Cellular and biochemical responses to environmental and experimentally induced stress in sea urchin coelomocytes

Coelomocytes are considered to be immune effectors of sea urchins. Subpopulations of coelomocytes can be purified from a total cell suspension. The proportion of each cell type can vary not only among species, but also between individuals of the same species, according to their size and physiological conditions. We tested the hypothesis that coelomocytes play a role in defense mechanisms activated by adverse external conditions. Total coelomocytes from control and stressed (temperature, pollution, and injuries) sea urchins were analyzed for their expression of the 70 kDa heat shock protein (hsp70), a well recognized stress marker. Further analysis was performed by separation of coelomocytes into subpopulations by step gradients. We demonstrated that sea urchin coelomocytes respond to temperature shock and to polluted seawater by the upregulation of hsp70. Among coelomocytes certain cells, known as red spherula cells, showed a great increase in number in animals collected from polluted seawaters or subjected to "accidental" injury. The present study confirms the immunological function of sea urchin coelomocytes, as indicated by the upregulation of the hsp70 molecular marker, and suggests that sea urchin coelomocytes can be utilized as sensitive bio-indicators of environmental stress.     

Coelomocytes and post-traumatic response in the common sea star Asterias rubens

Coelomocytes are recognized as the main cellular component of the echinoderm immune system. They are the first line of defense and their number and type can vary dramatically during infections or following injury. Sea stars have been used as a model system to study the regeneration process after autotomy or predation. In the present study we examined the cellular and biochemical responses of coelomocytes from the European sea star Asterias rubens to traumatic stress using immunochemical and biochemical approaches. In terms of trauma and post-traumatic stress period, here we consider the experimental arm amputation and the repair phase involved in the first 24 hours post-amputation, which mimicked a natural predation event. Four cell morphotypes were distinguishable in the coelomic fluid of both control and post-traumatic-stressed animals (phagocytes, amoebocytes, vibratile cells, hemocytes), but phagocytes were the major components, accounting for about 95% of the total population. Thus, the effects measured relate to the overall population of coelomocytes. A modest increase in the total number of freely circulating coelomocytes was observed 6 hours post-amputation. Interestingly, a monoclonal antibody (McAb) to a sea urchin embryo adhesion protein (toposome) cross-reacted with isolated sea star coelomocytes and stained the coelomic epithelium of control animals with an increase in trauma-stressed arms. In addition, coelomocytes from trauma-stressed animals showed a time-dependent increase in Hsp70 levels, as detected by both immunocytochemistry and immunoblotting within 24 hours after arm tip amputation, with a peak at 6 hours after amputation. Our findings indicate a clear role for coelomocytes and classic stress molecules in the post-traumatic stress associated with the early repair phase of regeneration.     

Studies on heat shock proteins in sea urchin development

Work on stress proteins in sea urchin embryos carried out over the last 20 years is reviewed and the following major results are described. Entire sea urchin embryos, if subjected to a rise in temperature at any postblastular stage undergo a wave of heat shock protein (hsp) synthesis and survive. If subjected to the same rise between fertilization and blastula formation, they are not yet able to synthesize hsp and die. Four clones coding for the major hsp, hsp70, have been isolated and sequenced; evidence for the existence of a heat shock factor has been provided, and a mechanism for the developmental regulation of hsp synthesis discussed. Intraembryonic and intracellular hsp location has been described; and a mechanism for achievement of thermotolerance proposed. A chaperonine role for a constitutive mitochondrial hsp56 has been suggested, as well as a role for the constitutive hsp70 in cell division. Heat shock, if preceded by 12-O-tetradecanoylphorbol-12-acetate (TPA) treatment causes apoptosis.     

