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The Journal of Immunology, 2007, 179, 5927-5935
Copyright © 2007 by The American Association of Immunologists, Inc.
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Cyclosporin A Suspends Transplantation Reactions in the Marine Sponge Microciona prolifera1

Clarissa Sabella*,{dagger}, Ellen Faszewski*,{ddagger}, Lisa Himic*,{dagger}, Katherine M. Colpitts{dagger}, Jane Kaltenbach*,{dagger}, Max M. Burger*,§ and Xavier Fernàndez-Busquets2,*,¶

* Marine Biological Laboratory, Woods Hole, MA 02543; {dagger} Mount Holyoke College, South Hadley, MA 01075; {ddagger} Wheelock College, Boston, MA 02215; § Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland; and Bioengineering Institute of Catalonia, and Nanoscience and Nanotechnology Institute, Barcelona Science Park, University of Barcelona, Barcelona, Spain


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Sponges are the simplest extant animals but nevertheless possess self-nonself recognition that rivals the specificity of the vertebrate MHC. We have used dissociated cell assays and grafting techniques to study tissue acceptance and rejection in the marine sponge Microciona prolifera. Our data show that allogeneic, but not isogeneic, cell contacts trigger cell death and an increased expression of cell adhesion and apoptosis markers in cells that accumulate in graft interfaces. Experiments investigating the possible existence of immune memory in sponges indicate that faster second set reactions are nonspecific. Among the different cellular types, gray cells have been proposed to be the sponge immunocytes. Fluorescence confocal microscopy results from intact live grafts show the migration of autofluorescent gray cells toward graft contact zones and the inhibition of gray cell movements in the presence of nontoxic concentrations of cyclosporin A. These results suggest that cell motility is an important factor involved in sponge self/nonself recognition. Communication between gray cells in grafted tissues does not require cell contact and is carried by an extracellular diffusible marker. The finding that a commonly used immunosuppressor in human transplantation such as cyclosporin A blocks tissue rejection in marine sponges indicates that the cellular mechanisms for regulating this process in vertebrates might have appeared at the very start of metazoan evolution.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Ancestors of the MHC have recently been pushed back to urochordates (1, 2, 3), thus opening the question as to whether self-nonself recognition developed simultaneously with animal multicellularity. Sponges are the oldest extant metazoans (4) and, hence, are ideally suited for studying the evolutionary origins of allorecognition (5). The sessile lifestyle of sponges and the limited dispersal of their offspring leads to the close proximity of conspecifics, promoting frequent tissue contacts between relatives that elicit rejection responses mediated by complex cell recognition processes (6). When tissues from different individuals of a given sponge species are brought into contact, they either fuse or reject through cellular events similar to those observed in vertebrate grafts (7). Sponges possess elements involved in transplantation immunity that are key components of mammalian innate immunity (8). These include molecules containing scavenger receptor cysteine-rich domains (9), TLRs (10), the 2',5'-oligoadenylate synthetase (11), cytokine-like molecules (12), and also factors similar to those produced by cytokine-responsive macrophage molecules such as the allograft inflammatory factor 1 (13). In addition, hallmarks of adaptive immune systems such as Ig-like domains have been identified in sponges (14, 15), in some cases exhibiting intraspecific polymorphism (16, 17).

One of the characteristics of self-nonself recognition systems is their high specificity conferred by loci with as many as 200 alleles (18). The frequency of graft rejection in sponges is very high (19), requiring an extensive genetic polymorphism at the locus or loci controlling graft acceptance and rejection. Sponge cell adhesion is based on extracellular matrix (ECM)3 proteoglycans termed aggregation factors (AFs) (20). AFs are necessary and sufficient to aggregate sponge cells species-specifically, but the observation that sponges possess a wide variety of eumetazoan cell adhesion molecules hints that an AF-only model might be incomplete (21). In contrast, the elevated polymorphism of AF proteins (19, 22) suggests that some of them represent diverging forms adapted to a different function. It is therefore possible that the cell adhesion machinery can be shared in sponges, at least to a certain degree, with an incipient histocompatibility system (6). In parabiotic grafting experiments it has been shown that the intraspecific variability of AFs rivals that of the vertebrate MHC; any two individuals rejecting each other have different genomic sequences for AF proteins as indicated by restriction fragment length polymorphism analysis (19, 22). Other molecules involved in cell-ECM interactions such as {alpha} and beta integrins and the hyaluronan receptor CD44 have been found to be differentially expressed in sponge isografts and allografts (7, 23). Elevated integrin expression in the absence of appropriate ligands can promote apoptosis under otherwise permissive growth conditions (24). This observation indicates that integrins have a biosensory role, regulating cell death or survival in tissue remodeling events as a function of ECM composition. Apoptosis in sponges was first demonstrated by TUNEL assay in hibernating sponges that had undergone tissue regression as a naturally occurring winter event (25). Biochemical and immunohistochemical methods were later used to study apoptotic cell death in sponge extracts where caspase 3 activity was found to be greatly increased in allografts when compared with isografts (26, 27). Higher expression of other molecules potentially related to apoptosis has been detected in allografts of several sponge species (28, 29).

