© 2003 by The Society for Integrative and Comparative Biology
Origin of the Metazoan Immune System: Identification of the Molecules and Their Functions in Sponges1
1 Institut für Physiologische Chemie, Abteilung Angewandte Molekularbiologie, Universität, Duesbergweg 6, D-55099 Mainz; Germany
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During the evolutionary transition to Metazoa, cell-cell- as well as cell-matrix recognition molecules have been formed, which made a further step in evolution possible, the establishment of an immune system. Sponges [Porifera] represent the oldest still extant metazoan phylum and consequently testify to major features of the common metazoan ancestor, the Urmetazoa. Most studies with respect to evolution and phylogeny in sponges have been performed with the marine demosponges Suberites domuncula and Geodia cydonium. These animals possess effective defense systems against microbes and parasites which involve engulfment of bacteria into specific cells, but also signal transduction pathways which actively kill bacteria. Among those is the LPS-mediated pathway, with the stress-responsive kinases. In addition, sponges are provided with an interferon-related system, with the (2–5)A synthetase as controlling enzyme. Transplantation studies have been performed on tissue, as well as at the cellular level ("mixed sponge cell reaction assay") which demonstrate the complex molecular strategy by which sponges respond to allogeneic- and/or autogeneic signals. Among the molecules involved in histo(in)compatibility response of sponges, cytokines e.g., the allograft inflammatory factor 1, have been identified which control rejection of allografts. Furthermore, transcription factors, with Tcf-like factor as an example, have been identified which very likely control gene expression during histocompatibility reactions. The immune reactions in sponges can be modulated by FK506, a drug which has been successfully used as immunosuppressant in humans. One further surprising finding is the fact that G. cydonium has several molecules containing polymorphic Ig-like domains of the variable type. It is concluded that the successful evolutionary transition to the Metazoa, with the sponges as the oldest still extant phylum, and the subsequent rapid radiation into the other metazoan phyla, became possible because of the acquisition of modular molecules, involved in cell adhesion and the immune system.
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Research activity focused on the elucidation of the cellular and molecular basis of immunity in sponges (phylum Porifera) was hampered by the fact that the phylogenetic position of these animals was uncertain. In 1899 (DéLage and Hérouard, 1899
) the Porifera had been grouped with the Metazoa, restricting them to a taxonomic position, the Spongiaires, separate from the rest of the multicellular animals. Prior to this attempt the sponges had been regarded as Corallines (Ellis, 1756; 92), Plant-Animals (Esper, 1794; vol. 2, p. 165) or as Animal-Plants (Pallas, 1787; vol. 2, p. 212). Intermittently, the Porifera were classified to the Protista/Protozoa (Haeckel, 1866; and Spencer, 1864; vol. 1, p. 302). Unfortunately the view that sponges are almost animals, Parazoa, is still even today often seen in textbooks (e.g., Pechenik, 2000), since sponges lack a nervous system, a "true" musculature and the basal lamina. It even remained unclear if Porifera share one common ancestor with other metazoan phyla (Christen et al., 1991). After analyzing protein sequences from sponges, especially those which comprise receptors, it was established that all Metazoa, including Porifera, are of monophyletic origin (Müller et al., 1994; Müller, 1995). In addition, it became obvious in the last five years that sponges do contain nervous elements, e.g., neuronal receptor (Perovic et al., 1999), and contractile systems, e.g., dynamic elements (Lorenz et al., 1996), as well as the basic structures of the basal lamina, e.g., integrin and collagen (Pancer et al., 1997; Schröder et al., 2000). Thus, the question arises as to whether the existing differences in the nervous system (lack of neurons), the musculature (lack of connecting muscular fibrils) and the basal lamina (lack of cell junctions) justify division of the Porifera into a separate subkingdom. Considering especially the complex immune system of sponges, which is closely related to that of higher Metazoa, especially the Deuterostomia, (see this review) a neutral term for the hypothetical ancestor of all metazoan phyla, the Urmetazoa, was proposed (Müller, 2001). This grouping is supported by the fact that the extracellular matrix molecules, as well as their corresponding cell surface receptors (Müller, 1997), had been the prerequisites for the evolution of an efficient immune system in Metazoa (Müller et al., 1999a).
