Allograft rejection, autograft fusion and inflammatory responses to injury in Callyspongia diffusa (Porifera; Demospongia)

Sponges exhibit a variety of swift, cellular defence responses to protect self integrity. The sponge Callyspongia diffusa has been used to characterize the cytological changes that occur during allograft rejection, autograft fusion, and inflammation. Allogeneic contact results in fusion of the two exopinacoderms followed by an infiltration of mesohyl cells into the graft zone. As mesohyl cells accumulate, they form tissue bridges that span the graft interface. After a few days, the tissue bridges and the nearby cellular infiltrate become necrotic and slough off, which separates the allogeneic tissues. Autograft fusion begins similarly but cellular infiltration does not follow exopinacoderm fusion. Contacted exopinacocytes are redistributed, the endopinacoderms and choanosomes come into contact, and the grafted sponge tissues merge. Tissue damage exposes internal regions of the sponge to the external environment. In many areas of injury, exposed choanosome is sealed by infiltrating mesohyl cells. In other areas, exposed endopinacoderm appears to serve as new exopinacoderm. Cellular debris is removed by phagocytic archaeocytes and new exopinacoderm is regenerated over the damaged choanosome. No scars remain once the inflammatory infiltrate has dispersed. In general, mesohyl cells are involved in defence responses without an observed enrichment of any specific cell type. However, archaeocytes from rejecting sponges appear to line both sides of the allogeneic interface.     

Continuous cell movements rearrange anatomical structures in intact sponges.

Time-lapse cinemicrography was used to record the active movements of cells in living intact sponges. Each of the three main cell types (pinacocytes, mesohyl cells, and choanocytes) continuously moved and rearranged themselves so that the internal anatomy of the sponge was continuously remodeled. The shape and appearance of the sponges anatomical structures often changed substantially within a few hours. The most motile were the mesohyl cells, with many moving as fast as one cell-length per minute (15 microns/min). Mesohyl cell locomotion was often accompanied by displacements of spicules, canals, and choanocyte chambers; the patterns of these displacements suggested that the mesohyl cells were providing the motive forces for these rearrangements. The locomotion of the pinacocytes varied according to position: those along the outer sponge margins were most active, whereas those in other parts of the surface moved relatively little. Choanocytes were never observed to undergo independent locomotion but were always found grouped together in choanocyte chambers. These choanocyte chambers interacted with pinacocytes and mesohyl cells to form excurrent canals, which continuously moved, fused with, and branched from one another. These observations suggest that the experimental phenomenon of sponge cell-reaggregation and reconstitution, discovered by H. V. Wilson, represents an extreme version of morphogenetic processes that normally go on continuously within intact sponges. The results from the present study also suggest that these cellular rearrangements are controlled by active cell movements and behavioral responses that include but are not limited to selective cell adhesion.     

The vacuolar cells of Oscarella lobularis (Porifera,Demospongiae): Ulatrastructural organization,origin, and function

This work is concerned with the ultrastructural organization and some histochemical features of the vacuolar cells of the demosponge Oscarella lobularis. Vacuolar cells are characterized by large clear vacuoles containing an aqueous fluid. They are commonly found in the mesohyl of this sponge and tend to constitute a sort of parenchyma in the choanosome. Mobile cells of the mesohyl appear to differetiate into vacuolar cells through the progressive formation of wide cytoplasmic lacunae. We have identified four types of cells showing progressive transformation toward the vacuolar cell type. Precursors (types 1–4) of the vacuolar cells probably derive from endopinacocytes, since they share several histochemical and ultrastructural characteristics with them. Our data support the notion that vacuolar cells are involved in the synthesis of collagen, act as a mechanical support of the sponge body, and are eventually extruded from the sponge through the canals of the aquiferous system.     

Cyclosporin A suspends transplantation reactions in the marine sponge Microciona prolifera

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.     