Sea urchin coelomocytes as a novel cellular biosensor of environmental stress: a field study in the Tremiti Island Marine Protected Area,Southern Adriatic Sea,Italy

The aim of the present study was to investigate on the suitability of the sea urchin as a sentinel organism for the assessment of the macro-zoobenthos health state in bio-monitoring programmes. A field study was carried out during two oceanographic campaigns using immuno-competent cells, the coelomocytes, from sea urchins living in a marine protected area. In particular, coelomocytes subpopulations ratio and heat shock protein 70 (HSC70) levels were measured in specimens of Paracentrotus lividus (Lamark, 1816) collected in two sampling sites, namely Pianosa and Caprara Islands, both belonging to the Tremiti Island Marine Protected Area (MPA) in the Southern Adriatic Sea, Italy. By density gradients separation performed on board the Astrea boat, we found an evident increase in red amoebocytes, a subpopulation increasing upon stress, in those specimens collected around Pianosa (strictly protected area with no human activities allowed), unlike those collected around Caprara (low restrictions for human activities). Likewise, we found higher HSC70 protein levels in the low impacted site (Pianosa) by Western blots on total coelomocyte lysates. The apparent paradox could be explained by the presence in the Pianosa sampling area of contaminating remains from Second World War conventional ammunitions and a merchant boat wreck. Metal determination performed using sea urchin gonads by inductively coupled plasma atomic emission spectrometry (ICP-AES) revealed higher Fe and lower Zn levels around Pianosa with respect to Caprara, in accordance with the persistent contaminating metal sources, and thus calling for remediation measures. Taken all together, our results confirm the feasibility of using sea urchin coelomocytes as biosensors of environmental stress.     

Cadmium induces the expression of specific stress proteins in sea urchin embryos

Marine organisms are highly sensitive to many environmental stresses, and consequently, the analysis of their bio-molecular responses to different stress agents is very important for the understanding of putative repair mechanisms. Sea urchin embryos represent a simple though significant model system to test how specific stress can simultaneously affect development and protein expression. Here, we used Paracentrotus lividus sea urchin embryos to study the effects of time-dependent continuous exposure to subacute/sublethal cadmium concentrations. We found that, between 15 and 24 h of exposure, the synthesis of a specific set of stress proteins (90, 72-70, 56, 28, and 25 kDa) was induced, with an increase in the rate of synthesis of 72-70 kDa (hsps), 56 kDa (hsp), and 25 kDa, which was dependent on the lengths of treatment. Recovery experiments in which cadmium was removed showed that while stress proteins continued to be synthesized, embryo development was resumed only after short lengths of exposure.     

Isolation and characterization of a sea urchin hsp 70 gene segment

Three clones containing Paracentrotus lividus sea urchin DNA sequences which cross-hybridize to Drosophila heat shock protein (hsp) 70 gene were isolated. The sequence arrangements in the three cloned DNA inserts were compared by restriction and cross-hybridization analysis. The results showed that they contain four different genes related to one Drosophila hsp 70 gene. One of these genes was subcloned, and two of the isolated fragments were shown to hybridize to genomic DNA and to RNA from heat-treated sea urchin embryo.     

Volume regulation in the coelomocytes of the blood wormGlycera dibranchiata

Summary Median volumes ofin vitro coelomocyte populations fromGlycera dibranchiata rapidly change in response to external differences of osmotic pressure (Fig. 3). Fifty per cent haemolysis occurred in just under 30% sea water, 285 mOSm·kg^–1. Hydraulic conductivities (L_p=0.92 to 2.78×10⁷ cm×s^–1·atm^–1) calculated from rates of osmotic swelling were similar to values for sea urchin eggs and squid axons. Coelomocytes show a slower partial return to their original volumes in hypotonic but not hypertonic media. This asymmetry is reflected in Ponder's R values of 0.787 and 0.987, respectively, determined after this regulatory phase is complete (Figs. 4 and 5). Evidence for an irreversible stress dependent leakage of osmotically active solutes when the coelomocytes are removed from the animal and diluted with sea water is presented.     