Despite this incipient knowledge at the molecular level of some components of the allogeneic recognition system in sponges (8), little is known about the actual underlying mechanism or the cells involved in the process. Sponges lack lymphocytes and the existence of immune cells in this phylum has been object of debate (30, 31). Among the several cellular types existing in Microciona prolifera, gray cells have been singled out as the immunocytes responsible for the allogeneic response observed in tissue transplantation experiments (7, 32, 33, 34).

In the present study, we examined the effect of the immunosuppressor cyclosporin A (CsA) on gray cells in allografts and isografts by using immunocytochemistry and in vivo fluorescence confocal microscopy as a first approach to unravel the intracellular pathways responsible for self-nonself recognition in marine sponges. Our data showing that CsA blocks gray cell motility are discussed in the frame of the existence of cell adhesion-related elements that could represent a basic scaffold for later evolved histocompatibility systems.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Materials and preparation of grafts

Marine sponges were collected by the Marine Resources Center of the Marine Biological Laboratory, Woods Hole, MA (www.mbl.edu), and maintained in a tank of running seawater. All chemicals were purchased from Sigma-Aldrich except where otherwise indicated. Two sponge papillae (elongated tissue branches ~2 cm in length) from the same individual were used for isografts and two papillae from different individuals of the same species for allografts. Papillae were cut only at one end and particular care was taken to not damage their epithelia. Each graft was held together with a no. 0 stainless steel insect pin, which was then pushed into the underside of a Styrofoam board floating in a tank of running seawater unless otherwise indicated. The entire grafting procedure was conducted under seawater.

Preparation of cell suspensions and 5-chloromethylfluorescein diacetate (CMFDA) treatment

Mechanical dissociation of cells to obtain cell suspensions was performed by squeezing sponge fragments through a 100-µm mesh cheesecloth submerged in seawater. For labeling with CellTracker Green 5-chloromethylfluorescein diacetate (CMFDA; Molecular Probes) before cell dissociation, the sponge fragments were incubated overnight at 16°C in seawater containing 5 µM CMFDA. Suspensions of cells (~3,800 cells/mm3 as determined by hemocytometer count) from two different individuals were mixed in 50-ml Falcon tubes and incubated with gentle shaking for 6 h at 16°C. A drop of the mixed cell suspension was then placed on a slide and stained with a 1% solution of trypan blue (Mallinckrodt) to detect dead cells. The trypan blue solution was prepared in seawater and after 30 min it was filtered. The sample was covered with a coverslip and cell-enriched areas were photographed either with bright field for trypan blue staining or fluorescence at the excitation wavelengths of 470 nm (CMFDA) and 420 nm (gray cells). The experiment was repeated three times and a total of 20 areas were analyzed for each case.

Cyclosporin treatment of grafts for immunohistochemistry

Before grafting, sponge tissue fragments from two different individuals were incubated in separate 50-ml Falcon tubes for 2 h at room temperature in 25 ml of seawater containing 10 µg/ml CsA. A stock solution was prepared by dissolving 10 mg of CsA in 2 ml of 100% DMSO and stored at –20°C. DMSO was present in the control experiments at the same concentration used in the CsA-containing assays. Control tissue fragments were incubated in 25 ml of seawater.

Immunohistochemical procedures

Grafts were removed from the seawater tank at the specified times and fixed overnight in 10% formalin prepared with Marine Biological Laboratory artificial seawater, washed in artificial seawater, and dehydrated in a series of ethanols (30, 50, and 70% in dH2O). Spicules in the grafts were dissolved by a 6-h treatment with 1.5 N hydrofluoric acid prepared in 80% ethanol. Grafts were further dehydrated in an ethanol series (80, 95%, and absolute), embedded in paraffin (pins removed with pliers as the paraffin hardened), sectioned at 7 µm, and the sections were affixed to slides. Sections on the slides were deparaffinized in xylene and hydrated to dH2O in a series of ethanols.

All primary Abs were obtained from BD Biosciences. Mouse monoclonal purified anti-mouse {alpha}3 integrin and rabbit anti-human active caspase 3 were used at a final concentration of 10 µg/ml. Rat anti-mouse CD44 was used at a final concentration of 2.5 µg/ml. The Abs used against Microciona cell surface molecules have been described as detecting single bands in Western blots of cell membranes (CD44) (7) and in whole cell extracts (integrin) (35). Biotinylated secondary Abs, rabbit anti-mouse, goat anti-rabbit, and rabbit anti-rat (Vector Laboratories), were used at a concentration of 28.8 µg/ml. For controls, 10 mM PBS (pH 7.4) was used in place of the primary Ab.

