of approximately 2-5 cm thickness were cut from the surface of animals (Fig. 1, A and I). Size-matching pieces were attached to each other and loosely fixed by rubber bands (studies with G. cydonium;Fig. 1B) or with nylon fibers (experiment with S. domuncula;Fig. 1J). One day later, the rubber bands/fibers could be removed, as the grafts/parabionts already adhered to each other.
Applying the insertion technique (with G. cydonium), tissue pieces removed with a cork drill from one specimen (diameter of 1 cm; approximate length of 4 cm) were inserted into holes in the recipients, which had a slightly narrower diameter (0.9 cm) as described (32; Fig. 1E). All autografts fused, and no boundary line was seen finally (Fig. 1F). Allografts initially fused together (Fig. 1G); after approximately 2-3 days, the rejected graft tissue formed a pronounced demarcation boundary and underwent necrotic degeneration (Fig. 1, G and H) and finally resorption (Fig. 1H; 32).
S. domuncula occurs in nature in red, orange, whitish, blue, or as a mixture of shades (Fig. 1I). Autografts from this species started to fuse approximately 2 days after grafting; after 6 days, the zone where the two grafts attached was no longer visible, either by eye inspection (Fig. 1K) or by microscopical analysis after sectioning and staining. In contrast, allografts started to reject each other after an initial period of fusion (duration: 2-3 days); a gap became visible ≈4 days after grafting (Fig. 1L). A similar pattern was seen with grafts from G. cydonium. Tissue samples from 20-60-cm large animals (Fig. 1A) were removed; autografts or allografts were tied together (Fig. 1B). Again, the autografts fused after ≈2 days; no contact zone was seen after 6 days (Fig. 1C). Allografts fused transiently after 2-3 days but then formed a border between the two transplants (Fig. 1D). Histological analysis revealed that, in the contact zone of the autografts, an organized canal-like water-current system was formed. In contrast, in allografts, a demarcation zone became visible, composed of collagenous bundles (6).
CELL CULTURE AS A MODEL SYSTEM FOR THE STUDY OF IMMUNITY IN SPONGES
In a classical study, Moscona (4) demonstrated that the AF-mediated cell reaggregation is species-specific. Applying the same approach, we demonstrated that also cells from the two related species, Thoosa istriaca and Thoosa mollis, form-in the presence of their AFs-species-specific aggregates (33; Fig. 1P).
A further step to elucidate the immune system in sponges on a cellular level has been achieved recently. Culture conditions required for the formation of multicellular aggregates from S. domuncula starting from dissociated single cells (Fig. 1M) have been described (18, 19). These aggregates (Fig. 1M) termed primmorphs and showed a tissue-like appearance (Fig. 1N). Cross-sections through the primmorphs reveal an organized zonation into a distinct unicellular epithelial-like layer of pinacocytes and a central zone composed primarily of spherulous cells. Important is the finding that a major fraction of the cells in the primmorphs underwent DNA synthesis and hence had the capacity to divide (Fig. 10). Applying the BrdU (bromodeoxyuridine) labeling and detection system, it was demonstrated that up to 33.8% of the cells in the primmorphs were labeled with BrdU after an incubation period of 12 hr.
ELEMENTS OF THE INNATE IMMUNE SYSTEM IN SPONGES
The nonspecific, innate immune system in mammals is based on (i) phagocytosis, (ii) the complement system, and (iii) macrophage-derived cytokines. Related molecules have been identified in sponges.
Phagocytosis
In mammals, macrophages are the first cells that encounter non-self material, especially bacteria, and engulf and subsequently degrade them, using hydrolytic enzymes and oxidative attack. Macrophages express several receptors, scavenger receptors, which bind to bacteria or their constituents and hence act as key molecules in innate immunity. Among them is the type I macrophage scavenger receptor, which comprises highly conserved scavenger receptor cysteine-rich (SRCR) domains (reviewed in 34). The SRCR domain consists of the approximately 100-aa (amino acid) motif with conserved spacings of six to eight cysteines, which apparently participate in intradomain disulfide bonds. SRCRs are subdivided according to the number of cysteines into two groups; group A contains six Cys, while most of those in group B possess eight Cys. Group A SRCRs are, as examples, present in the type I macrophage scavenger receptor, macrophage M130 antigen, and complement factor I. Group B SRCRs are found in WC1, M130, CD6, and CD5 antigens, which are likely to be involved in immunological reactions, e.g., WC1 in mixed lymphocyte reactions, CD6 in antigen-specific cytotoxicity, or CD5 in hyperresponsivity of T lymphocytes.