It was the contribution of Metchnikoff (1892) who described the phagocytotic activity of sponge cells, archaeocytes, as a mechanism to eliminate non-self particles and, even more advanced, to encapsulate the foreign material within cell aggregates of the sponge prior to the elimination by "ablation" (p. 56–57). These abilities of sponges had been discussed by Metchnikoff in the context of inflammation processes, that proceed in Metazoa during infection. The major step in the elucidation of the cellular mechanisms by which the sponges eliminate non-self and accept self came from elegant experimental transplantation studies. In their extensive review Smith and Hildemann (1986) have grouped sponge alloimmune responses into two major rejection processes. Some species may form barriers to separate from non-self tissue; e.g., the marine sponge Axinella verrucosa (Buscema and van de Vyver, 1983) or the freshwater sponge Ephydatia muelleri (Mukai, 1992), while others may react by cytotoxic factors which destroy the transplant; e.g., the marine sponges Callyspongia diffusa (Hildemann et al., 1979) or Geodia cydonium (Pfeifer et al., 1992). Finally, the breakthrough in the discovery that immune mechanisms in sponges are highly similar to those, found in other metazoan phyla, came again after the application of molecular biological techniques (see Müller et al., 1999a).
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Almost all marine demosponges contain bacteria. Until now no conclusive data were available to say which bacterial strains are symbiotic and which are parasitic. At least one report suggests that the number of bacterial strains that are symbiotic or commensal is limited (Althoff et al., 1998
). All specimens of the marine demosponge Halichondria panicea collected from the Baltic Sea, the North Sea, as well as in the Mediterranean Sea were found to harbor one defined bacterium which belongs to the taxon Roseobacter/Rhodobacter. Based on this finding it was suggested that this bacterial strain has at least a commensal relationship with the host.
However, the first data are now available which help to gain further insight into the molecular mechanism by which the host (sponge) might discriminate between symbiotic or commensal and parasitic bacteria. First, it was demonstrated that defined bacterial strains can be engulfed by specific sponge cells, the bacteriocytes (Böhm et al., 2001). Furthermore, it was shown that protein synthesis in tissue from S. domuncula is inhibited after incubation with the bacterial endotoxin lipopolysaccharide (LPS; Böhm et al., 2001). Since serine-threonine directed mitogen-activated protein (MAP) kinases are essential components of the LPS-mediated pathway, evidence of activation of these kinases in response to LPS was sought (Böhm et al., 2001). Molecular biological and immunological studies confirmed that these pathways also exist for the Porifera, indicating that such defense pathways are highly conserved between sponges and humans. This conclusion was also strengthened by earlier results, which revealed that another stress-responsive kinase, KRS (Kruse et al., 1997; Taylor et al., 1996), which is involved in the phosphorylation of MAP kinases, is also present in Demospongiae.
Molecules involved
One powerful mechanism to eliminate microbes is intracellular digestion. This cellular defense mechanism against foreign invaders is well developed from sponges to insects and humans. Sponges possess specialized amoeboid cells, the archaeocytes (Metchnikoff, 1892), which have in the past been regarded as macrophages of sponges (Van de Vyver, 1981).