繁茂膜海绵原细胞富集细胞团培养过程中的细胞迁移规律

海绵是重要的生物活性物质来源, 近10年来, 从海绵中发现的具有生物活性的新化合物占海洋生物来源的30%以上, 并且大多具有显著的抗肿瘤, 抗艾滋病病毒的活性。但是, 由于海绵生物量不能满足这些活性物质进一步研究和商业化的需求, 目前仅有一种活性物质被成功的商业化, 这不仅是商业开发的损失, 也是提高人类生活质量活动的一种损失。为了解决海绵供给不足的问题, 人们进行了包括化学合成、海绵养殖以及海绵细胞培养在内的多种尝试,目前的研究结果表明, 海绵细胞离体培养技术是最有可能彻底解决海绵供给不足的途径之一。但是由于海绵自身的特殊性, 还没有人成功的建立起海绵细胞系以满足生产需要。人们发现, 海绵细胞的相互接触对于离体海绵细胞长期培养至关重要。经过多年的探索, 大连化物所海洋生物产品工程组建立了开发出了海绵原细胞富集细胞团培养技术, 通过对海绵组织内的原细胞进行富集来获得可长期培养的海绵细胞。海绵原细胞是海绵组织内的“干细胞”, 具有很强的分化、增殖潜力, 同时也是海绵组织内负责消化的主要细胞类型。为了探索海绵原细胞的增殖、分化规律, 本研究基于海绵原细胞富集细胞团培养体系, 构建了海绵细胞培养实时观测平台, 对繁茂膜海绵原细胞、领细胞、上皮细胞3类主要海绵细胞类型在海绵细胞团形成及生长的全过程进行观察, 了解不同类型细胞迁移规律的变化。通过对视频记录进行分析,发现离散的海绵细胞与细胞团内的海绵细胞具有截然相反的运动规律, 海绵细胞的运动具有很强的协同性。伴随原细胞在细胞团内不停息的迁移, 还观察到海绵细胞团内新生骨针的迁移以及细胞间进行颗粒物质的传递。这些信息的获得, 将有助于进一步了解不同细胞的功能与作用, 也有助于在此基础上探索海绵细胞的增殖、分化控制规律。     

Characterization of natural killer activity in sponge matrix allografts

NK cell activity, defined by the ability of infiltrating host cells to lyse the YAC-1 tumor target, can be detected in sponge matrix allografts across all genetic barriers tested. Nonspecific tumor cell killing cannot be detected either within bulk populations of host-infiltrating cells or in populations enriched for non-adherent lymphocytes. NK activity is also detected in cells infiltrating a syngeneic sponge matrix graft although to a much lesser extent than an allogeneic graft. NK cell functional activity at the graft site precedes the appearance of alloimmune CTL by several days. The surface phenotype of the NK cell is Thy-1.2+ and L3T4- as determined by depleting the various subpopulations with antibody and C. Systemic treatment of sponge-bearing animals with repeated injections of anti-asialo GM1 (AGM1) results in inhibition of both NK activity and CTL activity recovered from the graft on days 5 to 9 after grafting, but on days 11 to 13 after grafting both NK activity and CTL activity are found within the sponge graft. Treatment of sponge-associated cells with anti-AGM1 in vitro or intrasponge injection of anti-AGM1 at various times after grafting eliminates NK activity more readily than alloimmune CTL activity. The intimate association observed between NK cells and alloimmune CTL at the graft site prompts further investigation into the role of NK cells in the allograft response.     

Microscopic anatomy and pigment characterization of coral-encrusting black sponge with cyanobacterial symbiont, Terpios hoshinota

Terpios hoshinota is a black-colored sponge encrusting both live and dead corals. On examination, we determined that in the mesohyl, unicellular cyanobacteria were distributed extracellularly in dense populations; roughly half of the area in the histological sections was occupied by the cyanobacterial cells in the mesohyl of the inner zone. Pigment extraction tests of whole sponges and microspectrophotometrical analysis of cyanobacterial symbionts showed that the black coloration of the sponge is attributable to extremely densely packed cyanobacteria expressing R-phycoerythrin. Spermatic follicles were found in the mesohyl of specimens collected in summer, indicating seasonal reproduction, which is likely to play a crucial role in long-distance dispersal. We also found a possible oocyte that did not contain cyanobacteria in the ooplasm.     