All staining steps were conducted at room temperature, each followed by a wash with PBS unless otherwise noted. To block endogenous peroxidase activity, sections were treated with 3% hydrogen peroxide for 3 min. To block nonspecific staining, sections were treated first for 30 min with 1% normal serum from the same type animal as that used for the secondary Ab and then with 2% BSA (immunohistochemical grade; Vector Laboratories) for 30 min with no PBS rinse in between. These steps were followed by incubation of sections with a primary Ab in a moist chamber for 2 h and a secondary Ab for 15 min. Ab stock solutions were prepared in normal serum and diluted in PBS for final concentrations. Sections were then treated with an avidin-biotin-peroxidase complex (Vectastain Elite ABC Kit; Vector Laboratories), and the primary Ab reaction was visualized as a brown precipitate by oxidation of the peroxidase substrate 3,3'-diaminobenzidine-H2O2 for ~3 min. Sections were dehydrated in a series of ethanols, cleared in xylene, and coverslips were mounted with Permount (Fisher Scientific).

For double staining of {alpha}3 integrin and caspase 3 or {alpha}3 integrin and CD44, two staining procedures were conducted sequentially. Active caspase 3 or CD44 was visualized by a second staining on the same section already treated for integrin as described above. After blocking with the appropriate 1% normal serum for 30 min, incubation with primary Abs for caspase 3 or CD44 was done overnight at 4°C. After incubation with biotinylated secondary Abs for 15 min, sites of the primary Abs were visualized as bright red fluorescence by the application of avidin-labeled alkaline phosphatase (Vectastain ABC-AP kit) and the phosphatase substrate Vector Red for 20 min. A rhodamine excitation and emission filter system was used for fluorescence photography.

Microphotography, colocalization analysis, and cell counts

Digital images were obtained with a Zeiss Axioplan 2 microscope equipped with a Zeiss Axiocam digital color camera and Zeiss Axiovision version 4.2 software. For colocalization analysis, bright field integrin images were converted to grayscale pictures and pixelated in the green channel. Caspase 3 and CD44 Vector Red fluorescent images in the red channel were used as such. Green and red channels were finally merged in the same image using Adobe Photoshop version 6.0 software. Cells were counted in a 292-mm2 area centered on the zone of contact. One to three different sections of tissue were counted and the average of three independent counts was calculated.

Transmission electron microscopy

Mechanically dissociated M. prolifera cells were fixed for 10 min with 3% paraformaldehyde/Sörensen phosphate buffer (pH 7.4). The cells were then dehydrated in a series of graded ethanols and cleared in propylene oxide before embedding in Spurr low viscosity resin. Sections were contrasted in a mixture of methylcellulose and uranyl acetate and observed with a JEOL 1010 transmission electron microscope.

Cell viability assays

Cell viability was determined by measuring mitochondrial activity. Sponge cells were chemically dissociated as previously described (36) and diluted in artificial Ca2+- and Mg2+-free seawater (prepared according to Marine Biological Laboratory standards) to a final concentration of ~3,800 cells/mm3 (determined by hemocytometer count). To 1 ml of this cell suspension were added 4 µl of either 100% DMSO as negative control (0.4% final concentration), different dilutions of CsA in DMSO, or SDS to a final concentration of 0.5% as a positive control. For assays with intact tissue, freshly excised M. prolifera papillae were placed into 1 ml of seawater containing 4 µl of either 100% DMSO or 5 mg/ml CsA in DMSO (20 µg/ml final CsA concentration). After incubation at room temperature for the times indicated, 100 µl of WST-1 reagent (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate; Roche Diagnostics) was added to determine cell viability and after 30 min of incubation the samples were read in a Beckman DU640 spectrophotometer at A440. In intact tissue assays, the papillae were weighed at the end of the experiment and the absorbance of each sample was normalized for equal tissue mass. Samples were prepared in triplicate for each experiment.

Fluorescence confocal microscopy

Allografts and isografts were made immediately before the start of the experiment and placed in petri dishes filled with either seawater or seawater containing 2.5 µg of CsA per milliliter. Grafts were scanned with the confocal microscope at 20°C for a 12-h period. Upon visualization of the zone of contact, fluorescent images were collected using a z scan consisting of 22–27 sections through the zone every 15 min. The excitation wavelength was set in the UV range (below 380 nm) with a two-photon laser and the yellow autofluorescence of gray cells was recorded at 570 nm. A Zeiss LSM-510 Meta NLO confocal microscope equipped with Zeiss LSM software was used. The autofluorescence of gray cells has been used to purify them by fluorescence-assisted cell sorting to >95% purity (37), indicating that this method tracks essentially gray cells and not other cell types.