SRCR (group A) molecules. Until the isolation of the proteins from the sponge G. cydonium, only one molecule was known from invertebrates that contains SRCR domains, the speract (sperm egg peptide receptor) from the sea urchin (Strongylocentrotus purpuratus) (35). However, in molecules from G. cydonium, both groups of SRCRs are found. Group A SRCR domains are present in the putative AR (antibodies raised against the recombinant protein inhibit cell reaggregation; 36, 37). Amazing is the fact that this sponge molecule is present in at least three forms of alternatively spliced transcripts of 6.5, 4.9, and 3.9 kb. The largest form, SRCR-SCRm, is a cell-surface receptor of 220 kDa, the putative AR; the second form, SRCRm, is also a putative receptor of 166 kDa, while the third form, SRCRs, is a soluble molecule of 129 kDa (Fig. 2A). The SRCR-SCRm molecule consists of 14 SRCR domains, six short consensus repeats (SCR), a C-terminal transmembrane domain, and a cytoplasmic tail. The SRCR repeats of SRCR-SCRm show the typical pattern of group A SRCR: six Cys residues; the consensus pattern reads Cys2-X12-Cys3-X30-Cys5-X9-Cys6-X9-Cys7-X9-Cys8(34)(Fig. 2B).
Molecules featuring SRCR/SCR domains from the sponge G. cydonium. (A) Putative sponge AR from G. cydonium. Models of the three deduced proteins belonging to the SRCR and the SCR SRCR-SCR domain superfamilies (group A) are shown. The model illustrates (i) the SRCR-SCRm molecule (a membrane-bound form), which contains the 13 domains and the 6 SCR segments as well as the stalk-like Ser and Pro-rich segment; (ii) the SRCRm (a membrane-bound form) with 12 SRCR repeats, and the stalk segment, the C-terminal transmembrane domain, and the cytoplasmic tail; and (iii) SRCRs, which lacks the two C-terminal SRCR repeats and the transmembrane domain. (B) Alignment of one representative SRCR repeat from members of group A of the SRCR superfamily: GCSRCR R9 (sponge putative SRCR, 9th repeat); MARCO (murine macrophage bacteria-binding receptor; U18424); MRSE (bovine macrophage type I scavenger receptor; P21758); CyCAP (murine cyclophilin-C-binding protein; A48231); MAC2 (human MAC2-binding protein; A47161); SPERACT R2 (sea urchin sperm egg peptide receptor, second repeat; P16264); CFI (human complement factor I; P05156). Residues conserved among all seven sequences are shown in inverted type, while residues conserved in at least five of the sequences are shaded. The location of six Cys residues characteristic of group A is marked and numbered. (C) Putative "multi-adhesive protein" from G. cydonium. Three modules have been identified; the fibronectin (FN3), the SRCR (group B), and the SCR module. (i) The deduced aa sequences of FN3 modules displayed highest similarity to those found in deuterostomes and protostomes FN3. (ii) The SRCR scavenger module is characterized by high similarity to those modules found in polypeptides of lymphocytes and macrophages. (iii) The SCR module is highly related to modules in proteins from the human complement system. While fibronectin is known to interact with integrin, both the SRCR-rich polypeptides and the SCR-rich proteins are involved in immune reactions.
SRCR (group B) molecule. In addition, a cDNA was cloned (38) that encodes a putative "multi-adhesive protein," which comprises three modules: (i) a fibronectin module, (ii) a SRCR module, and (iii) a SCR module (Fig. 2C). The fibronectin module of the deduced sponge protein shows the characteristic topology and aa found in fibronectin type-III (FN3) elements (39). It is the first invertebrate molecule displaying the cysteine pattern for group B SRCR receptors, with eight C aa residues (C1-C8). A phylogenetic analysis revealed (38) that the sponge group B SRCR domain displays high similarity to the mammalian WC1 surface antigens, human CD6 antigen, human CD5 surface glycoprotein, as well as human M130 antigen.
Sponges have been shown to live, very likely, in symbiosis with bacteria (40); in addition to those, "non-self" bacteria are also ingested, which will certainly be killed by both an oxidative and a nonoxidative (enzymatic) mechanism. To date, several cDNAs coding for lysosomal enzymes, e.g., cathepsin, which is abundantly present in G. cydonium(41), have been isolated from sponge cDNAs.
Complement System: Prophenoloxidase Activating System
A further key system in vertebrates, active in innate immunity and inflammation, is the complement system. Proteins that are structurally related to vertebrate complement proteins have been identified in crustaceans, e.g., the α2-macroglobulin related molecule (reviewed in 42).
SCR. The SCR repeats conserve well the general consensus of four Cys residues, two Gly, one Trp, one Phe, and two Pro; they are typical for the mammalian complement control protein superfamily (43). According to Nonaka et al. (44), SCR modules are classified into class I (e.g., in human coagulation factor XIII-b), class II (coagulation factor H), class III (coagulation factor XIII-b), and class IV (β2-glycoprotein I). The finding that the complement control protein module SCR (45) already exists in a sponge molecule was unexpected. The sponge SCR, present in the putative AR (Fig. 2A)(37) and the "multi-adhesive protein" (Fig. 2C)(38), are best classified as class III modules. Database homology searches with the sponge SCR repeats region (370 aa) revealed the highest similarity to mammalian selectins (mouse e-selectin); slightly less similar were mammalian complement receptors (mouse complement receptor type 2) and the invertebrate SCR proteins (Limulus clotting factor C), all of which are characterized by four conserved cysteine residues in each repeat.