Efficient induction of primary and secondary T cell-dependent immune responses in vivo in the absence of functional IL-2 and IL-15 receptors

IL-2 and IL-15 are thought to be important cytokines for T cell-dependent immune responses. Mice deficient in IL-2, IL-2Ralpha, and IL-2Rbeta are each characterized by a rapid lethal autoimmune lymphoproliferative disorder that complicates their use in studies aimed at investigating the role of these cytokines and receptors for immune responses in vivo. We have previously characterized a novel transgenic (Tg) mouse on the IL-2Rbeta-/- genetic background (Tg-/- mice) that lacks autoimmune disease but still contains peripheral T cells that are nonresponsive to IL-2 and IL-15. In the present study, these mice were used to investigate the extent by which IL-2 and IL-15 are essential for T cell immunity in vivo. Tg-/- mice generated near normal primary and secondary Ab responses to OVA, readily mounted first and second set allogeneic skin graft rejection responses, and developed primary and recall CD8 T cell responses to vaccinia virus. However, Tg-/- mice generated a slightly lower level of IgG2a Abs to OVA, exhibited a somewhat delayed first set skin graft rejection response with lower allo-specific CTL, and developed a significantly lower number of IFN-gamma-producing vaccinia-specific CD8+ T cells. Thus, although T effector function is somewhat impaired, T cell immunity is largely functional in the absence of IL-2- and IL-15-induced signaling through IL-2Rbeta.     

Effect of localized cytokine dysregulation: accelerated rejection of IL-2-expressing skin grafts

Transgenic mice were created in which a sheep keratin promoter directed the expression of IL-2 into the dermis. These KIL-2 transgenic mice were used to investigate the effects of localized IL-2 dysregulation on immune responses. Peripheral tolerance to skin antigens was not broken by in situ IL-2 expression because syngeneic KIL-2 skin grafts were not rejected. However, MHC Class I-disparate skin grafts from KIL-2 donors were rejected faster (median survival time (MST) 12 days) than grafts of non-transgenic littermate skin (MST 18 days). In contrast, the kinetics of KIL-2 H-Y-disparate skin graft rejection (MST 14 days) did not differ significantly from controls (MST 16 days), suggesting that upregulation of IL-2 at the effector site could affect CD4+ T cell- independent, but not CD4+ T cell-dependent, responses. No effect on rejection kinetics was observed when wild type allogeneic skin was grafted onto transgenic mice that expressed bcl2 constitutively in their lymphocytes (MST of 14 days, both sets), indicating that this was not simply due to increased longevity of T cells within the IL-2 expressing graft. We therefore suggest that aberrant expression of IL-2 can accelerate helper-independent CD8+ T cell responses by increasing proliferation and/or differentiation of cytolytic T cells at the effector site.     

Rapid conversion of effector mechanisms from NK to T cells during virus-induced lysis of allogeneic implants in vivo

Viral infections can strongly stimulate both NK cell and allospecific CD8 T cell responses, and these same effector cells can lyse allogeneic cell lines in vitro. However, the impact of viral infections on the effector systems mediating rejection of allogeneic tissues in vivo has not been fully explored. Using in vivo cytotoxicity assays, we evaluated the effector systems mediating the rejection of CFSE-labeled allogeneic splenocytes after an infection of C57BL/6 (B6) mice with lymphocytic choriomeningitis virus. Naive B6 mice predominantly used a NK cell-effector mechanism to reject allogeneic splenocytes because they rejected BALB/C (H2(d)) splenocytes but not CBA (H2(k)) splenocytes, and the rejection was prevented by immunodepletion of NK1.1(+) or Ly49D(+) NK cells. This rapid and efficient in vivo cytotoxicity assay recapitulated the specificity of NK cell-mediated rejection seen in longer duration in vivo assays. However, as early as 1 day after infection with lymphocytic choriomeningitis virus, a CD8 T cell-dependent mechanism participated in the rejection process and a broader range of tissue haplotypes (e.g., H2(k)) was susceptible. The CD8 T cell-mediated in vivo rejection process was vigorous at a time postinfection (day 3) when NK cell effector functions are peaking, indicating that the effector systems used in vivo differed from those observed with in vitro assays measuring the killing of allogeneic cells. This rapid generation of allospecific CTL activity during a viral infection preceded the peak of viral epitope-specific T cell responses, as detected by in vivo or in vitro cytotoxicity assays.     

Membrane-bounded nucleoids in microbial symbionts of marine sponges

In thin sections of resin-embedded samples of glutaraldehyde- and osmium tetroxide-fixed tissue from five genera of marine sponges, Stromatospongia, Astrosclera, Jaspis, Pseudoceratina and Axinyssa, cells of a bacteria-like symbiont microorganism which exhibit a membrane-bounded nuclear region encompassing the fibrillar nucleoid have been observed within the sponge mesohyl. The nuclear region in these cells is bounded by a single bilayer membrane, so that the cell cytoplasm is divided into two distinct regions. The cell wall consists of subunits analogous to those in walls of some Archaea. Cells of the sponge symbionts observed here are similar to those of the archaeal sponge symbiont Cenarchaeum symbiosum.     