Mammalian macrophages are the first cells to encounter non-self material. They express several receptors, termed scavenger receptors, that 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 SRCR domains (reviewed in: Resnick et al., 1994). With regard to sponges, molecules comprising SRCR domains have been first cloned in G. cydonium (Pancer et al., 1997; Blumbach et al., 1998). It was surprising to find that these sponge molecules are present in at least three alternatively spliced forms. The largest form, SRCR-SCRm, is a cell-surface receptor of Mr 220 kDa, the putative AR; the second form, SRCRm, is also a putative receptor with Mr 166 kDa, while the third form, SRCRs, is a putative soluble molecule of Mr 129 kDa. The SRCR-SCRm molecule consists of fourteen SRCR domains, six SCR repeats, one C-terminal transmembrane domain and a cytoplasmic tail. In addition, a cDNA was cloned (Pahler et al., 1998a) that encodes a putative "multiadhesive protein," which comprises three modules: (i) a fibronectin-, (ii) a SRCR-, and (iii) a SCR-domain. A phylogenetic analysis revealed that the sponge SRCR domain present in the "multiadhesive protein" displays high similarity to the mammalian WC1 surface antigens, e.g., from bovine, the human CD6 antigen, the human CD5 surface glycoprotein, as well as the human M130 antigen.
These data strongly suggest that sponges comprise SRCR-domain containing cell-surface molecules which might be involved in the recognition of bacteria. In addition, it is likely that the ingested "non-self" bacteria are killed by both an oxidative and a nonoxidative (enzymatic) mechanism. Several cDNAs coding for lysosomal enzymes, e.g., cathepsin which is abundant in G. cydonium (Krasko et al., 1997), have been isolated from sponges.
The interferon-related system: (2–5)A synthetase
Very recently, a further (putative) defense system against invading bacteria and/or viruses has been detected in Demospongiae: the (2–5)A (2'–5') oligoadenylate synthetase ([2–5] A synthetase) system (see: Rebouillat et al., 1999). In mammalian systems, the (2–5)A synthetase(s) catalyzes the synthesis of a series of 2'–5'-linked oligoadenylates, termed (2–5)A [=pppA(2'p5'A)n [pnAn] from ATP (Hovanessian, 1991). In turn, (2–5)A acts as an allosteric activator of a latent endoribonuclease, the RNase L, which degrades single-stranded, viral or cellular RNA (Zhou et al., 1993). In mammalian organisms the (2–5)A system is activated by interferons (Pestka et al., 1987).
The first sponge species studied that was found to display higher levels of (2–5)A oligoadenylate synthetase and its products than vertebrate cells (Kuusksalu et al., 1995) was G. cydonium. The sponge (2–5)A synthetase was cloned (Wiens et al., 1999). Interestingly enough, this enzyme as well as its products are present in sponges and in the deuterostome lineage, but are absent in protostomes (Wiens et al., 1999). Recently, functional assays were performed to elucidate the role of the (2–5)A synthetase in sponges, especially with respect to a potential infection with foreign, pathogenic microorganisms. The sponge cellular system, which proved to be suitable for this approach are the sponge primmorph—special cultured aggregates of sponge cells (Custodio et al., 1998; Müller et al., 1999b). The experiments showed that primmorphs synthesized (2–5)A in larger amounts if they were incubated with LPS, suggesting an activation of the synthetase through a LPS-initiated pathway. To clarify if LPS also caused increased expression of the gene on the transcriptional level, cDNA encoding the (2–5)A synthetase was cloned from S. domuncula (Grebenjuk et al., 2002). The three sponge (2–5)A synthetases cloned, from G. cydonium, from S. domuncula as well as from H. panicea (Fig. 1A) share high sequence similarity with the corresponding vertebrate enzymes (Fig. 1B). The H. panicea putative enzyme comprises 296 amino acids, and has a calculated Mr of 33,783 (Müller, unpublished).
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Sig-1 and |It is interesting that the level, as well as the activity, of the (2–5)A synthetase increases after bacterial infection. For these studies the recombinant synthetase was prepared and antibodies raised. Using these as tools in Western blot experiments, it was demonstrated that animals which had been kept for over 12 months under low bacterial load, also show a low level of expression (Fig. 2A; lane c). The (2–5)A synthetase level in animals analyzed immediately after collection from the sea (field) is slightly higher (lane d). However, a drastic increase (10-fold) is seen in animals which were infected with bacteria of the genus Vibrio in the aquarium (lane b). Surprisingly, the antibodies recognized two (2–5)A synthetase species, reflecting earlier observations that two different enzymes are present in S. domuncula (Grebenjuk et al., 2002
).
