Sea urchin immune cells as sentinels of environmental stress ☆

Annalisa Pinsino *, Valeria Matranga **Consiglio Nazionale delle Ricerche, Istituto di Biomedicina e Immunologia Molecolare “A. Monroy”, Via Ugo La Malfa 153, 90146 Palermo, Italy

A R T I C L E I N F O

Article history:Received 19 September 2014Revised 14 November 2014Accepted 17 November 2014Available online 24 November 2014

Keywords:Paracentrotus lividusImmuno-toxicityCellular modelBiomarkersCoelomocytes

A B S T R A C T

Echinoderms, an ancient and very successful phylum of marine invertebrates, play a central role in themaintenance of ecosystem integrity and are constantly exposed to environmental pressure, including:predation, changes in temperature and pH, hypoxia, pathogens, UV radiation, metals, toxicants, and emerg-ing pollutants like nanomaterials. The annotation of the sea urchin genome, so closely related to humansand other vertebrate genomes, revealed an unusually complex immune system, which may be the basisfor why sea urchins can adapt to different marine environments and survive even in hazardous condi-tions. In this review, we give a brief overview of the morphological features and recognized functions ofechinoderm immune cells with a focus on studies correlating stress and immunity in the sea urchin. Immunecells from adult Paracentrotus lividus, which have been introduced in the last fifteen years as sentinelsof environmental stress, are valid tools to uncover basic molecular and regulatory mechanisms of immuneresponses, supporting their use in immunological research. Here we summarize laboratory and field studiesthat reveal the amenability of sea urchin immune cells for toxicological testing.

© 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Echinoderms, an ancient and very successful phylum of marineinvertebrates, represent a morphologically varied group consistingof around 7000 living members with unique shapes and colours. Theextant phylum is divided into five main classes: crinoids (feather stars),asteroids (sea stars), ophiuroids (brittle stars), echinoids (sea urchins)and holothurians (sea cucumbers). Crinoids are considered the mostprimitive class, while echinoids and holothurians the most ad-vanced. However, recent molecular studies support an ophiuroid/asteroid clade (Asterozoa) based on either convergent evolution ofthe pluteus or reversals to an auricularia-type larva occurring in as-teroids and holothurians (Telford et al., 2014). It may not be obvioushow animals like sea stars, sea urchins, sand dollars or sea cucum-bers are all related, but despite their various shapes they possesscommon characteristics: i) adult radial symmetry, ii) a water vascu-lar system, iii) a calcite endoskeleton with a specific three-dimensionalstructure (stereom), and iv) benthic lifestyle. Echinoderms play a keyrole in the maintenance of ecosystem integrity (Hereu et al., 2005)and are constantly exposed to environmental pressure, including: pre-

dation, changes in temperature and pH, hypoxia, pathogens, UVradiation, free radicals, metals, toxicants and emerging pollutants. Thekeys for their success include a few survival strategies, such as a spinyphysical defence structure, an effective immune defence system, atoxin producing equipment, and an amazing regeneration capabili-ty, which provide them with protection, robustness, resistance andstemness. Echinoderms appeared 520 million years ago, prior to theCambrian explosion, and are globally distributed in the oceans inalmost all depths, latitudes, temperatures and environments(Bottjer et al., 2006; Iken et al., 2010). What we now call immunedefence appeared early in the evolution of these marine inverte-brates through the invention of the innate immune response, mediatedby a vast repertoire of recognition molecules (immunome), and thestress response, mediated by a subset of stress-sensing gene fami-lies and pathways (defensome). These protective mechanisms are usedby the echinoderm immune cells to recognize both biotic and abioticstressors and to sense, transform and eliminate many potentiallynoxious materials.

2. Echinoderm immune cells

Echinoderm immune cells, also known as coelomocytes, are aheterogeneous population of freely moving cells found in all coe-lomic spaces, including the perivisceral coelomic cavities and thewater-vascular system (Glinski and Jarosz, 2000; Smith et al., 2010).They are also present sparsely in the connective tissue and amongsttissues of various organs (Munõz-Chápuli et al., 2005; Pinsino et al.,2007). Molecular studies have suggested the presence of phago-cytic cells in the major organs and tissues, including the axial organ,

☆ This article is handled by Dr. Lynn Courtney Smith.* Corresponding author. Consiglio Nazionale delle Ricerche, Istituto di Biomedicina

e Immunologia Molecolare “A. Monroy”, Via Ugo La Malfa 153, 90146 Palermo, Italy.Tel.: +390916809526.

