Suppression of allograft rejection in the sponge Suberites domuncula by FK506 and expression of genes encoding FK506-binding proteins in allografts
1 Institut für Physiologische Chemie, Abteilung Angewandte Molekularbiologie, Universität, Duesbergweg 6, D-55099 Mainz, Germany,
2 Max-Planck-Institut für Züchtungsforschung, Carl-von-Linné-Weg 10, D-50829 Köln, Germany and
3 Center for Marine Research, Ruder Boskovic Institute, HR-52210 Rovinj, Croatia
*e-mail: WMUELLER{at}mail.UNI-Mainz.DE
Accepted April 25, 2001
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Summary |
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Key words: allograft, autograft, FK506, FK506-binding protein, sponge, Suberites domuncula.
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Introduction |
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Sponges are among the longest-studied recognition models in the Metazoa. The earliest studies were performed by Wilson (Wilson, 1907) who described that cells of only the one-species type reaggregate. Until recently, studies on immune-like histoincompatibility reactions were performed solely at the tissue and cellular levels. They conclusively revealed that histoincompatibility reactions between allogeneic donor tissue/cells from Callyspongia diffusa resulted in a cytotoxic reaction that occurs over a week for animals, over days for tissue and over hours for cells (Yin and Humphreys, 1996). Elements of the adaptive immunity system comparable with that found in higher vertebrates were identified in grafting experiments performed by Hildemann et al. (Hildemann et al., 1979), who succeeded in showing that after grafting for the second time the sponges react with an elevated rate of rejection in one species over the short term (2 weeks), but failed to do so in another sponge Hymeniacidon sinapium (Smith and Hildemann, 1984). The absence of adaptive immunity was reported by Van de Vyver (Van de Vyver, 1980). It has recently been established, by using molecular biological techniques, that sponges possess, in addition to their innate immunity, elements of an adaptive immunity (for a review, see Müller et al., 1999a).
A closer insight into the phylogenetic relationships between the immune systems of the oldest extant metazoan phylum (the Porifera) and the existing immune mechanisms in Protostomia and Deuterostomia became possible after identification of the potential function of molecules, possibly involved in the immune response, in sponges. Our group has focused on molecules involved in transplantation immunity in sponges, more specifically in the two demosponge species Suberites domuncula and Geodia cydonium. The data gathered hitherto are reviewed by Müller et al. (Müller et al., 1999a). The results showed that sponges possess key molecules of innate immunity, such as the (2',5')oligoadenylate synthetase (Kuusksalu et al., 1995; Kuusksalu et al., 1998; Wiens et al., 1999), which is induced in mammals by the cytokine interferon or by cytokine-like molecules, and also factors similar to those synthesized by cytokine-responsive macrophage molecules, which are presumably involved in the inflammatory response associated with human cardiac transplant rejection (Utans et al., 1995), such as the allograft inflammatory factor 1 (Kruse et al., 1999). In addition, precursors of an adaptive immune system have been identified in sponges, e.g. one molecule that is related to the human pre-B-cell colony-enhancing factor (Müller et al., 1999b) and molecules that contain immunoglobulin-like domains and are highly polymorphic (Pancer et al., 1996; Pancer et al., 1998). These immunoglobulin-like domains have been grouped with the class of variable immunoglobulin-like domains (for a review, see Williams and Barclay, 1988).
Expression of these potential immune molecules could be studied using the methods of parabiosis (Pancer et al., 1996; Müller et al., 1999a) or the insertion technique (Pancer et al., 1996). It was found that almost all autografts fused, whereas the allografts were rejected as a result of a coordinated interaction between proapoptotic molecules (e.g. a polypeptide that comprises two DEATH domains) and potential anti-apoptotic molecules (e.g. Bcl-2 homologous proteins; Wiens et al., 2000a; Wiens et al., 2000b). In addition, the data revealed that the level of transcripts for these factors changes during histocompatibility reactions in both autografts and allografts (for a review, see Müller et al., 1999a).
S. domuncula occurs in nature as red, orange, whitish or blue, or as a mixture of these colors (Arndt, 1935). Hence, graft experiments can, as a first approach, be performed without labelling the transplants. Parabionts attach firmly to each other in the initial phase after transplantation, irrespective of whether autogenic or allogenic tissue is used. After approximately 3 days, autografts fuse, while allografts reject each other and usually remain separate under the conditions used here (Müller et al., 1999a). The initial firm contact between allografts has also been reported from the sponge Callyspongia diffusa (Yin and Humphreys, 1996).
The aim of the present work was to identify, in allografts of S. domuncula, genes that are expressed in the zone of attachment. The technique of differential display of mRNA by means of the polymerase chain reaction (PCR) was employed. This strategy resulted in the isolation of a fragment and subsequently of the complete cDNAs encoding putative FK506-binding proteins, FKB1_SUBDO and FKB2_SUBDO. Finally, it was demonstrated that the immunosuppressant macrolide lactone FK506, also termed tacrolimus (Kino et al., 1987), which has been successfully applied clinically to prevent graft-versus-host diseases (for a review, see Jacobson et al., 1998), also effectively prevents allograft rejection in this sponge.
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Materials and methods |
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FK506 (tacrolimus) was a gift of Fujisawa Pharmaceutical Co. Ltd (Osaka, Japan). A stock solution of 2mgml-1 was prepared in dimethyl sulphoxide (DMSO).