70-Kilodalton Heat Shock Protein Induction in Cerebellar Astrocytes and Cerebellar Granule Cells In Vitro: Comparison with Immunocytochemical Localization After Hyperthermia In Vivo

Induction of the 70-kDa heat shock protein, hsp70, was evaluated in cultured cerebellar astrocytes and granule cell neurons subjected to a hyperthermic stress, using a monoclonal antibody and an oligonucleotide probe that selectively recognize stress-inducible species of hsp70-related proteins and RNAs, respectively. Immunoblots of cultures enriched in either granule cells or astrocytes, and immunocytochemical localization studies in cocultures of these cell types, demonstrated that hsp70 induction was restricted to the astrocyte population. Amino acid incorporation experiments showed little difference in the loss and recovery of overall protein synthesis activity in these two cell types following transient hyperthermic stress. RNA blot hybridizations confirmed the preferential glial induction of hsp70. In vivo immunocytochemical studies in brains of adult rats following hyperthermia were consistent with earlier observations that suggested a primarily glial and vascular localization of the heat shock response in most brain regions, although the intense immunoreactivity in the cerebellar granule cell layer suggests that there is induction of hsp70 in these neurons under in vivo conditions. These results suggest the potential value of such defined cell cultures in identifying mechanisms responsible for differences in the heat shock response of various cell types in vitro, and in revealing factors that may account for the apparent absence of the stress response in cultured cerebellar granule cell neurons.     

Phylogeny of natural cytotoxicity: cytotoxic activity of coelomocytes of the purple sea urchin, Arbacia punctulata.

Coelomocyte-mediated nonspecific cell cytotoxic activity against human and murine target cells by the purple sea urchin Arbacia punctulata was investigated in vitro. Cytotoxic activity toward target cells was shown to be mediated by different coelomocyte populations isolated by discontinuous density gradient centrifugation. The population of phagocytic amebocytes showed the strongest cytotoxic activity and the highest binding to human NK markers by cytometry analysis. Our immunophenotypic studies showed that A. punctulata phagocytic amebocytes are CD14(+), CD56(+), CD158b(+), CD3(-), CD4(-), CD8(-), and CD16(-). The cytotoxic activity was independent of experimental incubation temperatures, required viable effector cells, and required cell-cell contact between the effector and target cells. Sodium azide significantly decreased coelomocyte cytotoxicity, indicating that cytotoxicity is metabolically dependent, and EDTA reduction of cytotoxic activity is consistent with the involvement of divalent cations in the cytotoxic process. These data describe a population of sea urchin coelomocytes (the phagocytic amebocyte) that are CD14(+), CD56(+), and CD158b(+), with cytotoxic activities.     

Intermediate filament proteins in echinoderm coelomocytes

The presence and organization of intermediate filament (IF) proteins in petaloid coelomocytes from two species of echinoderms, the sea urchin Strongylocentrotus droebachiensis and the sea cucumber Cucumaria frondosa, were studied. Two monoclonal antibodies (IFA and Ah6) and one polyclonal antibody (W3-1) that together recognize invertebrate as well as vertebrate IF proteins were used to probe coelomocytes by immunofluorescence and immunoblotting methods. All three antibodies cross-reacted with a single M_r 68 000 sea urchin lamin, as well as two putative lamin isoforms of approximately M_r 70 000 and 68 000 in sea cucumber coelomocytes. Both IFA and Ah6 labeled granular material in the cytoplasm of sea urchin coelomocytes; by contrast, IFA labeling revealed a striking network of reticular material irregularly arrayed within the central regions of the sea cucumber coelomocyte cytoplasm. In addition, foci of Ah6-positive material were present in coelomocyte nuclei from both species. Comparison of immunoblotting patterns among whole cell and isolated nuclear preparations suggest that the cytoplasmic IF-like material is composed of M_r 46 000 and 58 000 polypeptides, while M_r 215 000 and 185 000 proteins are candidates for the immunoreactive nuclear foci. Further study of the functions of these non-filamentous arrays of IF proteins may furnish valuable insights into the evolution of IF function within vertebrate cells, particularly with respect to certain cytoplasmic and nuclear regulatory functions with which IF proteins have been speculated to be involved.     