E-mail address: annalisa.pinsino@ibim.cnr.it (A. Pinsino).** Corresponding author. Consiglio Nazionale delle Ricerche, Istituto di Biomedicina

e Immunologia Molecolare “A. Monroy”, Via Ugo La Malfa 153, 90146 Palermo, Italy.Tel.: +390916809551.

E-mail address: valeria.matranga@ibim.cnr.it (V. Matranga).

http://dx.doi.org/10.1016/j.dci.2014.11.0130145-305X/© 2014 Elsevier Ltd. All rights reserved.

Developmental and Comparative Immunology 49 (2015) 198–205

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pharynx, esophagus, intestine and gonads (Majeske et al., 2013b).Cell type composition has been postulated to depend on the species,as well as on patho-physiological conditions of each individual. Basedon morphological criteria echinoderm immune cells have been clas-sified into at least six cell types, but not all six have been identifiedin all classes/species. Names used to describe them in the pastinclude phagocytic amoebocytes, phagocytes, amoebocytes, spher-ule cells, vibratile cells, haemocytes, crystal cells and progenitor cells(Smith, 1981). It is well recognized that echinoderm immune cellscarry out functions similar to those of the vertebrate blood cells,such as clot formation, phagocytosis, encapsulation, clearance of bac-teria or other foreign materials, oxygen transport (Matranga et al.,2005). It is not the purpose of this review to unravel the morpholo-gies, roles and functions of the different cell types for each class ofechinoderms; rather this report will centre mostly on the speciesof interest, namely the sea urchin Paracentrotus lividus, and will de-scribe the results that correlate environmental stress and immunity(see sections 4–6). Although echinoderms have been the focus ofclassical studies that defined animal cellular immunity (Metchnikoff,1891), only recent studies have addressed immune functions in thesea urchin.

The coelomic fluid in which the immune cells reside and moveis a key factor governing immunological capabilities, as it con-tains essential trophic and activating factors produced by immunecells themselves (Matranga, 1996; Matranga et al., 2005; Smith et al.,2010). Echinoderms lack a distinct directional closed circulatorysystem; on the contrary, they possess an open water vascular system(WVS), which is structurally and physiologically specialized to carryout several functions typical of the higher vertebrate vascular system(Smith, 1981) (Fig. 1). In addition, the WVS serves to generate,distribute and control the hydrostatic pressure necessary for loco-motion, respiration, feeding, reproduction, and excretion (Nichols,1972).

The coelomic fluid, which can be considered similar to seawater witha dense population of immune cells and a high concentration of factors,has functions similar to the blood of higher animals. Thus, by being indirect contact with internal cells and tissues, it can provide an overallprofile of the physio-pathological state of the organism. The loss of coe-lomic fluid can affect the behaviour and the physiological functions ofechinoderms. Thus, an efficient mechanism to plug and repair acci-dental or pathological leaks in the body wall becomes crucial to prevent

infections and maintain homeostasis. In echinoderms, the immunesystem evolved as a defence strategy not only against external insults,but also against internal pathological threats. In fact, echinoderms donot show variations in metabolic functions and fertility over time, andno cases of cancer, immune and age-related diseases have been re-ported (Bodnar, 2009). In accordance, recent analysis of oxidative damageand proteomic studies in three sea urchin species with different lifespansrevealed that the sea urchin is a promising tool for investigations of oxi-dative cell damage, senescence, and longevity (Bodnar, 2013; Du et al.,2013).

3. The relationship between stress and immune response

A less restrictive definition, perhaps more applicable to inver-tebrates in general and to echinoderms in particular, defines anantigen as any chemical substance capable of stimulating theimmune system to respond by one or a combination of several re-actions, including phagocytosis, cell-mediated immune responses,and the cell stress response. Recent studies have shown that pro-teins eliciting the cellular stress responses, including heat shock-,ER stress- and DNA damage-responses, interact with and regulatethe signalling pathways involved in the activation of both innateand adaptive immunity (Muralidharan and Mandrekar, 2013). Inhumans, the regulation of innate immune cell activation by cell stresspathways is essential in host defence. In fact, this interaction is rel-evant to the control of diseases that are characteristic of aberrantimmune responses, such as chronic inflammatory diseases, auto-immune disorders, allergic disorders and cancer. The immune-signalling cascades that are linked to cellular stress responses arestimulated by an accumulation of unfolded proteins within theimmune cells (Fig. 2), which serves as a signal amplification cascadefavouring cytokine production (Cláudio et al., 2013).