Prophenoloxidase activating system. This system, perhaps an evolutionary forerunner to the complement proteins, acts as major recognition and defense pathway in invertebrates, especially in insects and crustaceans (reviewed in 46 and 47). The prophenoloxidase activating system comprises a complex cascade of factors, among them also serine proteases. The final product of the phenoloxidase activity is melanin, which is ubiquitously present throughout the metazoan kingdom. The enzyme is activated by serine protease(s) from its inactive form, the prophenoloxidase(s) (48). Melanin and/or its reactive intermediates have been shown to display fungistatic, bacteriostatic, and antiviral activity (reviewed in 46).
It has been suggested that melanization also occurs during allorecognition tissue responses (49). Melanin is a polymer formed via the phenoloxidase(s) from tyrosine, an aa that is converted from phenylalanine by the phenylalanine hydroxylase (PAH) (see 50). The PAH is the rate-limiting enzyme in the pathway catabolizing phenylalanine. Recently, we showed that after allo-transplantation in the marine sponge G. cydonium, PAH is up-regulated in the grafts (51). Enzyme determination studies revealed that PAH activity increases by 3-fold 2 days after transplantation and reaches its maximum after 3 days (by 3.7-fold). This finding was supported by determining the steady-state level of the mRNA for PAH. The cDNA encoding this enzyme was isolated from G. cydonium. Its deduced aa sequence codes for a protein of 51 kDa. Alignment studies indicate that the sponge PAH shares the consensus pattern as well as one characteristic pterin-binding site with the biopterin-dependent aromatic aa hydroxylases. It is concluded that, in the sponge model system, G. cydonium allogeneic rejection involves an up-regulation of PAH, an enzyme initiating the pathway to melanin synthesis (51).
Macrophage-derived Cytokine-like Molecule in Sponge
Allorecognition and rejection in vertebrates involves both cytotoxic and inflammatory mechanisms (52, 53). One factor, which has been described in rats (54), was identified as a cytokine-responsive macrophage molecule termed allograft inflammatory factor 1 (AIF-1). AIF-1 is highly expressed in rejecting allografts (54); it was later found that AIF-1 may be involved in inflammatory response associated with human cardiac transplant rejection (55).
The cDNA encoding the putative AIF-1 like molecule from S. domuncula has recently been cloned3 and termed SDAIF (accession number Y18439). The putative size (Mr) of the protein is 16,637. Like the vertebrate molecules, also the sponge AIF-1-related protein has the EF-hand (Ca2+)-binding) motif, which is found between aa 55 and 67 (Fig. 3A). This domain precedes the internal 39-residue-long motif characteristic of peptide hormones; it is framed by KR-GK, aa that are putative cleavage sites for prohormone convertases and peptide amidation enzymes. Within the potential peptide hormone, the typical KK motif is found. Immediately adjacent to the C terminus, one nuclear targeting signal sequence is located. This hitherto unique invertebrate AIF-1 related sequence from S. domuncula, AIF_SUBDO, is very similar to the vertebrate AIF-1 molecules from rat and carp (Fig. 3A); the degree of identity (similarity) between the AIF_SUBDO and the vertebrate sequences is ≈45% (≈70%).
Sponge AIF-1-related molecule. (A) Alignment of the aa sequence, AIF_SUBDO with the related sequences from rat (AIF1_RAT; U49392) and the carp, Cyprinus carpio (AIF_CARP; AB012309). Within the sponge AIF-1-related protein, the location of the EF-hand-like motif (-EF-), the sequence motif characteristic of posttranslational processing of peptide hormones (〈PH〉) with the internal KK motif (•), as well as the potential nuclear targeting sequence (+∼∼NTS∼∼+) are indicated. (B) Northern blot analysis to estimate the level of expression of the gene encoding the S. domuncula AIF-1-related protein. Tissue either from controls samples (Con) or from the contact zones of allografts and autografts were analyzed. Five μg of total RNA was extracted from the contact zones at day 2, 4, and 6 after starting the autograft (a) or allograft experiments (b). RNA was size-separated; after blot transfer, hybridization was performed with the SDAIF probe. Control samples were taken from a non-treated animal. The intensity of the transcript for SDAIF is correlated with the expression of β-tubulin.
The expression of the sponge genes encoding the putative AIF-1 and GPX (see below) proteins was determined both in autografts and allografts from _S. domuncula._3 The expression of the putative AIF-1 in S. domuncula is low in controls; an intensity of the 0.8-kb band of ≈0.2-fold, with respect to the β-tubulin expression, was measured. In autografts, the low level of expression remained unchanged during the 6-day observation period (Fig. 3B, a). However, in allografts, the expression increased strongly and was significantly higher (with respect to the controls) after a 2-day incubation period; the steady state level further increased drastically during the 6-day grafting period (Fig. 3B, b). These findings indicate that the strong expression of the AIF-1-like protein occurs only in allografts and not in autografts of S. domuncula, suggesting a possible function in activating "immunocytes" to alloimmune rejection. This finding is the first experimental documentation for a putative cytokine-mediated alloimmune response in invertebrates at a molecular level.