Sponge
Live specimens of Suberites domuncula (Porifera, Demospongiae, Hadromerida) were collected by SCUBA near Rovinj (Croatia) from depths between 20 and 35m. The sponges were kept in Mainz (Germany) in aquaria (103l) at 17°C under continuous aeration for more than 1month before use in the experiments (Fig.1A,B). The specimens used for the allograft experiments were collected from different areas (23km apart), 5km west of Porec or 8km west of Rovinj. Control experiments revealed that none of the allografts fused (N=63).
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Tissue samples from the contact zones between allografts were taken 0, 10 or 12h after grafting for the isolation of RNA to identify the level of expression of the genes coding for FK506 binding proteins. In parallel, tissue samples were taken for histological analysis. For each experiment/treatment, at least seven parallel replicates were performed.
Incorporation studies of single cells
The procedure to dissociate cells from S. domuncula has been described previously (Müller et al., 1999b; Müller et al., 1999c). In brief, tissue cubes (45 cm3) were dissociated in Ca2+- and Mg2+-free artificial sea water (CMFSW). The cell suspension obtained was centrifuged (3000g; 5min) and washed twice with CMFSW. The cells were then resuspended in natural sea water supplemented with antibiotics (penicillin [100 i.u.ml-1] and streptomycin [100µgml-1]) and placed into culture Petri chambers (Falcon; diameter 9 cm).
After 2 days in sea water, the cells were incubated with different concentrations of FK506 for 24h as follows. Samples (5ml) of 3x106 cells were incubated with 925x106Bq of the labelled DNA precursor [3H]deoxythymidine (dThd) for 24h. Subsequently, the samples were analyzed for radioactivity in the acid-insoluble (DNA) fraction as described previously (Munro and Fleck, 1966; Müller et al., 1977). The amount of radioactivity incorporated was correlated with the amount of protein isolated from the cells used for the measurement.
The protein content was determined using two established methods (Lane, 1957; Lowry et al., 1951) with bovine serum albumin as standard. The values obtained by the two methods differed only by 8%.
Differential display
Total RNA was extracted from control tissue (non-graft tissue) and from the zone of attachment of allografts 1 day after parabiosis (attachment of two grafts), using TRIzol Reagent (GibcoBRL), as recommended by the manufacturer. Following procedures described previously (Liang and Pardee, 1992; Lohmann et al., 1995; McClelland et al., 1995), total RNA (5µg) was reverse-transcribed using T11AC as the 3' primer (Levi et al., 1997). The resulting cDNA was added to the polymerase chain reaction (PCR) using the arbitrary primer GTGATCGCAGG, the T11AC primer in the assay prepared as described previously (Levi et al., 1997) and -[32P]dATP. After 30 cycles of the PCR reaction at an annealing temperature of 45°C, the amplified radioactive fragments were separated on a 5% polyacrylamide sequencing gel (Pancer et al., 1998) and autoradiographed. One fragment of 185 nucleotides, which was expressed only in the contact zone of allografts and not in control tissue, was selected and used to screen the cDNA library. Three separate experiments were performed, and all gave the same results.
Library screening and isolation of two cDNAs encoding putative FK506-binding proteins
The 185 nucleotide fragment expressed only in the contact zone of allografts was used to isolate two related complete cDNAs, SUBDOFKB1 and SUBDOFKB2, encoding the two sponge FK506-binding proteins FKB1_SUBDO and FKB2_SUBDO. The S. domuncula cDNA library (Kruse et al., 1997) was used to screen (under lower-stringency hybridization conditions) plaques lifted from 3x105plaque-formingunits on nitrocellulose. Filters were hybridized at 37°C overnight in 35% formamide, 5x SSC (1x SSC is 0.15moll-1 sodium chloride, 0.015moll-1 sodium citrate), 0.02% SDS, 0.1% N-laurylsarcosine and 1% blocking reagent (Boehringer Mannheim), containing 10ng ml-1 of DIG-labelled probe. Filters were washed twice in 2x SSC, 0.1% SDS (5min, 22°C), followed by two additional washes in 0.1x SSC, 0.1% SDS (15min, 42°C). Positive clones were detected with an alkaline-phosphatase-conjugated anti-DIG antibody using BCIP/NBT (5-bromo-4-chloro-3-indolyl-phosphate/4-nitroblue tetrazolium chloride) as substrate (Blake et al., 1984).
The clones obtained were sequenced using an automatic DNA sequencer (Li-Cor 4200). The longest insert obtained for the shorter species, SUBDOFKB1, was 474 nucleotides long [excluding the poly(A) tail] and that for SUBDOFKB2, the longer form, was 683 nucleotides long.
Sequence comparisons
The sequences were analyzed using the sequence similarity algorithm Blast (Altschul et al., 1990). Multiple alignment was performed with CLUSTAL W Ver. 1.6 (Thompson et al., 1994), and graphics were prepared with GeneDoc (Nicholas and Nicholas, 1997). The phylogenetic tree was constructed on the basis of amino acid sequence alignments by neighbour-joining, as implemented in the Neighbor program from the PHYLIP package (Felsenstein, 1993). The distance matrices were calculated using the Dayhoff PAM matrix model, as described previously (Dayhoff et al., 1978). The degree of support for internal branches was further assessed by bootstrapping (Felsenstein, 1993).
Northern blotting
Sponge tissue samples from the contact zones (from both auto- and allografts) were pulverized in liquid nitrogen, and RNA was extracted using TRIzol reagent. A sample of 5µg of total RNA was electrophoresed through a 1% formaldehyde/agarose gel and blotted onto Hybond N+ membrane following the manufacturers instructions (Amersham; Little Chalfont, Buckinghamshire, UK) (Wiens et al., 1998). Hybridization was performed at lower stringency (two washes with 2x SSC, 0.1% SDS and two additional washes with 0.2x SSC, 0.1% SDS at room temperature 20°C) with two probes: (i) the complete SUBDOFKB1 cDNA to identify transcripts encoding the FKB1_SUBDO and FKB2_SUBDO molecules; and (ii) as a control, the complete sequence (1.5 kilobase-pairs, kb) of S. domuncula ß-tubulin (SDBTUB; M. Kruse and W. E. G. Müller, manuscript in preparation) was used as a probe for the northern blot experiments.