Sequence of a sea urchin hsp70 gene and its 5' flanking region

We report the nucleotide sequence of a 4470-bp fragment derived from a sea urchin genomic clone containing part of a heat-shock protein 70 (Hsp70)-encoding gene. This fragment, named hsp70 gene II, contains 1271 bp of the flanking region and 3299 bp of structural gene sequence interrupted by five introns and encoding the N-terminal 371 amino acids (aa) of the protein. The 5' flanking region contains a putative TATA element, two CCAAT boxes, four heat-shock consensus sequence elements (hse) and one consensus sequence for binding of Sp1. Remarkable homologies were observed for deduced aa sequence and intron-exon organization between hsp70 gene II and rat hsc73 gene.     

Morphological and ultrastructural characterization of sea urchin immune cells

The free circulating coelomocytes in the coelomic cavity of echinoderms are considered to be immune effectors by phagocytosis, encapsulation, cytotoxicity, and by the production of antimicrobial agents. Although echinoderms (especially sea urchin embryo) have been used as a model organisms in biology, no uniform criteria exist for classification of coelomocytes in echinoderms, and few studies have reported about the biological functions of their coelomocytes. Hence, we study the coelomocytes in the echinoid sea urchin, Paracentrotus lividus, and describe their morphological and ultrastructural features using light and transmission electron microscopes. We classify the coelomocytes of P. lividus into red spherule and colorless spherule cells, small cells, vibratile cells, and phagocytic cells; petaloid and filopodial cells. To our knowledge, this is the first report describing ultrastructural details of the coelomocytes of P. lividus. J. Morphol. 276:583–588, 2015. © 2015 Wiley Periodicals, Inc.     

Cellular biomarkers to elucidate global warming effects on Antarctic sea urchin Sterechinus neumayeri

Global warming is a reality and its effects have been widely studied. However, the consequences for marine invertebrates remain poorly understood. Thus, the present study proposed to evaluate the effect of elevated temperature on the innate immune system of Antarctic sea urchin Sterechinus neumayeri. Sea urchins were collected nearby Brazilian Antarctic Station ??Comandante Ferraz?? and exposed to 0 (control), 2 and 4°C for periods of 48?h, 2, 7 and 14?days. After the experimental periods, coelomic fluid was collected in order to perform the following analyses: coelomocytes differential counting, phagocytic response, adhesion and spreading coelomocytes assay, intranuclear iron crystalloid and ultra structural analysis of coelomocytes. The red sphere cell was considered a biomarker for heat stress, as they increased in acute stress. Besides that, a significant increase in phagocytic indexes was observed at 2°C coinciding with a significant increase of intranuclear iron crystalloid at the same temperature and same time period. Furthermore, significant alterations in cell adhesion and spreading were observed in elevated temperatures. The ultra structural analysis of coelomocytes showed no significant difference across treatments. This was the first time that innate immune response alterations were observed in response to elevated temperature in a Polar echinoid.     

Picosecond rotation of small polar fluorophores in the cytosol of sea urchin eggs.