Other Potential Sponge Cytokine-like Molecules
Two additional cytokine-like molecules have been identified in sponges; molecules related to the mammalian endothelial-monocyte-activating polypeptide (EMAP) type II and the glutathione peroxidase (GPX).
Sponge endothelial monocyte-activating polypeptide-related molecule. The EMAP type II causes cell activation, e.g., increase of cytosolic free Ca2+ concentration, release of tissue factors, alteration of cell migration, and expression of adhesion molecules in endothelial cells, as well as in monocytes and granulocytes from human and mouse (56), and it causes angiogenesis (57). The putative EMAP-related polypeptide was cloned from the marine sponge G. cydonium; it has a deduced molecular mass of 16,499 Da and shows high sequence similarity to the human and murine EMAP (58). It is interesting to note that the size of the deduced EMAP polypeptide from human and mouse is approximately 34 kDa but is released from the cells as a mature, approximately, 18-kDa polypeptide. Until now, no functional studies with the EMAP-related polypeptide have been completed in sponges.
Glutathione peroxidase. In early phases of inflammation, occurring during graft recognition or during wound healing (59) in mammals, reactive oxygen species (ROS) are formed. One of the major enzymes involved in the detoxification of ROS during these processes is the GPX (60). GPX enzymes use glutathione to reduce hydrogen peroxides and other ROS (60). Recently one novel non-selenium GPX, also termed antioxidant protein 2 (AOP2), was identified and cloned from mice, which was found to be up-regulated during the early inflammatory phase of wound healing (61). Evidence was presented that GPX is induced by the cytokine keratinocyte growth factor in hyperproliferative epithelium at the wound edge (62).
The cDNA encoding the putative sponge GPX was cloned from S. domuncula (SDGPX; Y18438).3 The deduced protein has a calculated mass of 24,131 Da (Fig. 4A). An alignment has been performed with the mouse AOP2 sequence (Fig. 4A). At aa level, the sponge sequence was found to be similar to the vertebrate sequences, with a degree of ≈75%.
Sponge GPX-related polypeptide. (A) The protein sequence from S. domuncula (GPX_SUBDO), deduced from the cDNA SDGPX, is aligned with the mouse GPX/AOP2 (AOP2_MOUSE; Y12883). (B) Levels of expression of the gene in S. domuncula encoding GPX. RNA was isolated from autografts (a) as well as from allografts (b). After extraction, RNA was size-separated, blotted, and hybridized with the SDGPX probe.
As in the previous experiment using the AIF-1-like molecule from S. domuncula, the expression of the gene encoding the GPX-related protein is also low in the controls compared with the level measured for β-tubulin; a value of ≈0.2 relative to the intensity of the β-tubulin band was measured (Fig. 4B, a). In autografts, the expression of SDGPX increased gradually and reached a maximal level of 6.5-fold at day 6 during the grafting period. In contrast, in allografts, the increase of SDGPX expression was only transient; at day 4, a 9-fold higher level, compared to the controls, was measured, while after the 6-day grafting period, a value only twice as high as in the controls was found (Fig. 4B, b). This finding suggests that during graft fusion and rejection in sponges, ROS are generated, as in cytokine-activated macrophages in vertebrates, that amplify the immune response (63).
(2-5)A Synthetase
The cytokine interferon mediates in mammals innate immunity and protects them against viral infections. One of the key functions of interferon is the induction of the (2′-5′)oligoadenylate synthetase ((2-5)A synthetase), whose product (2′-5′)oligoadenylate ((2-5)An) activates the endoribonuclease L, which then degrades viral RNA (reviewed in 64). In mammalian cells, (2-5)An is found after infection by viruses (reviewed in 65). Three isoforms have been described in mammalian systems (66, 67); (2-5)A synthetase I corresponding to 40-46 kDa, (2-5)A synthetase II with 69 kDa, and (2-5)A synthetase III with 100 kDa.
Until recently, (2-5)A synthetases have been identified enzymatically or by molecular cloning only in vertebrates. In non-vertebrate systems, the product of the (2-5)A synthetase, (2-5)An, has been unequivocally characterized in the sponge G. cydonium both by biochemical and immunological methods (68) and lately also by matrix-assisted laser desorption-ionization time-of-flight and nuclear magnetic resonance analyses (69).
We have now described the cloning of the first invertebrate (2-5)A synthetase from the marine sponge _G. cydonium._4 The deduced aa sequence shows the signatures characteristic for (2-5)A synthetase form I. Phylogenetic analysis of the sponge (2-5)A synthetase indicates that it is the ancestral molecule of the hitherto known members of the vertebrate (2-5)A synthetases I. In addition, bootstrap analysis revealed that the sponge (2-5)A synthetase is phylogenetically older than the (2-5)A synthetases II; the data suggested that the latter ones derived from it by duplication. A calculation, based on the rates of aa substitutions, revealed that the sponge sequence branched off from a common ancestor ≈520 MYA. Later in evolution, the ancestral molecule duplicated and gave rise to the group of (2-5)A synthetases II, a process that occurred 310 MYA. The C-terminal half of the (2-5)A synthetases II is highly similar to the present-day (2-5)A synthetases I; the time of divergence is 270 MYA (Fig. 5). At present, we suggest that this system in sponges might be involved primarily in growth control, including control of apoptosis; in vertebrates, the (2-5)A system then acquired its additional function in innate immune responses.