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37 kDa recombinant protein (lane a; rec) but also the enzyme present in control animals (kept in the aquarium for over 12 months; lane c, cont) as well as also in animals, analyzed immediately after collecting from the sea/field (lane d; sea). In addition, it is shown that the expression of the enzyme is upregulated in animals which had been infected with a bacterium of the genus Vibrio (lane b; inf). Equal amounts of protein were loaded onto the gels (20 µg/lane). B. Autoradiogram of a thin layer chromatography of [14C]ATP labeled 2'–5' oligoadenylate products synthesized by the enzyme in tissue from S. domuncula (specimens kept for over 12 months in the aquarium; lane a, aqua) as well as by the enzyme from a field animal (lane b, sea). In parallel, the product from the (2–5)A synthetase from G. cydonium was taken as a reference tissue (Geodia; lane c), from which it had been described earlier that they produce the oligoadenylate products p3A2 and p3A3 (Grebenjuk et al., 2002In an earlier study it was demonstrated using several techniques that the product of the (2–5)A synthetase from G. cydonium is indeed (2–5)A (Kuusksalu et al., 1995
and 1998). The G. cydonium enzyme was found to synthesize both the dimer (p3A2) and the trimer (p3A3), if analyzed by TLC (Fig. 2B; lane c). Using these authentic products as reference it was shown that the enzyme from S. domuncula synthesizes only dimers, and only in animals that were immediately taken from the field (lane b). The wild (newly collected) animals were found to harbor commensal or toxic bacteria in addition to the putative symbiotic bacteria (Böhm et al., 2001). In contrast, no product could be measured in specimens which were kept for over 12 months in an environment of low bacteria load in the aquarium (Böhm et al., 2001) (lane a). In line with this investigation, we postulate that in the sponge system the expression or activity of the (2–5)A synthetases is augmented after bacterial infection, via the jak/STAT pathway, as occurs in vertebrate cells (reviewed in Justesen et al., 2000). |
HISTO(IN)COMPATIBILITY RESPONSES IN SPONGES |
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Transplantation: tissue level
Studies of histo(in)compatibility response in sponges have been performed for 30 years. Initially it was reported that sponges have only a low capacity for allorecognition (Moscona, 1968
; Van de Vyver, 1970). However, after defining the system, it became apparent that sponges have a very high degree of precision when discriminating between self/self and self/non-self (see Hildemann et al., 1979 and 1980; Neigel and Avis, 1985).
The use of histology and light microscopy to observe the detection of autograft fusion and allograft rejection in sponges was superceded by the introduction of molecular biological techniques. The two marine demosponges S. domuncula (Fig. 3A-a) and G. cydonium (Fig. 3B-a) have primarily been used for those studies. This new approach led to the perhaps unexpected discovery that there are immune molecules in the Porifera which share high sequence similarity to those of higher metazoan phyla, and especially to deuterostomes (see Müller et al., 1999a). It was established for both sponge species that, under controlled conditions, practically all autografts/syngrafts fused, while the allografts were rejected (Pancer et al., 1996; Müller et al., 1999a). For the transplantation studies specimens collected from distances of at least 20 km around Rovinj (Croatia) were used. It remains to be determined which genetic markers are suitable for discriminating between the individuals.
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Two transplantation techniques were applied: the insertion technique for G. cydonium (Fig. 3B-b) and the parabiosis method (primarily for S. domuncula; Fig. 3A-b) (Pancer et al., 1996
). From G. cydonium tissue pieces were removed with a cork borer from one specimen and were inserted into holes in the recipients (insertion technique); Fig. 3B-b. All autografts fused and eventually no boundary line was seen (Fig. 3B-c). Allografts initially fused together, but after approximately 3 to 5 days the rejected graft tissue formed a pronounced demarcation boundary and underwent apoptotic/necrotic degeneration (Fig. 3B-d) and finally resorption. For S. domuncula, which may be red, orange, whitish, blue or as a combination of those colors, the autografts started to fuse approximately 3 days after grafting; after 6 days the attachment zone of the two grafts was no longer evident, either by visual inspection (Fig. 3A-c) or by microscopical analysis after sectioning and staining. In contrast, allografts started to reject each other after an initial period of fusion (duration 3 days); Figure 3A-d.