The Bacterial Community of the Lithistid Sponge Discodermia spp. as Determined by Cultivation and Culture-Independent Methods

The marine lithistid sponge Discodermia spp. (Family Theonellidae) contains many types of associated bacteria visible in the mesohyl while biofilms cover the pinacoderm. This study determined the identity of bacteria associated with members of the genus Discodermia using microbial culture, 16S rRNA gene clone libraries and fluorescence in situ hybridization. Four samples of Discodermia spp. were collected at depths between 24–161?m near Grand Bahama Island and Cay Sal Bank, Bahamas. A total of 80 unique isolates and 94 different clone sequences from at least eight bacterial classes were obtained. It appeared that Discodermia spp. may have a core community of bacteria that is common to all sponges of this genus. Species of at least six different classes of bacteria were regularly found in most of the sponge specimens collected, irrespective of collection depth or location. This indicates that a diverse spectrum of bacteria is associated with lithistid sponges irrespective of the transient seawater community that enters the sponge.     

Dynamic structure of the mesohyl in the sponge Chondrosia reniformis (Porifera, Demospongiae)

The common demosponge Chondrosia reniformis possesses the capacity to undergo an unusual creep process which results in the formation of long outgrowths from the parent body. These shape changes, which have been interpreted as adaptive strategies related to environmental factors, asexual reproduction or localised locomotor phenomena, are due mainly to the structural and mechanical adaptability of the collagenous mesohyl. This contribution describes the morphological correlates of mesohyl plasticisation in C. reniformis. The microscopic anatomy of the mesohyl was examined when it was in different physiological conditions: (1) standard ”resting” condition, (2) ”stiffened” condition and (3) dynamic ”creep” condition. In this last case four representative regions of the sponge body were analysed: the parent region, the elongation region, the transition region and the propagule region. The results show that the histological modification of the sponge mesohyl during plasticisation is limited and localised. The most significant structural changes involve mainly cytological features of specific cellular components characterised by granule inclusions (i.e. the spherulous cells) and the arrangement and density of the collagenous extracellular framework, though the integrity of the collagen fibrils themselves is not affected. Morphological and functional aspects of mesohyl plasticisation invite comparison with the mutable collagenous tissue of echinoderms. Possible functional analogies between these two tissues are hypothesised. Accepted: 29 June 2001     

Dense populations of Archaea associated with the demosponge Tentorium semisuberites Schmidt, 1870 from Arctic deep-waters

The associated microbial community in the mesohyl of the Arctic deep-water sponge Tentorium semisuberites Schmidt, 1870 (Hadromerida, Demospongiae) is dominated by Archaea. This is the result of an integral approach applying analyses of microbial lipid biomarkers as well as microscopic investigations using differential fluorescence in situ hybridisation with universal probes and counterstaining with 4′,6′-diamidino-2-phenylindole (DAPI) on sponge sections based on samples collected in the Greenland Sea in 2001, 2002 and 2005. The distribution of isoprenoidal C₄₀ hydrocarbons of the biphytane series suggests that affiliates of both major archaeal kingdoms, the Crenarchaeota and the Euryarchaeota, are present in the choanosome of T. semisuberites. Positive signals using the oligonucleotide probe ARCH915 indicate high numbers of Archaea in the mesohyl of this sponge. Based on optical estimations 70–90% of all microbial DAPI signals accounted for archaeal cells. Archaea in these high proportions have never been described in an Arctic deep-sea hadromerid sponge, nor in any other demosponge species. Similar observations in specimens collected over a time scale of 4 years suggest permanent sponge-Archaea associations.     