A new fluorescence method to measure viscosity in cell cytosol [Fushimi, K., & Verkman, A. S. (1991) J. Cell Biol. 112, 719-725] has been applied to determine fluid-phase viscosity in sea urchin eggs. Freshly harvested eggs from Lytechinus pictus were loaded with the dyes 2,7-bis(2-carboxyethyl)-5-(and-6-)carboxyfluorescein (BCECF), 6-carboxyfluorescein (6CF), fluorescein, or calcein. Fluorescence lifetimes and anisotropy decay were measured in single eggs by multiharmonic, frequency-domain microfluorometry using a 1-2-micron focused laser spot and 25x air objective. In calibration solutions consisting of glycerol in pH 8 buffered sea water, probe lifetime was single exponential and probe rotation was isotropic with a single correlation time which increased linearly with viscosity in the range 1-3.6 cP. In eggs at 22 degrees C, there were single lifetimes (in nanoseconds) of 3.6 (BCECF), 3.4 (6CF), 3.2 (fluorescein), and 3.3 (calcein). Probe rotation in eggs had two components, a fast component (in picoseconds, mean +/- SE, 10-18 eggs) of 568 +/- 39 (BCECF), 311 +/- 21 (6CF), 313 +/- 15 (fluorescein), and 516 +/- 44 (calcein) and a slow component of 10-40 ns. The fractional amplitude of the fast component, corresponding to unbound dye, was 0.72-0.81. Apparent viscosities of fluid-phase cytoplasm (centipoises) given by the four different probes were in good agreement: 2.3 +/- 0.2 (BCECF), 2.1 +/- 0.1 (6CF), 2.5 +/- 0.1 (fluorescein), and 2.3 +/- 0.2 (calcein). The viscosity in cytosol of sea urchin eggs (2.1-2.5 cP) is thus relatively low, yet significantly greater than that of water (1 cP) or cytosol in cultured fibroblasts (1.2-1.4 cP).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of heat stress on development in vitro and in vivo and on synthesis of heat shock proteins in porcine embryos

The present study was conducted (1) to examine the effect of an acute increase in ambient temperature on the development of porcine day 6 embryos in culture and after transfer to recipient gilts, and (2) to analyze intracellular production of heat shock proteins (hsps). The viability of porcine day 6 embryos following a temporary acute elevation in ambient temperature (at 42°–45.5°C and for 10–180 min) was examined. Synthesis of 70 kDa hsp (hsp 70) and 90 kDa hsp (hsp90) was determined by SDS-PAGE and Western blot analysis in porcine day 6 embryos subjected to heat stresses. Nonheat-stressed embryos were considered as control. Significantly higher numbers of viable nuclei were observed in treatment groups of 42°C-10 min (236.6 ± 71.4; P < 0.05) and 43°C-30 min (276.8 ± 89.4; P < 0.005) compared to control (173.9 ± 53.9). The 42°C-180 min group (158.0 ± 27.1 μm) had a greater increase in diameter after 24 hr in culture following heat stress compared to control (82.5 ± 47.3 μm), while heat stress with 43°C for ≧60 min, 44°–44.5°C for ≧30 min, or 45°-45.5°C for ≧10 min impaired their survival, as assessed by differences in number of viable nuclei. The embryos subjected to heat stresses under the conditions of 42°C-180 min, 43°C-10 min, 43°C-30 min, 44°C-10 min, or 45°C-10 min developed to normal piglets after transfer to recipient gilts. Overall pregnancy rate was 75% (6/8), and farrowing rate 62.5% (5/8). Of heat-stressed embryos transferred, 59% (36/61) developed to normal piglets. Heat-stress conditions of 42°C for 180 min, 43°C for 30 min, 44°C for 10 min, and 45°C for 10 min were determined as critical with respect to the in vitro and in vivo survival of porcine embryos. Porcine day 6 embryos constitutively synthesized hsp70 even without heat stress, while hsp90 was detected only at trace level. Neither hsp70 nor hsp90 levels increased in the embryos subjected to heat stresses. In conclusion, porcine day 6 embryos could continue to develop in vivo or during in vitro culture after exposure to acute and temporary rise in temperature. However, no increase of hsp70 and hsp90 was observed in the heat-stressed porcine embryos, while hsp70 was detected in the nonheat-stressed porcine embryos. The precise mechanism of the thermotolerance was unclear. © 1996 Wiley-Liss, Inc.     