Proposed branching order of the sponge (2-5)A synthetase (2-5A SY) from a common ancestor of metazoan (2-5)A synthetases (ancestor: 2-5A SY); this process has been calculated to have happened ≈520 MYA. The ancestral (2-5)A synthetase underwent gene duplication ≈310 MYA. From this ancestor of the (2-5)A synthetases of group II, with the two termini, 2-5A SY-IIa (NH2-ter) and 2-5A SY-IIa (COOH-ter), (i) the present-day (2-5)A synthetases of group II (2-5A SY-II (NH2-ter)-2-5A SY-II (COOH-ter)) evolved, while (ii) the C-terminal half became ≈270 MYA the building block for the (2-5)A synthetases of group I (2-5A SY-I); both types are found in vertebrates. The emergence of the various (proposed) functions of the (2-5)A system(s) is indicated.
PRECURSORS OF AN ADAPTIVE IMMUNE SYSTEM IN SPONGES
In mammals, the innate immune system initiates and subsequently directs the adaptive immune system.
Recognition of Non-self in Sponges; Lymphocyte-derived Cytokine: PreB-cell Colony-enhancing Factor (CEF)
Since the first publication on the systematization of vertebrate- versus invertebrate allorecognition (70), it has been assumed until today (30) that these two animal taxa differ in their mode of allorecognition; it was accepted that invertebrate also is based primarily on active recognition of "self," whereas in vertebrates the process of allorecognition involves also reactions to "non-self" antigens. Non-self-recognition requires a phase of sensitization before the rejection process. However, functional data suggested that also in invertebrates, from sponges to echinoderms (28, 71), cytokines are formed that mediate immunoregulatory reactions. Now, molecular data have been presented supporting the existence of such mediators.
The first sponge cytokine was cloned using a degenerate primer, directed against a conserved segment within the human preB-cell CEF (72). This molecule was selected on the rationale that preB-cell CEF is a secreted protein without any typical signal sequence, its gene can be induced in lymphocytes by a lectin, it is not totally tissue-specific, and it comprises messenger-destabilizing sequences in the 3′-untranslated part of the mRNA (72). The cDNA obtained from S. domuncula, 1.9 kb in length, encodes an open reading frame spanning the sponge preB-cell CEF-related polypeptide.5 The deduced 472-aa-long sequence (Fig. 6A) has a putative size of 52,580. The degree of identity (similarity) between the sponge preB-cell CEF and the human molecule is high at ≈55% (≈73%).
Sponge preB-cell CEF related polypeptide. (A) Deduced aa sequence from the S. domuncula cDNA encoding the putative preB-cell CEF. The 472-aa sponge sequence is aligned with the related human preB-cell CEF (accession number P43490). The corresponding nt region towards which the degenerate primer was designed is underlined. (B) Induction of preB-cell CEF in S. domuncula primmorphs. The primmorphs were incubated with 5 μg/ml either S. domuncula or G. cydonium membranes. At time zero, or after 1 and 3 days, RNA was extracted from primmorphs. Northern blot analysis was performed with cDNA encoding the S. domuncula preB-cell CEF. Bands on the autoradiographs were quantitated and correlated with the level of expression at time zero.
The cDNA encoding the sponge preB-cell CEF related molecule was used as a probe to determine its level in homologous cells from S. domuncula in response to membranes from another species, G. cydonium. Membranes from G. cydonium were isolated (73) and added at a concentration of 5 μg/ml to primmorphs from S. domuncula. As a "control," membranes from S. domuncula were used. RNA was extracted from the primmorphs at time zero and 1 and 3 days after addition of the respective membrane fractions. After Northern blotting (hybridization with the S. domuncula preB-cell CEF probe), the intensities of the bands, corresponding to a size of 1.8 kb, were determined. The experiments revealed that homologous membranes caused only a slight increase from 1-fold (time zero) to 1.2-fold after 3 days (Fig. 6B). In contrast, if membranes from G. cydonium were added to the cells, a strong up-regulation of the expression of the sponge preB-cell CEF mRNA to 7-fold (after 3 days) was seen (Fig. 6B). From these data, we deduce that membranes from the non-homologous species initiate in primmorphs from S. domuncula a signal transduction mechanism, resulting in an induction of the gene encoding the preB-cell CEF. This result is the first indication that sponges are provided with a molecular mechanism to recognize non-self.
Ig-like Domains
Two groups of molecules have been identified in G. cydonium that contain immunoglobulin (Ig)-like domains: the receptor tyrosine kinase (RTK) and the sponge adhesion molecules (SAM).