Allogeneic stem cell transplantation for multiple myeloma

The sensitivity of myeloma cells to high dose chemotherapy has led to the use of allogeneic hematopoietic stem cell transplantation (HSCT) as a therapeutic modality in this disease. In addition to providing more effective chemotherapy, the transplantation of allogeneic stem cells also initiates the development of an allogeneic immune response directed against residual myeloma cells. Direct evidence for a graft vs. myeloma (GVM) effect is provided by the ability of donor lymphocyte infusion (DLI) to induce significant responses in 30-50% of patients with myeloma who have relapsed after allogeneic HSCT. Nevertheless, allogeneic stem cell transplantation is also associated with a high incidence of transplant related toxicities, including regimen-related toxicities, graft vs. host disease (GVHD) and opportunistic infections. DLI has been shown to enhance immune reconstitution after allogeneic HSCT in addition to inducing a GVM response. Current efforts are directed at reducing the toxicities associated with allogeneic HSCT, identification of the target antigens of GVM and the development of new strategies to selectively enhance the immune response to myeloma cells.     

Ultrastructural evidence of extruding exocytosis of residual bodies in the freshwater sponge Ephydatia fluviatilis

Exocytosis of residual bodies by choanocytes, archeocytes and endopinacocytes lining the aquiferous system of Ephydatia fluviatilis has been demonstrated using calibrated latex beads and Escherichia coli as tracers. In passing into the mesohyl or the lumen of the exhalant aquiferous canals, beads, and altered bacteria were enveloped by the plasma membrane of the cell containing them. The membrane constricted at a neck region to form extruding vacuoles. This process appeared first in choanocytes and later in other cell types. The occurrence of these buds increased with the length of incubation time, as did the number of particles they contained. Acid phosphatase activity was frequently associated with the particles budding from the cell membrane, confirming that this process followed digestive activity. Membranous vacuoles were recovered from the external medium and observed by TEM and those adhering to the substratum were seen by SEM. These observations proved that vacuoles were released from the sponges. This membrane-consuming mechanism of exoctyosis implies intense membrane replacement in the digestive cells of the sponge.     

Specific and nonspecific infiltration of sponge matrix allografts by specifically sensitized cytotoxic lymphocytes

Urethane sponges coated with allogeneic or syngeneic cells were implanted subcutaneously into mice and the cytotoxicity of infiltrating host cells was assessed in vitro. First-set allogeneic sponges attracted a population of lymphocytes enriched in cytotoxic T cells directed against the alloantigens in the sponge. If two sponges bearing cells of different H-2 specificity were grafted simultaneously to a single recipient, specifically sensitized cytotoxic cells (SSCL) were found in both sponges directed against both sets of alloantigens, although specific infiltration predominated. If a syngeneic and allogeneic sponge were transplanted, SSCL were found in both the syngeneic sponge and allogeneic sponge. These data are interpreted to suggest that chemotactic substances are elaborated at graft sites which can attract circulating SSCL into sites of inflammation and that those released at the specific site are more attractive for SSCL than are those elaborated at sites of nonspecific rejection or healing. In recipients who had previously been sensitized to alloantigens, second-set grafts were rapidly infiltrated by SSCL directed against the sensitizing antigen. First-set indifferent allografts in sensitized recipients were infiltrated by SSCL directed against the previous alloantigens as well as SSCL directed against its own alloantigens. Syngeneic grafts were not infiltrated by SSCL in presensitized recipients. These data suggest that any alloantigenic stimulus can induce the mobilization from lymphoid depots of preformed SSCL directed against another set of antigens; syngeneic grafts cannot. Once mobilized, however, circulating SSCL can respond to specific and nonspecific chemotactic factors elaborated by either healing or rejecting grafts.     

Maturation of cytotoxic T cells within sponge matrix allografts

Two parallel events may provide cytotoxic effector cells at the site of allograft challenge: 1) the migration of effector cells to the graft, and 2) the local maturation of effector cells. Various experimental conditions were manipulated to ascertain the relative role of local maturation of precytotoxic cells at the site of alloantigen challenge. A C57BL/6 allogeneic-coated sponge matrix allograft was placed in a BALB/c animal. The entire animal with the sponge was subjected to sublethal irradiation, or alternatively, just the animal was subjected to the same dose of irradiation, and the sponge was shielded. The data demonstrate that by day 5, precytotoxic cells directed against donor alloantigen appears within the sponge matrix allograft. These precytotoxic cells are in themselves x-ray sensitive, but in the absence of irradiation and in the absence of further contributions from the host can develop into mature cytotoxic cells with specificity limited to that of donor alloantigen. The relative role of local maturation vs continued influx of mature cytotoxic cells cannot be determined from these sets of experiments. These data, however, show that there is potential for local development of mature cytotoxic cells in the absence of other host influences.