Potentiation of heat stress-induced hsp70 expression in vivo by aspirin

Studies in cultured cells have demonstrated that non-steroidal anti-inflammatory agents can potentiate heat-induced hsp70 expression through activation of HSF1 to a DNA binding state. We investigated the influence of aspirin on hsp70 expression in intact rats subjected to heat stress. Rats were injected intraperitoneally either with aspirin (100 mg/kg) or vehicle alone, 60 min prior to their placement at 37°C or room temperature for 30 min. hsp70 mRNA expression was analyzed in lung, liver and kidney isolated from animals assigned to one of four different treatment paradigms; untreated controls, heat aspirin, and aspirin-plus-heat. Comparison of hsp70 expression in the treatment groups revealed that in all tissues examined, aspirin-plus-heat treatment resulted in 3–4 fold higher levels of hsp70 mRNA relative to those seen with heat treatment alone. Little or no hsp70 mRNA expression was detected in the unheated groups, regardless of aspirin treatment. In keeping with the mRNA expression, Hsp70 protein levels were also elevated in aspirin-plus-heat treated animals. Aspirin treatment did not alter hsp70 protein expression in the absence of heat. In contrast to in vitro observations, aspirin treatment in vivo did not alter HSF1 DNA binding properties. Core body temperature measurements revealed that aspirin pretreatment enhanced the rise in body temperature seen in response to heat treatment. This increased hyperthermic response to heat stress probably accounts for the potentiation of hsp70 expression observed in aspirin-plus-heat treated rats. Given the widespread use of aspirin in humans within a dose range comparable to that used here, our findings are likely to have important physiological consequences.     

Determination of sialic acids in immune system cells (coelomocytes) of sea urchin,Paracentrotus lividus,using capillary LC-ESI-MS/MS

Coelomocytes are considered to be immune effectors of sea urchins. Coelomocytes are the freely circulating cells in the body fluid contained in echinoderm coelom and mediate the cellular defence responses to immune challenges by phagocytosis, encapsulation, cytotoxicity and the production of antimicrobial agents. Coelomocytes have the ability to recognize self from non-self. Considering that sialic acids play important roles in immunity, we determined the presence of sialic acid types in coelomocytes of Paracentrotus lividus. Homogenized coelomocytes were kept in 2 M aqueous acetic acid at 80 °C for 3 h to liberate sialic acids. Sialic acids were determined by derivatization with 1,2-diamino-4,5-methylenediaoxy-benzene dihydrochloride (DMB) followed by capillary liquid-chromatography-electrospray ionization/tandem mass spectrometry (CapLC-ESI-MS/MS). Standard sialic acids; Neu5Ac, Neu5Gc, KDN and bovine submaxillary mucin showing a variety of sialic acids were used to confirm sialic acids types. We found ten different types of sialic acids (Neu5Gc, Neu5Ac, Neu5Gc9Ac, Neu5Gc8Ac, Neu5,9Ac₂, Neu5,7Ac₂, Neu5,8Ac₂, Neu5,7,9Ac₃, Neu5Gc7,9Ac₂, Neu5Gc7Ac) isolated in limited amounts from total coelomocyte population. Neu5Gc type of sialic acids in coelomocytes was the most abundant type sialic acid when compared with other types. This is the first report on the presence of sialic acid types in coelomocytes of P. lividus using CapLC-ESI-MS/MS-Ion Trap system (Capillary Liquid Chromatography-Electrospray Ionization/Tandem Mass Spectrometry).     

Identification of the eleocytes as the vitellogenin producing cells in nereids.

Coelomocytes of Nereis diversicolor synthesize and secrete vitellogenin in vitro. Using a monoclonal antibody which specifically recognized vitellogenin, we showed by immunocytochemistry that among the coelomocytes only a subpopulation, called eleocytes, contained vitellogenin. These results assert that eleocytes are the vitellogenin producing cells in nereids.