Transplantation: cellular level—mixed sponge cell reaction assay
Recently, a cellular assay was developed to allow analysis of the histo(in)compatibility reactions at a cellular level (Müller et al., 2002). The basis of the assay was developed following the establishment of the primmorph system, a three-dimensional aggregate system which comprises proliferating and differentiating cells (Custodio et al., 1998; Müller et al., 1999b). Under standard conditions, primmorphs (3 to 7 mm) are formed from a single cell suspension after incubation in seawater for approximately 5 days.
In the mixed sponge cell reaction (MSCR) assay dissociated cells either from the same individual (autogeneic MSCR) or from different individuals (allogeneic MSCR) were mixed at equal cell concentrations. After reaggregation in seawater for 12 hours small aggregates were formed. Then aggregates from the same individual or from two different specimens were allowed to react in the MSCR assays. After a period of five days primmorphs were formed from the aggregates. If cells from the same individual were mixed, autogeneic MSCR, 2 mm large aggregates were formed during the initial two days of incubation, which finally became 5 to 10 mm large primmorphs (Fig. 3D-a). In assays using cells from different specimens, they did not form single primmorphs but separated after two days (Fig. 3D-b), indicating that during the allogeneic MSCR the cells recognize non-self and form individual-specific aggregates. For a visual demonstration of an individual-specific aggregate formation, cells from white and red colored animals were used. If those aggregates are incubated for additional five days, white or red colored primmorphs were formed (Fig. 3D-c). This finding confirms an earlier study which also reported that aggregates from different individuals form, after a short transient contact period, different individual-specific aggregates (Humphreys, 1994).
FK506 as a tool to study immunosuppression in sponges
FK506, also termed Tacrolimus (Kino et al., 1987), is an immunosuppressant macrolide lactone which is successfully applied in clinics to prevent graft-versus-host diseases (reviewed in Jacobson et al., 1998). This drug was also shown to effectively prevent allograft rejection in S. domuncula (Müller et al., in 2002).
As outlined above tissue of autografts fused with each other, while allografted tissues rejected one another (Fig. 3C-a and -b). However, the presence of FK506 allows allograft fusion (Müller et al., 2002), at the non-toxic dose of 20 ng/ml FK506 the allografts fused with each other (Fig. 3C-c and -d). In addition, it was shown that at the attachment zone in untreated and in FK506-treated allografts (in which it is especially pronounced) the expression of the genes encoding the FK506-binding proteins is upregulated (Müller et al., 2001b).
FK506 causes also immunosuppression under in vitro conditions. Using the MSCR assay it was demonstrated that in the presence of 20 ng/ml of FK506 primmorphs from different specimens, allogeneic MSCR, fused (Fig. 3D-d), while in the control assay, in the absence of FK506, a clear separation of the primmorphs was maintained (Fig. 3D-b and -c) (Müller et al., 2002). The primmorphs in the autogeneic MSCR did not show rejection.