The chemiluminescence procedure was used to quantify northern blot signals (Stanley and Kricka, 1990); CDP-Star was used as substrate. The screen was scanned with the GS-525 Molecular Imager (Bio-Rad; Hercules, CA, USA).
Histological analysis
Fresh tissue was fixed in 4% (w/v) paraformaldehyde in Ca2+- and Mg2+-containing artificial sea water (Pancer et al., 1996). After dehydration, the samples were embedded in Technovit 8100 (Beckstead, 1985), according to the manufacturers instructions. Sections (9µm thick) were prepared and stained with 5% Indian ink [Pelikan, Hannover; no. 17 black (221143)] in 10% methanol (supplemented with 3% glacial acid) for 1h and then washed with water. The tissue was inspected using an Olympus AHBT3 microscope.
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Results |
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This 185 base pair (bp) fragment was used to screen the cDNA library. Two forms of cDNA were isolated that differed in sequence and length. The cDNA of the shorter form (474bp) is termed SUBDOFKB1 (GenBank accession number AJ278329) and that of the longer one (683bp) SUBDOFKB2 (GenBank accession number AJ278328). Northern blot analysis identified an RNA species of 0.62kb for SUBDOFKB1 and of 0.79kb for SUBDOFKB2 (see below), indicating that the full-length clones had been isolated.
The potential open reading frame (ORF) for the protein deduced from SUBDOFKB1, FKB1_SUBDO, starts at nucleotides 2931, ends at stop codon nucleotides 353355 and encodes a polypeptide of 108 residues (Fig.2A). On the basis of the presence of the two FKBP-type peptidyl-prolyl cistrans isomerase signatures, the first site is found at amino acid residues 2540 (VHYTGTLTNGKKFDSS) and the second at amino acid residues 5684 (VIRGWDEGVAKMSVGQRAKLTCSSDYAYG), so the polypeptide can be grouped with the FK506-binding proteins, which are cistrans peptidyl-prolyl isomerases (Harding et al., 1989; Fig.2A). The calculated Mr of FKB1_SUBDO is 11,645. Using the method of Rao and Argos (PC/GENE Data Banks CD Rom, 1995) no transmembrane helix was detected.
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The sequences were analyzed using the computer program Blast. The sponge sequences share more than 80% identity (and more than 90% similarity plus identity) with the related sequences from animals and more than 70% identity and and more than 80% similarity with yeast and plant sequences. Similarity to the sequences for monocellular eukaryotes (23% identity and 30% similarity; Trypanosoma cruzi, accession number Q09734) and to bacteria (23% identity and 30% similarity; Porphyromonas gingivalis AAD33931) is low. A phylogenetic tree, rooted with the plant FK506-related protein from Arabidopsis thaliana, revealed that this sequence, together with the yeast sequence, forms the basis of the tree, while the metazoan FK506-binding proteins form a separate branch with the sponge molecule as the common ancestor (Fig.2B).
These data show that S. domuncula contains two genes encoding FK506 polypeptides. In view of previous data which demonstrated that the drug FK506 blocks a common intermediate step required for the T-cell response to antigen presentation (Schreiber and Crabtree, 1992) and the findings that proliferation of yeast cells can be modulated by FK506 (Cardenas et al., 1994), we attempted to determine whether FK506 exerts (i) a toxic effect on sponge cells and/or (ii) an immunosuppressive effect on S. domuncula grafts.
Toxicity studies with FK506
Incorporation studies with [3H]dThd were performed to measure the effects of FK506 on DNA synthesis. The results revealed that cells incubated for 24h in the absence of the drug incorporated 742±78ctsmin-1mg-1protein. When FK506 was added at a concentration of 10ng ml-1, the amount incorporated did not change significantly (P0.1). However, when concentrations of drug above 100ng ml-1 were used, the amount incorporated dropped to 315±45ctsmin-1mg-1protein (Fig.3) (P<0.001). The concentration used for the transplantation studies, 20ng ml-1, was not inhibitory for incorporation of [3H]dThd into DNA fraction (788±85c.p.m.mg-1protein). At 200ng ml-1, the FK506 inhibition of incorporation is pronounced (373±65c.p.m.mg-1protein) (P<0.001). From these results, we conclude that concentrations below 20ng ml-1 of FK506 are not toxic for S. domuncula.
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To investigate whether this surprising effect, the fusion of allografts in the presence of low concentrations of FK506, is only a transient process and whether the allografts reject each other again later, the transplant incubation periods were extended for up to 10 days and, even after this longer incubation time, no non-fusion could be detected (results not shown).
Microscopic analysis
The attachment sites of autografts and of allografts were inspected microscopically at days 1 and 5 after transplantation. At day 1, the grafts attach to each other and close contact develops in the central part of the grafts. This contact is also caused by the slight pressure on the tissue by the fibres used to fix the parabionts (Fig.4A). A cleft was seen between the grafts at the rim of the parabionts, irrespective of whether the tissues were from the same or from different individuals (Fig.4A,C,E,G). In Fig.4A,C, the transition from the outer cleft-zone to the inner attachment zone is shown.