Receptor tyrosine kinase. The G. cydonium RTK molecule possesses in the putative polypeptide structure (i) the extracellular part with a Pro/Ser/Thr-rich region, and two complete immunoglobulin (Ig)-like domains, (ii) the transmembrane domain and (iii) the juxtamembrane region and (iv) the catalytic tyrosine kinase (TK) domain (6, 74, 75; Fig. 7A). Focusing on the Ig-like domains, allografting experiments have been performed to study the potential polymorphism within the Ig-like domains of this RTK (6). The experiments with six individual G. cydonium specimens revealed that the Ig-like domains are highly polymorphic (76); 14 distinct sequences out of 15 independent clones were recorded.
Structures of those molecules in G. cydonium that comprise Ig-like domains. (A) the receptor tyrosine kinase (GCRTK), (B) the sponge adhesion molecules (GCSAM), long form GCSAML, and (C) short form GCSAMS. The building blocks are: Pro-Ser-Thr(P/S/T)-rich domain, Ig-like domains 1 (Ig 1) and 2 (Ig 2), the transmembrane domain (TM), the juxtamembrane region (JM), and the TK domain (TK). The length of the stretches of the deduced aa domains is shown.
Sponge adhesion molecules. In a recent study, we searched for cDNAs that code for molecules containing only the Ig-like domain and not the TK domain (77). The rationale was to answer the question whether molecules related to hemolin or cell-surface associated molecules like N-CAM or CD2, all of which comprise Ig-like domains (reviewed in 78), are also present in sponges. The molecules were identified and operationally termed putative SAM.
Two SAM species have been isolated that do not encode a TK but two Ig-like domains (77). The deduced aa sequences were termed GCSAM. The longer form of the SAM, GCSAML (Fig. 7B), has an estimated size of 53,911. The short form of SAM, GCSAMS (Fig. 7C), comprises only 313 residues and has a calculated Mr of 33,987. Alignment of the deduced aa sequences GCSAML and GCSAMS with the corresponding sequences from the RTK cDNA, GCRTKc, and the RTK gene, GCRTKg, reveals that only the long form of the SAM comprises a Pro-Ser-Thr(P/S/T)-rich domain.
The Ig-like domains present in both GCSAML and GCSAMS can be homologized to the V-related set of Ig-like sequences. This decision is based on the consensus pattern known for this class of molecules (79, 80). Those aa residues that share the V-profile (81) are indicated in Figure 8 (A and B). In the sponge sequences, no C′ or C″ β-sheets, which are characteristic for the V-sets, could be predicted. However, as indicated for the Ig-like domains 1 of both GCSAML and GCSAMS (Fig. 8A), two aa residues (leucine and aspartic acid) are present between the C and the D β-sheets, which are also found in C′ β-sheets. This fact could indicate a relationship to the I-type of Ig-like domains, as described (81).
Polymorphism of aa sequences of the Ig-like domains from the RTK and the SAM molecules from G. cydonium. (A) Alignment of the deduced aa sequences of the Ig domain 1. The domains from GCSAML (GCSAML_IG1) and GCSAMS (GCSAMS_IG1) are compared with the Ig domain 1 in the RTK cDNA (GCRTKc_IG1) as well as the RTK gene (GCRTKg_IG1) and also with the two related Ig domains, the human immunoglobulin λ chain variable region (Ig_lambda, Z37377), and the human immunoglobulin λ chain V-VI region (WLT) (Ig_la/V-VI; P06318). (B) Relationships of Ig-like domains 2. The alignment was done with the Ig domain 2 of GCSAML (GCSAML_IG2), GCSAMS (GCSAMS_IG2), the RTK cDNA (GCRTKc_IG2) as well as with the RTK gene (GCRTKg_IG2) and the Ig domains from the human immunoglobulin heavy chain variable region (Ig_heavy, AF062133), cell adhesion molecule NCAM-180 from the Japanese common newt (NCAM_NEWT, D85084), the UNC-89/titin segment from C. elegans (UNC/TIT, U80022), the human titin (TITIN, 2136280), and the human basement membrane-specific heparan sulfate proteoglycan core protein precursor (Perlecan) (PERLECAN; P98160). The positions of the β-strands A to G within the Ig-like domains are indicated; the β-strands have been predicted using the method of Garnier (92). Substitutions of at least one aa between the corresponding Ig-like domains of the sponge sequences are marked (•). The aa residues that share the V-type are indicated by arrowheads. (C and D) Expression of GCSAML and GCSAMS during autografting. Northern blot analysis was performed under high stringency using 5 μg of total RNA, extracted from the fusion zones at time zero, and 2, 5, or 10 days after grafting. RNA was size-separated; after blot transfer, hybridization was performed either with the GCSAML probe (C) or the GCSAMS probe (D).
Polymorphism. The two Ig-like domains present in the GCSAM, in GCRTKc (cDNA for RTK) as well as in GCRTKg (gene for the RTK), are polymorphic. On the aa level, Ig-like domain 1 (Fig. 8A) of GCSAML shares 94% identical aa with GCRTKc, while GCSAMS has an even higher degree of identity (97%) within this region. Ig-like domains 2 (Fig. 8B) from both GCSAM show the same percentage of identical aa (97%) to the GCRTKc. On nt level, 13 substitutions between GCSAML and GCRTKc (3 substitutions for GCSAMS to GCRTKc) were recorded within the Ig-like domains 1; they lead to 6 aa substitutions (2 substitutions). Within the Ig-like domain 2 regions, 12 nt substitutions are present between GCSAML and GCRTKc (4 substitutions GCSAMS to GCRTKc), which result in 6 (2) aa exchanges.