Online supplemental material

Each of the five experiments presented in Fig. 7 as time frames is accessible as a video designated as follows: Fig 7video1.mov (control allograft); Fig 7video2.mov (CsA-treated allograft); Fig 7video3.mov (control isograft); Fig 7video4.mov (CsA-treated isograft); and Fig 7video5.mov (control isograft).4 We also make accessible an additional video showing a control allograft with a larger initial number of gray cells present (Fig 7video6.mov).


Figure 7
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  		FIGURE 7. In vivo fluorescence confocal microscopy study of the effect of CsA on M. prolifera grafts. A, Allogeneic or isogeneic grafts were prepared as described in Materials and Methods, placed in a petri dish containing either seawater or seawater containing 2.5 µg of CsA per milliliter, and examined with a confocal microscope for 12 h. Each frame corresponds to the projection of all the confocal sections collected for the chosen time points. Arrowheads indicate individual cells that can be seen immobilized in the videos presented as online supplemental material. B, Control isograft where the apposed tissues do not make contact. Note the incipient cell bridges (arrows) that start growing into the gap.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Contact reactions in sponges

Sponge tissue responds differently depending on the type of surface it makes contact with. In our grafting assays (Fig. 1A), sponge papillae are stacked on a pin that is placed in a tank with running seawater. After 72 h three different outcomes are evident depending on each pair of interacting surfaces. Xenogeneic contacts between tissues from different sponge species (e.g., Microciona/Cliona celata, M1/C) show an intense yellowish color in the contact zone resulting from extensive cell lysis (7, 33). Allogeneic interactions between different individuals from the same species (interface M1/M2) undergo more contained cell lysis that subsides after 24 h, making it difficult to distinguish the contact borderline in the 72-h graft presented here (see Fig. 4A for a better visualization of an allogeneic contact zone). Isogeneic contacts like M1/M1 merge without any indication of cell lysis, either by direct observation or by microscopical analysis of histochemical preparations (7). Finally, contact reactions with an inert material such as Styrofoam (M2/S) simply end up with the sponge tissue overgrowing the foreign object as it would when crawling on a rock or other nonliving substrate. Allograft contact zones are the result of the alignment of cells (Fig. 1B) that ultimately lay down a collagen barrier separating allogeneic tissues (6).


Figure 1
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  		FIGURE 1. Contact reactions in sponges. A, Tissue fragments of Cliona celata (C), two different M. prolifera individuals (M1 and M2), and a Styrofoam bead (S) were successively impaled on a pin according to the scheme on the upper left. The picture was taken after 72 h of incubation in seawater. Arrowheads indicate lighter colored areas in the zones of contact C/M1 and M1/M2. B, Allogeneic 24-h graft section stained with H&E. Arrows indicate the contact zone. C, A healthy, freshly excised V-shaped fragment of sponge tissue had one of its arms overlaid with cells from the same individual that provided the tissue (iso) and the other arm overlaid with cells from a different individual (allo) and was incubated in a tank of running seawater for 72 h. Note the necrotized tissue resulting from the allogeneic reaction.

 

Figure 4
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  		FIGURE 4. Immunohistochemical staining for {alpha}3 integrin (int), caspase 3 (cas), and CD44 in M. prolifera grafts. A, Setup of an allogeneic graft. Note the lightly colored zone of contact (arrowhead) due to cell lysis in this region. B, Low magnification image of an allograft section stained for {alpha}3 integrin. Note the intensity of the signal in the zone of contact (arrowhead) and the path of the needle that held the graft together (arrow). C and D, {alpha}3 integrin staining of an allograft (C) and an isograft (D). The arrowhead indicates the site of fusion of the two isogeneic grafted tissues. E–G, Double staining of an allograft section for {alpha}3 integrin (E) and for caspase 3 (F). G shows colocalizing pixels in images E and F. H–J, Double staining of an allograft section for {alpha}3 integrin (H) and for the hyaluronan receptor CD44 (I). J shows colocalizing pixels in images H and I. K and L, Higher resolution images of panels G and J, respectively, to show individual cell staining. All images are of 6-h grafts.

 
To explore whether allogeneic reactions require structured tissue, we studied the effect of depositing dissociated cells onto intact sponge papillae (Fig. 1C). M. prolifera cells were mechanically dissociated (~3,800 cells/mm3 as determined by hemocytometer count) and agglutinated by mixing them with an approximately equal volume of concentrated AF before application on the isogeneic or allogeneic tissue with a pipette. The origin of the AF (from the same individual that provided the cells or from a different individual) did not affect the outcome of the experiment. After 72 h the tissue treated with isogeneic cells was healthy whereas the tissue treated with allogeneic cells was heavily necrotized. This result indicates that allogeneic reactions in sponges are triggered by cell-cell contacts that do not require organized tissues.