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Expression of the FK506-binding proteins
From an earlier study with monkey cells, it is known that expression of the FK506-binding protein can be modulated by glucocorticoids, e.g. dexamethasone (Reynolds et al., 1998), which are known to act also as potent suppressors of the cellular immune response in allografts/xenografts (Hudde et al., 1999; Nielsen, 2000). Therefore, the level of expression was determined in tissue from the attachment zone 12h after grafting. The sizes of the two transcripts, SUBDOFKB1 and SUBDOFKB2, were determined by northern blotting (at low stringency); the blot of SUBDOFKB1 showed a band at 0.62kb, and the blot of SUBDOFKB2 showed a band at 0.79kb (Fig.5A). The level of expression of the shorter form of the FK506-binding protein (SUBDOFKB1) was approximately five times higher (Fig.5Biii; comparison between the signal intensities of FKB1 and FKB2) than that observed for the longer form (SUBDOFKB2), which we attribute to the fact that SUBDOFKB1 cDNA (shorter form) was used as the probe for the hybridization experiments.
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These data indicate that in allografts FK506 induces expression of the two sponge FK506-binding proteins.
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Discussion |
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In the present study, the technique of differential display was chosen to identify molecules that are newly expressed in the attachment zone of allografts. These molecules were identified as FK506-binding proteins on the basis of their high sequence similarity to related molecules from the Metazoa, yeast and plants. These molecules function in those organisms as peptidyl-prolyl cistrans isomerase, an enzyme that is assumed to be involved in the folding of proteins (Liu et al., 1990). Furthermore, the peptidyl-prolyl cistrans isomerase(s) apparently carry out a series of other biological functions that can be predicted also to be crucial for sponge metabolism, channel gating, association with cellular receptors, association with receptors involved in immune response, gene transcription or cell cycle control (as summarized by Galat and Rivière, 1998). In addition, in vertebrates, it has been shown that FK506 binds to this isomerase and it was therefore termed FK506-binding protein or immunophilin (Mouzaki and Rungger, 1994; Shaw et al., 1995). The complex formed (FK506/FK506-binding protein) inhibits the phosphorylase activity of calcineurin (Liu et al., 1991), thus preventing translocation of the nuclear factor of activated T-cells (NFAT) into the nucleus. In this way, FK506 inhibits interleukin-2 gene expression and prevents the transformation of precursor helper T lymphocytes into antigen-conditioned helper T lymphocytes (Mouzaki and Rungger, 1994; Shaw et al., 1995; for a review, see Jacobson et al., 1998).
Two genes encoding the FK506-binding proteins have been isolated from S. domuncula and extend our understanding of the role of FK506. The polypeptides deduced from the two genes comprise two characteristic peptidyl-prolyl cistrans isomerase signatures. The sequences display an overall high sequence similarity to mammalian, yeast and plant polypeptides (>70% identity and >80% similarity). In S. domuncula, two forms of FK506-binding protein are present: one that has no transmembrane region and is apparently a cytosolic protein, and a second that contains a transmembrane segment. The existence of more than one form of FK506-binding proteins has also been reported for other metazoan organisms, such as human (Jin et al., 1991). Future studies must clarify whether the two sponge genes have different roles; at present, however, on the basis of the same expression pattern in the graft attachment zone, we have no evidence of different functions, at least with respect to the immune response.
It is now accepted that all metazoan phyla, including the Porifera, evolved from one common ancestor (Müller, 1995); this view is based on the fact that sponges comprise homologous cellcell/matrix adhesion systems, e.g. the integrin-mediated adhesion receptor (Wimmer et al., 1999a; Wimmer et al., 1999b), and possess molecules likely to be involved in immune reaction, e.g. the cytokine-related molecules and the polymorphic immunoglobulin domains (for a review, see Müller et al., 1999a), which are very similar to those of higher metazoan animals. We wished to determine whether immune reactions in sponges can be modulated by drugs that have successfully been applied in higher metazoan phyla, and especially in humans. We therefore investigated whether allograft rejection in sponges is suppressed, as in mammalian systems, by the drug FK506. This macrolide lactone compound was selected since it was the most promising candidate for potential binding to the cloned FK506-binding proteins and because it might initiate immunosuppression.
The results presented show that, at concentrations above 100ng ml-1, FK506 causes a significant inhibition (P<0.001; Fig.3) of DNA synthesis in vitro in S. domuncula cells. At higher doses, toxic effects have been reported in clinical transplantation studies (Pizzolato et al., 1998). This toxic effect is attributable not to inhibition of the isomerase but to adverse effects on the calcineurin-mediated signal transduction system, resulting in reduced cell survival and T-cell activation (for a review, see Cardenas et al., 1999).
The effect of FK506 on the process of fusion of allografts in S. domuncula has been studied within the concentration range used for the in vitro experiments. The results show that, at concentrations of FK506 in the surrounding medium above 200ng ml-1, all allografts were rejected. At the high concentration of 2000ng ml-1, toxic effects on the grafts were also detected; a decrease in DNA content of the tissue was measured, which can be taken to indicate a loss of cells (data not shown). However, if the non-toxic concentration of 20ng ml-1 was applied, fusion of the allografts occurred, and persisted for at least 10 days. Within such a long transplantation period, untreated allografts are either split into two separate graft units (for a review, see Müller et al., 1999a), or one graft undergoes apoptotic death as a result of downregulation of the expression of anti-apoptotic genes such as Bcl-2 (Wiens et al., 2000a; Wiens et al., 2000b). It has recently been shown in the Geodia cydonium allograft system that the grafts undergo apoptosis as a result of upregulation of the genes coding for DEATH domain-containing protein and for an enzyme involved in inflammation, the LTB4 12-hydroxy-dehydrogenase (Wiens et al., 2000b).