Expression of Ig-like domains during grafting. Autografting experiments were performed with G. cydonium using the "insertion technique." The sponges were kept up to 10 days and were subsequently analyzed. Cross-sections revealed that the grafts almost completely fused with the host during this period. Staining the sections with a polyclonal antibody against the Ig-like domains from RTK (RepairGenics Mainz, Mainz, Germany), the fusion zone is brightly stained (77). Additionally, Northern blot analyses have been performed to support the immunohistochemical analyses. Analyzing the same amount of RNA from the fusion zones at different periods after grafting revealed that the expression of GCSAML as well as GCSAMS is up-regulated after grafting. Setting the intensity of the signal at time 0 to 1-fold, the intensity of the 2.1-kb band, reflecting GCSAML, increased in strength to 12-fold at day 10 after grafting (Fig. 8C). The increase of the 1.5-kb GCSAMS signal is also considerable but less strong than that after hybridization with GCSAML; after a 10-day observation period, the increase reached a level of 5-fold, compared to controls at day 0 (Fig. 8D; 77).
CONCLUSION: ORIGIN OF THE INNATE AND ADAPTIVE IMMUNE SYSTEM IN PORIFERA
Since the start of systematic analyses of potential immune molecules in G. cydonium and S. domuncula at a molecular level, it has become increasingly clear that the evolutionary oldest metazoans, the Porifera, already have key molecules that are used in mammals for their innate and adaptive immune system. It is likely that some of those molecules acquired a dual function during evolution, first as cell adhesion receptors or growth control factors and second as immune molecules required for self/self- and self/non-self-recognition. By combination of molecular- and cell/tissue biological approaches, it has now become possible to test basic questions about the potential involvement of candidate immune molecules in the immune response in sponges. In the past, conclusions relied on grafting experiments only and allowed no comparative analyses of the factors identified in sponges with molecules acting in immune responses in mammals.
However, at present, it cannot be stated with certainty whether molecules, found in lower invertebrates or even in the earliest metazoan phylum (the sponges), which share high sequence similarities with immune molecules of mammals, have a similar or a different function in sponges. One example might be the rhesus (Rh)-like protein (cDNA) of Mr 57,000 that was isolated from G. cydonium(82). Both the hydropathy profile of the sponge Rh-like protein and its high similarity of the aa sequence clearly show that the sponge molecule shares a common ancestor with the human as well as the rhesus monkey Rh30 antigen as well as with the Mr 50,000 Rh-like polypeptides from human (83, 84) and Caenorhabditis elegans. It might well be that after having elucidated the function of a given sponge molecule in the homologous system, this finding will also lead to the discovery of new functions of the homologous molecules in mammals. The Rh blood group polypeptides might be an example; it was recently suggested that they are related to NH4+ transporters (85).
Innate Immune System in Porifera
It is well established that sponges are provided with a natural, innate immune system. Only as a result of the existence of this basic type of immune strategy could metazoans have evolved. As in mammals, the "humoral" (secretory) immunity can be operationally separated from cellular immunity.
The humoral immunity (or, in order not to confuse it with the antibody-mediated immune reaction, the secretion-dependent immunity), is likely to include (i) lectins, (ii) cytokine(s), and (iii) bioactive compounds. It was suggested by Olafsen (86) that lectins are "immune" molecules also in sponges. One galectin was isolated and cloned from G. cydonium(87), which was shown to be down-regulated at zones adjacent to grafts (31). This finding might suggest that the function of this lectin as an immune molecule is only indirect at best. Second, a tumor necrosis factor-like molecule was identified by immunohistochemical analysis in G. cydonium in allografts undergoing necrosis/apoptosis (27). Until now, we have failed to identify the gene for this potential sponge cytokine. However, recently we have cloned a Fas death domain-containing molecule from G. cydonium, suggesting the presence of a tumor necrosis factor-related receptor. The implication of the cytokine-related molecules, especially of the allograft inflammatory factor 1 and glutathione peroxidase, in self/self- and non-self-recognition is strong (see above). Third, sponges are rich sources for low-molecular-weight bioactive compounds (88), and their involvement in defense against foreign invaders has been proposed (12).
Phagocytosis can-at present-be considered as the major, cellular defense system in sponges. It has been proposed that the archeocytes, the "macrophage"-like amoeboid cells, are those cells that digest invading non-self particles (89). The potential receptors for non-self-recognition (SRCR molecules) as well as the proteolytic enzyme are mentioned above.
Origin of an Adaptive Immune System in Porifera
Grafting experiments led to the conclusion that sponges have simple mechanisms required for an adaptive immunity. The principles of this immune strategy (21) are (i) synthesis of immunoglobulins in lymphocytes after activation by antigens, (ii) clonal selection of lymphocytes, and (iii) somatic recombination.