Participation of gray cells in cytotoxic contact reactions

Gray cells (Fig. 2A) are large, highly motile cells commonly found in growing regions of the sponge and in areas undergoing disorganization (30). They contain many dense autofluorescent cytoplasmic granules (38) and homogeneous, osmophilic small inclusions. A second cell type that has been demonstrated to move toward allograft contact zones in M. prolifera is the archaeocyte (Fig. 2B), the sponge stem cell (7). However, it is likely that this migration of archeocytes has as a final outcome their differentiation into gray cells (Fig. 2C) rather than a direct action of their own (7).


Figure 2
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  		FIGURE 2. Sponge cell types involved in allograft rejection. Transmission electron microscope images of a M. prolifera gray cell containing characteristic cytoplasmic vesicles and dense small glycogen inclusions (A), an archaeocyte with its nucleolate nucleus, abundant mitochondria, and well-developed Golgi and rough endoplasmic reticulum (B), and an intermediate cell type between the archaeocyte and the gray cell (C). G, Golgi apparatus; m, mitochondria; n, nucleolus; N, nucleus; rER, rough endoplasmic reticulum; v, vesicle. Bar, 1 µm.

 
As a first approach to study the possible participation of gray cells in cytotoxic allogeneic reactions, we performed mixed cell reaction assays (Fig. 3). Mechanically dissociated cells from one sponge individual previously loaded overnight with CMFDA were mixed with nonloaded cells from a different individual. After an incubation time of 6 h, a drop of the allogeneic cell suspension was placed on a microscope slide, stained with trypan blue to detect areas of cell death, and examined under a fluorescence microscope. Of 20 examined cell aggregates having weak trypan blue staining, like those in Fig. 3A, all of them turned out to contain cells from the same individual when CMFDA fluorescence was recorded (Fig. 3B). The endogenous fluorescence of Microciona gray cells indicated their presence in some of the clumps (Fig. 3C). Of 20 examined cell aggregates having strong trypan blue staining (Fig. 3D), all of them contained apposed groups of cells derived from the two different individuals (Fig. 3E). CMFDA fluorescence was intense in the extracellular medium as a result of abundant cell lysis in these regions. In all these 20 trypan blue-strong aggregates the cell groups from both individual sponges contained gray cells (Fig. 3F).


Figure 3
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  		FIGURE 3. Mixed cell reaction assay. Cells dissociated from one sponge individual were loaded with CMFDA before mixing them with nonloaded cells from a second individual. The mixed cell suspension was stained with trypan blue to reveal areas of cell death. A and D show bright field images of two different cell aggregates exhibiting little and intense trypan blue staining, respectively. The same areas are examined for the fluorescence of CMFDA (B and E) and autofluorescent gray cells (C and F). Arrowheads indicate selected cell clumps within the aggregates.

 
Allograft but not isograft interfaces stain positive for cell adhesion and apoptotic markers

Upon visual inspection, 6-h allografts showed the characteristic lightly colored line (Fig. 4A) resulting from extensive cell lysis in the area (7). Cells in the zone of contact of allografts exhibited intense integrin staining (Fig. 4, B and C), whereas isograft interfaces did not stain for integrin (Fig. 4D). Double stainings for integrin and caspase (Fig. 4, E–G) and for integrin and CD44 (Fig. 4, H–J) revealed colocalization of both signals in the zone of contact of allografts. Higher magnification images show that the target molecules are coexpressed in the same cells of a narrow line ~10-µm wide (i.e., one cell layer) on each side of the allograft contact zone (Fig. 4, K and L).

Unspecific faster reaction times in sponge allografts

To investigate the existence of a memory component in Microciona grafts, a fragment of sponge tissue was sensitized in a first-set graft and its reactions to second-set and third-party grafts were observed (Fig. 5, A and B). Second-set and third-party grafts had an equally faster reaction time; the lightly colored contact line appeared after 2.71 ± 0.30 and 2.67 ± 0.30 h, respectively, vs 3.67 ± 0.65 h for first-set grafts (n = 12). The third-party graft shown in Fig. 5B is necrotic, but evaluation of a large number of events indicated that first-set, second-set, and third-party grafts were equally susceptible to undergo necrosis in about a 5% of cases (n = 100). Preliminary cell counts from the zones of contact in immunostained memory grafts (Fig. 5, C–H) showed that the numbers of integrin- and caspase-positive cells in second-set and third-party grafts fixed 3 h after graft setup (Fig. 5, D, E, G, and H) were similar among them and not significantly different (p > 0.05) from first-set grafts fixed 4 h after graft setup (Fig. 5I). These times correspond to equal intensities of the lightly colored contact lines, i.e., to equivalent points in the rejection process.