Molecules involved in histocompatibility response of sponges
Using transplantation models from both G. cydonium and S. domuncula (Müller et al., 1999a) it was established that macrophage-derived cytokine-like molecules are activated during allograft rejection. Among those sponge cytokines activated is the allograft inflammatory factor 1 (AIF-1), a factor which has been described in rats (Utans et al., 1995) and was identified as a cytokine-responsive macrophage molecule. In mammalian systems, AIF-1 is highly expressed in rejecting allografts (Utans et al., 1995); later it was also found that AIF-1 may be involved in inflammatory response associated with human cardiac transplant rejection (Utans et al., 1996). The cDNA encoding the putative AIF-1 like molecule from S. domuncula has been cloned (Kruse et al., 1999). The relationship of the sponge molecule to the vertebrate (human) AIF-1 protein was much higher with an alignment score (in bits) of 160 and an "Expect value" (E [Coligan et al., 2000]) of 7e–40 than to a corresponding molecules of C. elegans (63 bits; E = 7e–11) or D. melanogaster; the alignment to the human AIF-1 molecule is shown in Figure 4A, reflecting 51% identity and 70% of similarity between the two sequences. The sponge AIF-1 has been found to be involved in graft rejection/fusion processes. A strong upregulation has been determined in the rejection zone from allografts (Kruse et al., 1999). In parallel with this change in expression, a second characteristic molecule was identified which resulted in increased expression of the Tcf-like transcription factor (TCF) after transplantation in S. domuncula (Müller et al., 2002). Also the sponge TCF polypeptide shares highest similarity to those protostome and deuterostome transcription factors that are involved in diverse developmental processes (Wegner, 1999; Cho and Dressler, 1998); the deduced protein comprises the characteristic high-mobility group (HMG) box (Soullier et al., 1999).
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After having established that both, the AIF-1 and the TCF gene are upregulated in vivo after tissue transplantation, especially in allografts, it was determined whether this reaction can also be monitored in vitro using the autogeneic- as well as allogeneic MSCR assay system. The results showed that the two AIF-1 and TCF genes are also induced during allogeneic MSCR (Müller et al., 2002
). In a further approach to define the MSCR system, the drug FK506 was applied. In the presence of 20 ng/ml of FK506 the expression of both AIF-1 and TCF was totally abrogated. In addition, it was shown that the recombinant sponge AIF-1 polypeptide caused an upregulation of the expression of TCF. Therefore, we conclude that the AIF-1 and TCF genes are upregulated in sponges during histoincompatibility reactions. The data support the view that sponges are provided with immune systems composed of complex elements closely related to those found in mammalian systems. A scheme of the proposed role of the AIF-1 cytokine and the TCF transcription factor is given (Fig. 4B).
Further molecules/factors very likely involved in histo(in)compatibility reactions are glutathione peroxidase and endothelial-monocyte-activating polypeptide (EMAP). In vertebrates EMAP (type II) causes cell activation and expression of adhesion molecules in endothelial cells as well as in monocytes and granulocytes from human and mouse (Kao et al., 1994) resulting in angiogenesis (Yoshida et al., 1997). The putative EMAP-related polypeptide was cloned from the marine sponge G. cydonium; it has a deduced molecular mass of 16 kDa and shows high sequence similarity (again) to the human and murine EMAP (Pahler et al., 1998b).
The glutathione peroxidase (GPX) is activated in humans/vertebrates during the early phases of inflammation that occur during graft recognition (Shiraishi et al., 1997) or during wound healing in mammals when reactive oxygen species (ROS) are formed. It is the major enzyme involved in the detoxification of ROS during these processes. The cDNA encoding the putative sponge GPX is known from S. domuncula (Kruse et al., 1999). 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. However in the zones between grafts (the attachment zones), the expression of SDGPX increases gradually with time, and reaches a maximal level of 6.5-fold. This finding suggests again that during graft fusion and rejection in sponges, ROS are generated which amplify the immune response, as they do in cytokine-activated macrophages in vertebrates.
Finally a pre-B-cell colony-enhancing factor has also been found in S. domuncula (Müller et al., 1999c). In the primmorph system of S. domuncula, the expression of the gene encoding this cytokine-like molecule was found to increase after exposure of the cells to membranes from another species, such as those from G. cydonium. This result provides a further indication that sponges have a molecular mechanism for the recognition of non-self.