The concentration of FK506 used clinically to suppress allograft rejection ranges from 300 to 20ngkg-1 (for a review, see Laskow et al., 1998). In the studies presented here, it was found that the rejection/non-fusion process could be abolished by addition of 20ng ml-1 FK506 to the medium surrounding the grafts. In humans, the drug is unevenly distributed among organs (for a review, see Jacobson et al., 1998), so that a higher overall drug concentration is required. In contrast in sponges, which are not compartmented into organs (Simpson, 1984), FK506 is probably distributed homogeneously.
In addition, we found that, in the attachment zone of FK506-untreated and FK506-treated allografts, the genes encoding the two binding proteins for FK506 are upregulated. In the absence of the drug, this expression is low, but the steady-state levels of the transcripts for the FK506-binding proteins 1 and 2 in the allograft zones are elevated and reach levels of 30% of the levels of ß-tubulin transcripts. At present, we explain this observation by assuming that, in the presence of the drug, the FK506-binding proteins become upregulated, perhaps to support cell survival and/or to promote immune activation of the cells. It is conceivable that overexpression of the FK506-binding proteins causes a reduction/abolition of the intracellular inhibitory response to FK506 and perhaps even an augmentation of calcineurin-mediated cell activation. However, calcineurin in mammalian cells, or in yeast, fulfils several roles that are under intense investigation (for a review, see Cardenas et al., 1999). In the yeast system, it has been elegantly demonstrated that inhibition of the FK506-mediated calcineurin signal-transduction system results in reduced cell survival during cation stress (for a review, see Cardenas et al., 1998).
In earlier studies using the sponge Callyspongia diffusa, it was reported that, when tissue pieces from different specimens are attached to each other, they form a contact zone in which cytotoxic reactions proceed (Yin and Humphreys, 1996). The dominant cell types involved in this cytotoxic recognition process are the gray cells. These authors propose the following series of events that result finally in the histoincompatibility reactions: recognition of transplants; generation of signals that suppress cell aggregation; accumulation of gray cells at the attachment zone; initiation of cytotoxic processes. Our results support this observation, indicating that, shortly after transplantation, an accumulation of cells occurs at the attachment zone between allografts. We have not yet tried to identify this cell type unequivocally since the characterization of sponge cells is difficult. However, on the basis of the intense fluorescence of the cells within the attachment zone, a characteristic feature of gray cells (Kuhns et al., 1980), it appears likely that they are indeed gray cells (data not shown).
There are intriguing recent findings indicating that, in contrast to the reaction in the attachment zone of autografts of the sponge S. domuncula, the expression of genes for two cytokines is upregulated in allografts. Expression of the allograft inflammatory factor 1, which displays high sequence similarity to the corresponding factor from vertebrates, increases strongly during the first days after transplantation in allografts; in autografts, no change in expression was observed (Kruse et al., 1999). The expression of a second cytokine, the endothelial-monocyte-activating polypeptide (Pahler et al., 1998), was also found to be upregulated in the attachment zone only of allografts of S. domuncula (M. Kruse and W. E. G. Müller, in preparation). In mammalian systems, these two factors are highly expressed in activated rat macrophages and monocytes (Schlüsener et al., 1999), an effect that was blocked by the glucocorticoid dexamethasone. Dexamethasone also suppresses allograft rejection in humans (Hudde et al., 1999). Studies are in progress to determine whether the expression pattern of these two cytokines can be modulated (a downregulation is expected) by FK506.
Taken together, these data show that sponges possess an immune system that shares high structural and functional similarities with those of higher metazoan phyla. Sponges contain molecules likely to be involved in immune reactions that share sequence similarity with those found in deuterostomian Metazoa, e.g. apoptotic molecules (DEATH domain-containing molecules; Wiens et al., 2000a; Wiens et al., 2000b), proteins involved in innate immunity, (2',5')oligoadenylate synthetase (Wiens et al., 1999), or cytokines (pre-B-cell related colony-enhancing factor) (Müller et al., 1999b). Here, we have shown that the drug FK506 used in human therapy for the prevention of allograft rejection functions in sponges in a similar or the same manner. This surprising finding supports a recent study showing that histoincompatibility in the sponge Microciona prolifera is suppressed by the immunosuppressant cyclosporin A (Humphreys, 1999). These new findings support earlier proposals that factors involved in human diseases, e.g. myotrophin in cardiovascular disease (Schröder et al., 2000), are highly conserved and occur in the phylogenetically oldest animal phylum. Our data now add the fact that a drug, FK506, successfully prevents a disease-related process in sponges. On the basis of these data, we propose that sponges, which branched off first from the common ancestor of all Metazoa (Müller, 1995), possess key pathways for disease control seen in mammals and current species, and respond to drugs successfully used to the treat disorders in higher mammals.
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Acknowledgments |
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References |
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---|
Altschul, S. F., Gish, W., Miller, W., Myers, E. W. and Lipman, J. (1990). Basic local alignment search tool. J. Mol. Biol. 215, 403410.[Medline]
Amano, S. (1990). Self and nonself recognition in a calcareous sponge, Leucandra abratsbo. Biol. Bull. 179, 272278.
Arndt, W. (1935). Porifera. In Tierwelt der Nord- und Ostsee (ed. G. Grimpe), pp. IIIa1IIIa139. Leipzig: Akademische Verlagsgesellschaft.
Beckstead, J. H. (1985). Optimal antigen localization in human tissues using aldehyde fixed plastic-embedded sections. J. Histochem. Cytochem. 9, 954958.