Figure 5
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  		FIGURE 5. Investigation of a memory component in M. prolifera allogeneic reactions. A, A graft was prepared between tissue from sponge individuals 51 and 50 (first-set graft denoted by black numbers). After allowing 12 h for sensitization of individual 51, second-set and third-party grafts were added by adjoining to individual 51 a second fragment of individual 50 (blue number denotes second-set graft) and a fragment of tissue from a third individual (individual 52; red number denotes third-party graft). B, The picture was taken 7 days after completion of the graft setup. C–H, {alpha}3 integrin (int) (C–E) and caspase 3 (cas) (F–H) staining of the contact zones of first-set (1st) (C and F), second-set (2nd) (D and G), and third-party grafts (3rd) (E and H). Arrowheads indicate the direction of the zone of contact. Microscope images correspond to samples fixed for 3 h (second-set and third-party grafts) and 15 h (first-set grafts) after graft setup. I, Cell counts from the zones of contact in memory grafts stained for integrin (int) and caspase (cas). Time of fixation after graft setup was 4 h for first-set and 3 h for second-set and third-party grafts.

 
Cyclosporin A inhibits allograft rejection and isograft fusion

To further explore the mechanism of allogeneic recognition mediated by gray cells, we exposed sponge grafts to the widely used immunosuppressor CsA. As shown in cell viability results (Fig. 6A), the concentrations of CsA used in this work are not toxic for papillae or chemically dissociated cells. Up to 12 h of incubation in the continued presence of 2.5 µg/ml CsA the viability of intact papillae was equal to that of the controls, ranging from 50 to 65% relative to the start of the experiment (data not shown). This indicates that the reduced cell viability is due to deterioration of the small volume of seawater in which papillae are incubated and not to the presence of CsA. Macroscopically, the effect of CsA on M. prolifera grafts was to inhibit both tissue fusion in isografts and the formation of the lightly colored zone of contact in allografts, indicative of cell lysis (data not shown). Immunohistochemical analysis of caspase 3 (Fig. 6B) indicated that cell accumulation in the contact zone of control allografts is detected by 2 h after grafting, peaking at ~6 h and receding after 12 h (Fig. 6C). In the presence of CsA, however, cell migration is delayed for ~4–6 h. This time difference is likely related to the time required for CsA to be washed away from the tissue after the graft has been placed in the tank. These results provide an additional confirmation that there is no lasting cytotoxicity, because rejection eventually takes place in CsA-treated grafts. Similar results were obtained when staining for {alpha}3 integrin (Fig. 6C).


Figure 6
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  		FIGURE 6. Immunohistochemical study of the effect of CsA on M. prolifera allografts. A, Viability analysis of M. prolifera dissociated cells and tissue papillae incubated for 2 h in the continued presence of CsA. B, Immunostaining for caspase 3 of the contact zone of allografts prepared with papillae that had been preincubated either in seawater (control) or in seawater containing 10 µg/ml CsA (cyclosporin A). Each graft was fixed at the indicated times after setup. Arrowheads indicate the direction of the zone of contact in those images where it cannot be easily deduced from the alignment of stained cells. C, Graph representing numbers of cells stained for integrin (int) and caspase (cas) in the zone of contact of control (ctrl) and CsA-treated grafts (CsA) at different hours after graft setup.

 
Cyclosporin A inhibits gray cell motility

In a final approach to assess the effect of CsA on the migration of cells toward graft interfaces, we took advantage of the autofluorescence of Microciona gray cells (38) and performed fluorescence confocal microscopy studies of live sponge grafts in the continued presence of CsA (Fig. 7). In control allografts, gray cells moved toward the zone of contact with the same spatio-temporal pattern previously described in our immunohistochemical results (see also Fig 7video1.mov and Fig 7video6.mov). The presence of CsA (2.5 µg/ml) clearly inhibited gray cell movements toward allograft interfaces (see also Fig 7video2.mov). In the videos provided as online supplementary material, individual gray cells appeared to be immobilized in a fixed position for over 1 h when CsA was present (the positions of some of these cells are indicated with arrowheads in Fig. 7). Tissue remodeling in the fusion zone of isografts involves a massive influx of gray cells that is already evident 1 h after graft set up (see also Fig 7video3.mov). We had previously reported that the contact zone of isografts does not stain for CD44 (7), which, together with autofluorescence data, indicates that this marker is not expressed by gray cells in isograft contact regions. CsA halted almost completely gray cell motility in isografts at a concentration much below its cytotoxic level (see also Fig 7video4.mov). In control isografts where the apposed tissues did not make contact (Fig. 7B), gray cells accumulated on both sides of the gap (see also Fig 7video5.mov), suggesting the existence in sponges of a diffusible extracellular marker of individual identity.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Sponge allogeneic reactions that establish a physical barrier between genetically distinct individuals likely arose in response to a need for keeping genomes separate. In the crowded marine environment, sedentary animals such as sponges develop strategies to maintain their individuality. To further understand such strategies, we have explored the cellular mechanisms underlying sponge self-nonself recognition and the effect of the immunosuppressant drug cyclosporine on this process.