Molecules in sponges comprising polymorphic Ig-like domains
The most striking similarity between molecules involved in the human adaptive immunity and sequences isolated from G. cydonium are among those which contain immunoglobulin (Ig)-like domains, the receptor tyrosine kinase (RTK) and the sponge adhesion molecules (SAM). The G. cydonium RTK molecule possesses in the deduced polypeptide structure two complete Ig-like domains (Müller and Schäcke, 1996). Two other SAM species have been isolated from this sponge, which do not encode a tyrosine kinase but also contain in the extracellular part two Ig-like domains (GC-SAM) (Blumbach et al., 1999). The longer form of the SAM, GC-SAML has an estimated size of size of 54 kDa, while the short form of SAM, GC-SAMS comprises only 313 residues and has a calculated Mr of 34 kDa.
The Ig-like domains found in GC-SAML and GC-SAMS as well as in the RTK display high sequence similarity to the V domain of mammalian immunoglobulin domains (Fig. 5A) (Blumbach et al., 1999; Du Pasquier, 2000). It is obvious that the key aa residues for the V frame are present in the sponge Ig-like domains, and also Chou-Fassman and Garnier-Robson strand predictions confirm the V nature of these molecules (Blumbach et al., 1999; Du Pasquier, 2000). Furthermore, the Ig-like domains of G. cydonium are polymorphic. At the amino acid level, Ig-like domain 1 (Fig. 5A) of GC-SAML (SAMLIG1_GC) shares 94% identical aa with GC-RTK (RTKIG1_GC); the Ig-like domains 2 from both RTK and GC-SAM show the same percentage of identical aa. At the nt level, 13 substitutions between Ig-like domains 1 of RTK and GC-SAMs were recorded; they lead to 6 aa substitutions. Within the Ig-like domain 2 regions 12 nt substitutions are present between RTK and GC-SAMs.
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In the mammalian Ig domains the ß-pleated sheets, which provide the framework region for binding to the antigens, are connected by hypervariable regions, termed complementary-determining regions (CDRs) (Kabat et al., 1991
). In a previous study (Pancer et al., 1996), the polymorphism among individuals with respect to the extracellular Ig-like domains of the RTK was documented.
Studies with the two G. cydonium genes GC-SAML and GC-SAMS were performed during auto- and allografting to estimate if those genes undergo a differential expression (Blumbach et al., 1999). RNA was extracted from the fusion zones from auto- and from allografts at time zero, and 2 days and 5 days after grafting. Northern blot analysis was performed using the respective cDNA probes. The blots demonstrated that both genes responded in autografts with an upregulation, while in the zone between allografts the strong increase in expression was seen only at day 2 (7 to 9-fold increase in the steady state level of transcripts) which during the longer course of the experiment dropped to zero; Figure 5B. This finding indicates that the two receptors, GC-SAML and GC-SAMS, are involved in histo(in)compatibility reactions, since additional experiments using tissue of different proliferation rates showed no alteration of the level of transcripts (Müller, unpublished). It is known that cell proliferation occurs in response to grafting.
Besides the high sequence similarity and high polymorphism of the Ig-like domains found in RTK as well as in the two SAMs, a further finding is remarkable. In the intracellular part of the GC-SAML molecule the immune receptor tyrosine-based inhibitory (ITIM)-motif is present. This motif, which shows the characteristic stretch VVpYEEVDG in the G. cydonium sequence (aa463 to aa470; Blumbach et al., 1999), is present in deuterostomes, especially mammalian inhibitory receptors (Bolland and Ravetch, 1999), which are found on natural killer cells. If phosphorylated, the ITIM-motif binds to tyrosine phosphatase(s), such as SHP-1 (reviewed in Bolland and Ravetch, 1999). In addition to the ITIM-motif-containing receptor an ITAM-motif-containing receptor (see: Howe and Weiss, 1995) has also been identified in sponges, the Rhesus (Rh) antigen-like protein (Seack et al., 1997).