Blake, M. S., Johnston, K. H., Russel-Jones, G. J. and Gotschlich, E. C. (1984). A rapid, sensitive method for detection of alkaline phosphatase-conjugated anti-antibody on Western blots. Analyt. Biochem. 136, 175179.[Medline]
Buss, L. (1987). The Evolution of Individuality. Princeton: Princeton University Press.
Cardenas, M. E., Cruz, M. C., Poeta, M. D., Chung, N., Perfect, J. R. and Heitman, J. (1999). Antifungal activity of the antineoplastic agents: Saccharomyces cerevisiae as a model system to study drug action. Clin. Microbiol. Rev. 12, 583611.
Cardenas, M. E., Lorenz, M., Hemenway, C. and Heitman, J. (1994). Yeast as model T cells. Persp. Drug Discovery Design 2, 103126.
Cardenas, M. E., Sanfridson, A., Cutler, N. S. and Heitman, J. (1998). Signal transduction cascades as targets for therapeutic intervention by natural products. Trends Biotechnol. 16, 427433.[Medline]
Curtis, A. S. G., Kerr, J. and Knowlton, N. (1982). Graft rejection in sponges. Genetic structure and rejecting populations. Transplantation 33, 127133.[Medline]
Dayhoff, M. O., Schwartz, R. M. and Orcutt, B. C. (1978). A model of evolutionary change in protein. In Atlas of Protein Sequence and Structure (ed. M. O. Dayhoff), pp 345352. Washington DC: Nat. Biomed. Res. Foundation.
Felsenstein, J. (1993). PHYLIP, ver. 3.5. Seattle: University of Washington.
Galat, A. and Rivière, S. (1998). Peptidyl-prolyl Cis/trans Isomerases. Oxford: Oxford University Press.
Harding, M. W., Galat, A., Uehling, D. E. and Schreiber, S. L. (1989). A receptor for the immunosuppressant FK506 is a cistrans peptidyl-prolyl isomerase. Nature 341, 758760.[Medline]
Hildemann, W. H., Johnston, I. S. and Jokiel, P. L. (1979). Immunocompetence in the lowest metazoan phylum: Transplantation immunity in sponges. Science 204, 420422.[Medline]
Hudde, T., Minassian, D. C. and Larkin, D. F. (1999). Randomised controlled trial of corticosteroid regimens in endothelial corneal allograft rejection. Br. J. Ophthalmol. 83, 13481352.
Humphreys, T. (1999). Regulatory mechanisms of immune cells in sponges. Mem. Queensland Mus. 44, 248.
Ilan, M. and Loya, Y. (1990). Ontogenetic variation in sponge histocompatibility responses. Biol. Bull. 179, 279286.
Jacobson, P., Ubert, J., Davis, W. and Ratanatharatorn, V. (1998). Tacrolimus: a new agent for the prevention of graft-versus-host disease in hematopoietic stem cell transplantation. Bone Marrow Transplantation 22, 217225.[Medline]
Jin, Y. J., Albers, M. W., Lane, W. S., Bierer, B. E., Schreiber, S. L. and Burakoff, S. J. (1991). Molecular cloning of a membrane-associated FK506- and rapamycin-binding protein, FKBP-13. Proc. Natl. Acad. Sci. USA 88, 66776681.[Abstract]
Kino, T., Hatanaka, H., Hashimoto, M., Nishiyama, M., Goto, T., Okuhara, M., Kohsaka, M., Aoki, H. and Imanaka, H. (1987). FK506, a novel immunosuppressant isolated from Streptomyces. I. Fermentation, isolation and physico-chemical and biological characteristics. J. Antibiotics 40, 12491255.[Medline]
Kozak, M. (1991). An analysis of vertebrate mRNA sequences: Intimations of translational control. J. Cell Biol. 115, 887903.[Abstract]
Kruse, M., Müller, I. M. and Müller, W. E. G. (1997). Early evolution of metazoan serine/threonine- and tyrosine kinases: identification of selected kinases in marine sponges. Mol. Biol. Evol. 14, 13261334.[Abstract]
Kruse, M., Steffen, R., Batel, R., Müller I. M. and Müller W. E. G. (1999). Differential expression of allograft inflammatory factor 1 and of glutathione peroxidase during auto- and allograft response in marine sponges. J. Cell Sci. 112, 43054313.
Kuhns, W. J., Bramson, S., Simpson, T. L., Burkhart, W., Jumblatt, J. and Burger, M. M. (1980). Fluorescence antibody localization of Microciona prolifera aggregation factor and its baseplate component. Eur. J. Cell Biol. 23, 7379.[Medline]
Kuusksalu, A., Pihlak, A., Müller, W. E. G. and Kelve, M. (1995). The (2-5)oligoadenylate synthetase is present in the lowest multicellular organisms, the marine sponges. Demonstration of the existence and identification of its reaction products. Eur. J. Biochem. 232, 351357.[Abstract]
Kuusksalu, A., Subbi, J., Pehk, T., Reintamm, T., Müller, W. E. G. and Kelve, M. (1998). (2'-5')Oligoadenylate synthetase in marine sponges: Identification of its reaction products. Eur. J. Biochem. 257, 420426.[Abstract]
Lane, E. (1957). Spectrophotometric and turbidimetric methods for measuring proteins. Meth. Enzymol. 3, 447454.