We had previously shown that the contact zone of allografts, but not of isografts, presented strong archaeocyte staining for AF proteins (7). The adhesion force exerted by AFs between sponge cells has been calculated to be very strong, comparable per surface area to that found in focal contacts (39). AF proteins have arginine-glycine-aspartic acid (RGD) sequences (22) that could act synergically with integrin, resulting in high binding forces upon the overexpression of both molecules. An inverse correlation between integrin up-regulation and cell motility has been described in systems other than the sponge (40). In some cells, the expression of integrins that bind fibronectin RGD is correlated with reduced cellular motility (41). The increased expression in sponge allogeneic contacts of cell adhesion markers like integrin and CD44 suggests a restriction of cell motility (Fig. 8A) as is indeed observed in our fluorescence confocal microscopy experiments that clearly show how gray cells are much more motile in isografts than in allografts. Slowing of aggregation was the earliest response to allogeneic contact observed in the marine sponge Callyspongia diffusa (34). In this scenario, allograft rejection could be in part the passive result of death by neglect as opposed to the active mechanism of vertebrate alloincompatibility. However, cell death is not observed during the first hours in the contact zone of CsA-treated allografts as assessed by the absence of both cell lysis and caspase-positive cells, indicating that death by neglect alone is not sufficient to explain sponge allogeneic reactions.


Figure 8
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  		FIGURE 8. Proposal for a sponge self-recognition-based histocompatibility system. In this model, the recognition of cell-bound nonvariant receptors would elicit an immune response (A) unless the simultaneous binding of membrane-bound or soluble self-markers acts as an inhibitory signal (B). The rejection mechanism would involve cytotoxic processes and a reduction in cell motility triggered by the increased expression of cell adhesion molecules. This scenario can have its molecular basis in the existence of a highly polymorphic family of pairs of cell adhesion coevolved ligand receptors (a1b1, a2b2, etc.), such that the probability of any given combination (e.g., 1 + 2 if each individual had two loci coding for polymorphic receptors) is sufficiently low to explain the very high percentage of tissue histoincompatibility that is found in sponges.

 
In the marine sponge Suberites domuncula, allograft rejection was suspended by the immunosuppressor FK506 (42), although in this case the inhibition happened to be allograft fusion. In S. domuncula grafts parabionts attach firmly to each other in the initial phase after transplantation irrespective of whether isogeneic or allogeneic tissue is used. After ~3 days isografts fuse whereas allografts reject and remain separate. The effect of FK506 is to block the allograft separation phase. This phenomenon is different from the early cellular events inhibited by CsA in M. prolifera parabionts, which are clearly observed as soon as 1 h after grafting. The clinical utility of CsA and FK506 stems from their ability to prevent graft rejection following organ transplantation, exerting their major therapeutic functions by arresting T cell activation (43). The major intracellular receptor for CsA is cyclophilin. In mammals, the CsA-cyclophilin complex binds and inhibits calcineurin, a calcium-calmodulin-activated protein phosphatase. The inhibition of calcineurin blocks the translocation of NFAT from the cytosol to the nucleus, thus preventing the transcription of genes encoding cytokines. NFAT proteins have not been described to date in invertebrates (44), thus suggesting that the observed effects of CsA on sponge transplantation reactions could be related to other pathways involving calcineurin, the existence of which in all metazoans has been clearly established (45). In the yeast Saccharomyces cerevisiae, inhibition of the calcineurin homologue by the complex between CsA and the cyclophilin A homologue Cpr1 prevents recovery from pheromone-induced growth arrest (46). In the human pathogenic fungus Cryptococcus neoformans, inhibition of the calcineurin homologue Cna1 by a complex of CsA with either of the cyclophilin A homologues Cpa1 or Cpa2 prevents growth at elevated temperatures (47, 48). Growth inhibition is a direct consequence of cell movement cessation, suggesting that one of the actions of immunosuppressors on invertebrate cells is to block cell migration.