In this context it is noteworthy that the phosphorylated ITAM motif binding Syk kinase, again an autapomorphic character of the Metazoa, is present in sponges (S. domuncula); Figure 6A. The deduced polypeptide, with a length of 567 aa and an Mr of 64,935, shares the characteristic domains of Syk kinases from higher metazoan phyla (Steele et al., 1999), the two SH2 domains (spanning aa9 to aa101 and aa163 to aa254) as well as the tyrosine kinase domain (aa278 to aa543); Figure 6A. Very significant is the finding that the Syk kinase, which is involved in the control of the activity of the receptors in the vertebrate lymphocytes, has so far been found in Hydra vulgaris and in deuterostomes but not in protostomes, e.g., C. elegans, (Steele et al., 1999). The finding, that the Syk kinase exists in sponges, underlines again the fact that at least some basic immune response pathways are conserved from sponges to human but have been lost in the protostomes (Müller et al., 1999a). This fact is also reflected in the phylogenetic tree (Fig. 6B) which shows that the Syk kinases, sponge (S. domuncula)—cnidarian (H. vulgaris)—vertebrate (human Syk-Zap70), cluster together and are separated from Non-Syk kinases, e.g., that from Drosophila melanogaster (SRC41) and Caenorhabditis elegans (v-ABL).
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This discovery, that both ITIM-receptors and ITAM-receptors, two counter-functional receptors of the adaptive immune system, and the tyrosine kinase Syk, which is involved in the modulation of their activities are present not only in mammalian but also in sponges once more suggests that allorecognition is an evolutionarily old system.
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CONCLUSION: THE IMMUNE SYSTEM OF PORIFERA AS ONE MAJOR EVOLUTIONARY NOVELTY OF URMETAZOA |
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Based on the analyses of protein sequences which control the cell-cell- and cell-matrix adhesion systems in sponges the monophyly of all metazoan animals could be established in 1994 (Müller et al., 1994
). This conclusion was confirmed later by analyzing the non-protein, rDNA sequences (Cavalier-Smith et al., 1996). Then came the surprising data which revealed that already in sponges a very complex immune system functions to allow histo(in)compatibility reactions as well as the defense against microbes (earlier review Müller et al., 1999a). The data contradict in a striking and an obvious manner the view, still stated in the present-day textbooks (e.g., Pechenik, 2000), that "cells from sponges are functionally independent."
In a recent review the hitherto known molecules involved in sponge immune response, have been analyzed with respect to their sequence similarity to both Protostomia (Drosophila melanogaster and Caenorhabditis elegans) and Deuterostomia (human) as well as to yeast (Saccharomyces cerevisiae), as the next closest related kingdom to Metazoa (Müller et al., 2001a). This comparison highlights three important aspects, that (i)—like in the case of the adhesion molecules—the immune molecules identified in sponges represent novelties of Metazoa and is not present in yeast, and (ii) the sequence similarity of the sponge molecules are higher to related ones in human than to those in D. melanogaster or C. elegans and (iii) several immune molecules which are found in sponges are absent in the two protostomian species. An example for the latter issue is the (2–5)A synthetase system, which has hitherto identified only in birds and mammals.
Taken together, the data presented strongly indicate that sponges are not primitive, but—in some aspects (e.g., with respect to the nerve system)—simple animals. Focusing on the immune system, one key mechanism which allowed the transition from functionally relatively independent cells to interacting cells in a complex metazoan organism, the high complexity and variety of pathways in sponges testifies that during the transition period from the common ancestor of yeasts and Metazoa to the Urmetazoa, the hypothetical ancestor of all Metazoa, major evolutionary novelties have been introduced. It might be speculated that the fast emergence of the different metazoan phyla, which evolved after the Porifera, was driven by the acquisition of modular molecules, involved in cell adhesion and the immune system, allowing the formation of complex Bauplans.
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ACKNOWLEDGMENTS |
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This work was supported by grants from the Deutsche Forschungsgemeinschaft [Mü/14-1], the Bundesministerium für Bildung und Forschung Germany [project: Center of Competence BIOTEC-MARIN] and the International Human Frontier Science Program (RG-333/96-M).
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FOOTNOTES |
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1 From the Symposium on Comparative Immunology presented at the Annual Meeting of the Society for Integrative and Comparative Biology, 2–6 January 2002, at Anaheim, California.
2 E-mail: WMUELLER{at}mail.UNI-Mainz.DE
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