Laskow, D. A., Neylan, J. F., Shapiro, R. S., Pirsch, J. D., Vergne-Marini, P. J. and Tomlanovich, S. J. (1998). The role of tacrolimus in adult kidney transplantation: a review. Clin. Transplantation 12, 489503.[Medline]
Levi, L., Douek, J., Osman, M., Bosch, T. C. G. and Rinkevich, B. (1997). Cloning and characterization of BS-cadherin, a novel cadherin from the colonial urochordate Botryllus schlosseri. Gene 200, 117123.[Medline]
Liang, P. and Pardee, A. B. (1992). Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 257, 967971.[Medline]
Liu, J., Albers, M. W., Chen, C. M., Schreiber, S. L. and Walsh, C. T. (1990). Cloning, expression and purification of human cyclophilin in Escherichia coli and assessment of the catalytic role of cysteines by site directed mutagenesis. Proc. Natl. Acad. Sci. USA 87, 23042308.[Abstract]
Liu, J., Farmer, J. D., Lane, W. S., Friedman, J., Weissmann, I. and Schriber, S. L. (1991). Calcineurin is a common target of cyclophilin-cyclosporin A and FKBPFK506 complexes. Cell 66, 807815.[Medline]
Lohmann, J., Schickel, H. and Bosch, T. C. G. (1995). REN display, a rapid and efficient method for nonradioactive differential display and mRNA isolation. BioTechniques 18, 200201.[Medline]
Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951). Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265275.
McClelland, M., Mathieu-Daude, F. and Welsh, J. (1995). RNA finger-printing and differential display using arbitrarily primed PCR. Trends Genet. 11, 242246.[Medline]
Moscona, A. A. (1968). Cell aggregation: properties of specific cell-ligands and their role in the formation of multicellular systems. Devl. Biol. 18, 250277.[Medline]
Mouzaki, A. and Rungger, D. (1994). Properties of transcription factors regulating interleukin-2 gene transcription through NFAT binding site in untreated or drug-treated naive and memory T-helper cells. Blood 84, 26122621.
Müller, W. E. G. (1995). Molecular phylogeny of Metazoa (animals): Monophyletic origin. Naturwissenschaften 82, 321329.[Medline]
Müller, W. E. G. and Müller, I. M. (2000). How was the Protozoa Metazoa threshold crossed: The Urmetazoa. Comp. Biochem. Physiol. 126B (Suppl. 1), S69.
Müller, W. E. G., Blumbach, B. and Müller, I. M. (1999a). Evolution of the innate and adaptive immune systems: Relationships between potential immune molecules in the lowest metazoan phylum [Porifera] and those in vertebrates. Transplantation 68, 12151227.[Medline]
Müller, W. E. G., Perovic, S., Wilkesman, J., Kruse, M., Müller, I. M. and Batel, R. (1999b). Increased gene expression of a cytokine-related molecule and profilin after activation of Suberites domuncula cells with xenogeneic sponge molecule(s). DNA and Cell Biol. 18, 885893.[Medline]
Müller, W. E. G., Seibert, G., Breter, H. J., Maidhof, A. and Zahn, R. K. (1977). Effect of cordycepin on nucleic acid metabolism in L5178y cells and on nucleic acid synthesizing enzyme systems. Cancer Res. 37, 38243833.[Medline]
Müller, W. E. G., Wiens, M., Batel, R., Steffen, R., Borojevic, R. and Custodio, M. R. (1999c). Establishment of a primary cell culture from a sponge: Primmorphs from Suberites domuncula. Mar. Ecol. Progr. Ser. 178, 205219.
Munro, H. N. and Fleck, A. (1966). Recent developments in the measurement of nucleic acids in biological fluids. Analyst 91, 7887.[Medline]
Nicholas, K. B. and Nicholas, H. B., Jr (1997). GeneDoc: A Tool for Editing and Annotating Multiple Sequence Alignments. Version 1.1.004. Distributed by the author; cris.com/ketchup/genedoc.shtml.
Nielsen, L. L. (2000). NK cells mediate the anti-tumor effects of E1-deleted, type 5 adenovirus in a human tumor xenograft model. Oncol. Rep. 7, 151155.[Medline]
Pahler, S., Krasko, A., Schütze, J., Müller, I. M. and Müller, W. E. G. (1998). Isolation and characterization of a cDNA encoding a potential morphogen from the marine sponge Geodia cydonium, that is conserved in higher metazoans. Proc. R. Soc. Lond. B 265, 421425.[Medline]
Pancer, Z., Kruse, M., Müller, I. and Müller, W. E. G. (1997). On the origin of adhesion receptors of Metazoa: cloning of the integrin subunit cDNA from the sponge Geodia cydonium. Mol. Biol. Evol. 14, 391398.[Abstract]
Pancer, Z., Kruse, M., Schäcke, H., Scheffer, U., Steffen, R., Kovács, P. and Müller, W. E. G. (1996). Polymorphism in the immunoglobulin-like domains of the receptor tyrosine kinase from the sponge Geodia cydonium. Cell Adhes. Commun. 4, 327339.[Medline]
Pancer, Z., Skorokhod, A., Blumbach, B. and Müller, W. E. G. (1998). Multiple Ig-like featuring genes divergent within and among individuals of the marine sponge Geodia cydonium. Gene 207, 227233.[Medline]
PC/GENE Data Banks CD-ROM (1995). Release 14.0. Inc. Mountain View, CA, IntelliGenetics.