The elevated polymorphism of AF genes (19) suggests that some allelic forms participate in functions other than species-specific cell adhesion. The extracellular nature of AFs makes them or closely related molecules good candidates to act as diffusible signals for isogeneic/allogeneic recognition. In addition, AF proteins have abundant calcium-binding motifs (22), potential transmembrane regions (49), and an elevated sequence homology with the intracellular loop of Na+-Ca2+ exchangers (50). It is conceivable then that some AF isoforms may be involved in intracellular Ca2+ signaling, a pathway network where cell motility and (in higher animals at least) immune responses converge, both through calcineurin-dependent and calcineurin-independent mechanisms (51). Interestingly, CsA has been shown to inhibit transport activity and surface expression of the Na+-Ca2+ exchanger NCX1 (52), indicating that CsA might exert a double check on inhibiting Ca2+ signaling by blocking both Ca2+ entry and the pathway itself.

In addition to the known participation of the gray cell in tissue remodeling areas (30), the data presented here support its role as the main cell type responsible for allogeneic/isogeneic recognition in M. prolifera (32, 33, 34). Are then sponge gray cells related to some vertebrate lymphocyte lineage? A major cell type involved in the innate immune response of vertebrates is the NK cell, a non-T, non-B granular lymphocyte that lacks Ag-specific receptors but plays an important role in the elimination of pathogens and tumors (53). NK cells have been proposed to be remnants of a primeval immune system that arose with animal multicellularity (54), and they bear some resemblance with invertebrate immunocytes. First, upon activation NK cells express high levels of the hyaluronan receptor CD44 (55) and destroy target cells by apoptosis and/or membrane damage (56); elevated CD44 expression, cell lysis, and apoptosis are present in sponge graft rejection where gray cells are the key players. Second, carbohydrates are crucial to the functions of NK cells (57, 58), which can be identified and subdivided into functionally distinct cell subsets on the basis of the expression of lectin-like receptors (59); similarly, the species-specific adhesion of sponge cells has a strict dependence on finely tuned carbohydrate interactions (6). Third, during development NK cells are the first lymphocyte lineage to appear (60) in accordance with the controversial concept that ontogeny recapitulates phylogeny; NK-like cells have been demonstrated in invertebrates like a mollusc (61), a tunicate (62), an earthworm (63), and the leech (64). Fourth and finally, the primary mechanism of invertebrate allorecognition is based on self-recognition, and any cell that lacks self Ags will be immediately recognized as foreign (5, 65) whereas the presence of self-MHC class I molecules protects target cells from lysis mediated by NK cells (66). NK cells use a series of surface receptors to recognize malignant cells. Following receptor ligation free intracellular Ca2+ increases, triggering a series of downstream events including the activation of calcineurin. It has been recently shown that CsA-treated NK cells exhibit an alteration of calcineurin activity that disrupts intracellular Ca2+ patterns and blocks NFAT translocation (67). What effects this has on later stages of NK cell activation are not well understood.

Comparison between first-set and second-set sponge grafts reveals a faster reaction time for the second exposure to a given tissue. However, the lack of difference between second-set and third-party reaction times indicates that the accelerated response is the result of an unspecific increased cell sensitization. This absence of anticipatory immune memory is consistent with a nonclonal, nonadaptive innate immunity characteristic of invertebrates (68). Our data are in agreement with a model for sponge histoincompatibility reactions that is based on self-recognition as opposed to the nonself recognition of the vertebrate MHC. Rejection in these circumstances could be explained by allelic disparity between the two grafted individuals whereby encounters between mature immunocytes and nonself cells result in an elicitor signal (Fig. 8A) that is not subdued by the engagement of histocompatibility receptors that otherwise behave as inhibitors in self-self encounters (Fig. 8B) (7, 33). Sponge allograft rejection and isograft fusion, then, are likely based on cells acting individually without the cytokine-mediated coordination that lies downstream from NFAT. This type of cellular anarchy is characteristic of sponges, which lack cells organized in tissues. On top of such a primeval recognition system, the elements defining T cell-based immunity could have been assembled during evolution.


   Acknowledgments
 
We thank Louie Kerr and Becky MacDonald from the Central Microscopy Facility at the Marine Biological Laboratory for assistance in microscopy and digital imaging.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grant 2005-SGR00037 from the Generalitat de Catalunya, Sweden. Back

2 Address correspondence and reprint requests to Dr. Xavier Fernàndez-Busquets, Bioengineering Institute of Catalonia, Barcelona Science Park, Josep Samitier 1-5, Barcelona E08028, Spain. E-mail address: xfernandez_busquets{at}ub.edu Back

3 Abbreviations used in this paper: ECM, extracellular matrix; AF, aggregation factor; CMFDA, 5-chloromethylfluorescein diacetate; CsA, cyclosporin A. Back

4 The online version of this article contains supplemental material. Back

Received for publication February 6, 2007. Accepted for publication August 20, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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