Immunoglobulins. The synthesis of antibodies in response to foreign antigens is a major contribution of lymphocytes to adaptive immunity. They are formed from the basic building blocks, the immunoglobulin (Ig) domains. These domains comprise a superfamily present in membrane receptors and also in the soluble Igs; they are grouped into several categories, among them the constant and variable sets (reviewed in 78). The variable domains are polymorphic and involved in binding of the Igs to the antigen (Fig. 9D). It is tempting to correlate this function of the Igs with the potential role of the Ig-like domains of G. cydonium; the latter are related to the variable sets of Igs and are polymorphic (Fig. 9, B and C).
Comparison of the arrangement of the polymorphic Ig-like domains in the sponge SAM (long form as well as short form) (B) and the RTK (C) with the Ig domains, which are present in the human T-cell receptor (A) and the immunoglobulin molecule (D); scheme. The variable region in the sponge molecule, the Ig-like domain 1 (Ig 1), which is closely related to the human IgG-light chain (VL-chain-λ) is in black, while the Ig-like domain 2 (Ig 2), which has homology to the human IgG-heavy chain variable region (VH-chain), is shaded. The constant domains of the human T-cell receptor and the IgG molecule are cross-hatched.
Somatic recombination; somatic hypermutation. The available cloning studies in the G. cydonium system give no hint for a rearranging molecular mechanism. However, the striking homology between the two sets of the polymorphic sponge Ig-like domains, present in the RTK and the SAM, allows the hypothesis that the polymorphism seen in G. cydonium is the result of somatic hypermutation; this can and will be tested in future. This assumption is already supported by previous data, which revealed that the aa substitutions within the Ig-like domains in G. cydonium are restricted to "hot spots" (6; Fig. 8, A and B).
Clonal selection of lymphocytes. Inasmuch as, in sponges, the tools (genes and cell cultures) required to answer the question of the existence of clonal selection of potential immunocytes have been elaborated only in the last 2 years, it is too early to provide an answer. It is known that all (or almost all) sponge cells have a high telomerase activity (90). It therefore appears to be unlikely that differentiated immunocytes exist in sponges.
Recognition of self in sponges. During the last few years, the classical studies on autografting in sponges (3, 4), which, like mammals, demonstrate immunological self-tolerance, have been supported by the identification of molecules involved in this recognition process. In autografts, integrin receptors are strongly up-regulated at the zones between the grafts, suggesting their functional involvement (20, 31). However, binding of this receptor to collagen or to fibronectin does not provide enough specificity to support the functional interactions of the cells in a multicellular individual. Hence, additional components allowing the fusion of self tissue must exist. The search for the possible involvement of molecules related to a T-cell receptor or to a major histocompatibility complex (MHC) has not yet begun. It may be noted that the T-cell receptor has a structure similar to that for the SAM from G. cydonium: two extracellular Ig-like domains and a short cytoplasmic tail (Fig. 9, A and B).
Recognition of non-self in sponges. This phenomenon is closely linked with the recognition of immunoglobulins by antibodies. The studies reported here demonstrate that cells from S. domuncula up-regulate a lymphokine-like molecule, the preB-cell CEF, in cells after exposure to membranes from G. cydonium. This results suggests that mechanisms for non-self-recognition exist in sponges. The receptor for non-self molecules in sponges is not yet known. Again, MHC-related gene clusters have to be identified in the future; some components, e.g., proteasome genes (91), have already been found.
FUTURE DIRECTIONS
In the last few years, sponge models, especially using the species G. cydonium and S. domuncula, have been elaborated to a suitable system for the study of immune reactions at both molecular and cellular levels. The first results are extraordinary because they show that molecules very likely to be involved in graft fusion and rejection are so similar and so highly conserved. By application of molecular biological techniques, it became possible only 5 years ago to establish the monophyly of Metazoa (12). Having now characterized the first potential immune molecules from these two sponge species, it appears very likely that all Metazoa have the basic structural elements for the innate and acquired immune system. The tools, the molecular biological techniques and cellular/tissue models, are now available.
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* Abbreviations: aa, amino acid; AF, aggregation factor; AIF-1, allograft inflammatory factor 1; AR, aggregation receptor; BrdU, bromodeoxyuridine; EMAP, endothelial-monocyte-activating polypeptide; GPX, glutathione peroxidase; Ig, immunoglobulin; kb, kilobase; MHC, major histocompatibility complex; MYA, million years ago; nt, nucleotide; PAH, phenylalanine hydroxylase; preB-cell CEF, preB-cell colony-enhancing factor; ROS, reactive oxygen species; RTK, receptor tyrosine kinase; SAM, sponge adhesion molecule; SCR, short consensus repeats; SRCR, scavenger receptor cysteine-rich domain; TK, tyrosine kinase.
3 Kruse M, et al. (manuscript submitted for publication).
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4 Wiens M, et al. (manuscript submitted for publication; accession no. Y18497).
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5 Müller WEG, et al. (manuscript submitted for publication; accession no. Y18901).
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