Perovic, S., Prokic, I., Krasko, A., Müller, I. M. and Müller, W. E. G. (1999). Origin of neuronal-like receptors in Metazoa: cloning of a metabotropic glutamate/GABA-like receptor from the marine sponge Geodia cydonium. Cell Tissue Res. 296, 395404.[Medline]
Pizzolato, G. P., Stajzel, R., Burkhardt, K., Megret, M. and Borisch, B. (1998). Cerebral vasculitis during FK506 treatment in a liver transplant patient. Neurology 50, 11541157.[Abstract]
Reynolds, P. D., Roveda, K. P., Tucker, J. A., Moore, C. M., Valentine, D. L. and Scammell, J. G. (1998). Glucocorticoid-resistant B-lymphoblast cell line derived from the Bolivian squirrel monkey (Saimiri boliviensis boliviensis). Lab. Anim. Sci. 48, 364370.[Medline]
Schäcke, H., Müller, W. E. G., Gamulin, V. and Rinkevich, B. (1994a). The Ig superfamily includes members from the lowest invertebrates to the highest vertebrates. Immunol. Today 15, 497498.[Medline]
Schäcke, H., Rinkevich, B., Gamulin, V., Müller, I. M. and Müller, W. E. G. (1994b). Immunoglobulin-like domain is present in the extracellular part of the receptor tyrosine kinase from the marine sponge Geodia cydonium. J. Mol. Recognition 7, 272276.
Schlüsener, H. J., Seid, K. and Meyermann, R. (1999). Effects of autoantigen and dexamethasone treatment on expression of endothelial-monocyte activating polypeptide II and allograft-inflammatory factor-1 by activated macrophages and microglial cells in lesions of experimental autoimmune encephalomyelitis, neuritis and uveitis. Acta Neuropathol. 97, 119126.[Medline]
Schreiber, S. L. and Crabtree, G. R. (1992). The mechanism of action of cyclosporin A and FK506. Immunol. Today 13, 136142.[Medline]
Schröder, H. C., Krasko, A., Batel, R., Skorokhod, A., Pahler, S., Kruse, M., Müller, I. M. and Müller, W. E. G. (2000). Stimulation of protein (collagen) synthesis in sponge cells by a cardiac myotrophin-related molecule from Suberites domuncula. FASEB J. 14, 20222031.
Shaw, K. T., Ho, A. W. and Raghavan, A. (1995). Immunosuppressive drugs prevent a rapid dephosphorylation of transcription factor NFAT1 in stimulated immune cells. Proc. Natl. Acad. Sci. USA 92, 1120511209.[Abstract]
Simpson, T. L. (1984). The Cell Biology of Sponges. New York: Springer-Verlag.
Smith, L. C. and Hildemann, W. H. (1984). Alloimmune memory is absent in Hymeniacidon sinapium, a marine sponge. J. Immunol. 133, 23512355.
Stanley, P. E. and Kricka, L. J. (1990). Bioluminescence and Chemiluminescence: Current Status. New York: John Wiley & Sons.
Thompson, J. D., Higgins, D. G. and Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 46734680.[Abstract]
Utans, U., Arceci, R. J., Yamashita, Y. and Russell, M. E. (1995). Cloning and characterization of allograft inflammatory factor-1: a novel macrophage factor identified in rat cardiac allografts with chronic rejection. J. Clin. Invest. 95, 29542962.[Medline]
Van de Vyver, G. (1980). Second-set allograft rejection in two sponge species and the problem of an alloimmune memory. In Phylogeny of Immunological Memory (ed. M. J. Manning), pp. 1526. New York: Elsevier.
Warburton, F. E. (1958). Reproduction of fused larvae in the boring sponge, Cliona celata. Nature 181, 493494.
Wiens, M., Koziol, C., Hassanein, H. M. A., Batel, R. and Müller, W. E. G. (1998). Expression of the chaperones 1433 and HSP70 induced by PCB 118 (2,3',4,4',5-pentachlorobiphenyl) in the marine sponge Geodia cydonium. Mar. Ecol. Prog. Ser. 165, 247257.
Wiens, M., Krasko, A., Müller, C. I. and Müller, W. E. G. (2000a). Molecular evolution of apoptotic pathways: cloning of key domains from sponges (Bcl-2 homology domains and death domains) and their phylogenetic relationships. J. Mol. Evol. 50, 520531.[Medline]
Wiens, M., Krasko, A., Müller, I. M. and Müller, W. E. G. (2000b). Increased expression of the potential proapoptotic molecule DD2 and increased synthesis of leukotriene B4 during allograft rejection in a marine sponge. Cell Death Differentiation 7, 461469.[Medline]
Wiens, M., Kuusksalu, A., Kelve, M. and Müller, W. E. G. (1999). Origin of the interferon-inducible (2'5')oligoadenylate synthetases: cloning of the (2'5')oligoadenylate synthetase from the marine sponge Geodia cydonium. FEBS Lett. 462, 1218.[Medline]
Williams, A. F. and Barclay, A. N. (1988). The immunoglobulin superfamily domains for cell surface recognition. Annu. Rev. Immunol. 6, 381405.[Medline]
Wilson, H. V. (1907). On some phenomena of coalescence and regeneration in sponges. J. Exp. Zool. 5, 245258.
Wimmer, W., Blumbach, B., Diehl-Seifert, B., Koziol, C., Batel, R., Steffen, R., Müller, I. M. and Müller, W. E. G. (1999a). Increased expression of integrin and receptor tyrosine kinase genes during autograft fusion in the sponge Geodia cydonium. Cell Adhesion Commun. 7, 111124.[Medline]
Wimmer, W., Perovic, S., Kruse, M., Krasko, A., Batel, R. and Müller, W. E. G. (1999b). Origin of the integrin-mediated signal transduction: functional studies with cell cultures from the sponge Suberites domuncula. Eur. J. Biochem. 178, 156165.
Yin, C. and Humphreys, T. (1996). Acute cytotoxic allogeneic histocompatibility reactions involving gray cells in the marine sponge, Callyspongia diffusa. Biol. Bull. 191, 159167.