The induction of proteins related to the cellular stress re-sponses does not necessarily indicate response to a stress. Instead,it can be an integral part of a selective transcription programme con-trolled by innate immune receptors (Hetz, 2012). For example, theextra-cellular 70-kDa heat shock protein (Hsp70), a cognate of thefirst stress protein described in the literature to respond to an in-crease in the temperature of the organism (De Maio et al., 2012;Ritossa, 1962), can function as a cytokine that acts on human mono-cytes, showing the ability to: i) bind with high affinity to the plasma

Fig. 1. Basic anatomy of the sea urchin. The schematic illustration points to the complex open water vascular system (WVS), captions in purple colour. Seawater entersthrough the madreporite on the aboral surface into a short straight canal, connected to a circular canal, the ring canal, which in turn is linked to the radial canals. Radialcanals bring the seawater to each ampulla and thereafter to the tube feet. (For interpretation of the references to colour in this figure legend, the reader is referred to theweb version of this article.)

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membrane, ii) activate NF-κB, and iii) up-regulate the expressionof a few pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α) (Aseaet al., 2000). Hsp70 can be released from cells, with a traffickingmechanism involving lysosomal lipid rafts (Hunter-Lavin et al., 2004).Growing evidence suggests that this mechanism also occurs in thesea urchin immune cells. In fact, Browne et al. (2007) showed thatextra-cellular mixtures of the constitutive and inducible forms ofthe Hsp70 (Hsc70 and Hsp70, respectively) are involved in the clot-ting reaction of hypotonically stressed sea urchin immune cells,probably promoting mitosis of dividing cells and inhibiting cellspreading. A shotgun proteomics analysis of the coelomic fluid ofthe purple sea urchin, Strongylocentrotus purpuratus injected withLPS identified 27 proteins belonging to the stress response and de-toxification classes (Dheilly et al., 2013), validating the key role ofstress sensing in the regulation of the sea urchin immune cellactivation.

The human innate immune response relies on recognition of evo-lutionarily conserved structures on pathogens, the pathogen-associated molecular patterns (PAMPs), through a limited numberof pattern recognition receptors (PRRs), of which the family of Toll-like receptors (TLRs) has been studied extensively (O’Neill et al.,2013). Analyses of the sea urchin genome revealed an unprece-dented complexity of innate immune recognition receptors,regulators, and effectors, of which the majority are closely relatedto human homologues (Hibino et al., 2006; Rast et al., 2006). Thecomplexity varies widely between sea urchin species with Lytechinusvariegatus having far fewer immune genes than S. purpuratus,Strongylocentrotus franciscanus and Allocentrotus fragilis (Buckley andRast, 2012).

To fight different pathogens, the sea urchin has generated arandom diversification and expansion of PRRs, perhaps by gene re-combination and/or gene duplication/deletion mechanismsgenerating receptor gene sequence diversity resulting from a con-stant, long-term evolutionary competition between high rates ofmutation and/or variation in antigens (Smith, 2010). Three classesof innate receptor proteins are particularly expanded in the seaurchin genome, which comprise vast families of TLRs, leucine-richrepeat (LRR) domain-containing proteins similar to the vertebrateNOD/NALP receptors (NLRs) and scavenger receptor cysteine-richdomains (SRCRs) (Hibino et al., 2006). These classes of receptors are

also present in vertebrates where they are represented in fewernumbers by a factor of ~10. These findings reveal an innate immunesystem of unprecedented complexity that is present in the sea urchin.Understanding the intensive selective pressure that moulded thesegene families that likely originated first during evolution could aidin dissecting the mechanisms that occurred to result in the appear-ance of adaptive immunity in higher vertebrates (Hibino et al., 2006).

4. P. lividus immune cells: morphological features andrecognized functions

P. lividus is a common echinoid with great ecological impor-tance due to its central role for the structure and function of theMediterranean rocky reef ecosystem assemblages. This keystone,ecologically relevant species also has a high commercial valuebecause its roe (gonads) are considered a delicacy and attracts a largemarket share of echinoderm sea food. It is a regular sea urchin havinga globular calcareous test, with long, sharply pointed spines andoccurs in a variety of colours (Fig. 3). Sometimes mistakenly calledthe purple sea urchin because some morphs are similar to the Pacificspecies, S. purpuratus, P. lividus is distributed throughout the Med-iterranean Sea and in the North-Eastern Atlantic Sea, from Scotlandand Ireland to Southern Morocco and the Canary Islands. P. lividusis a very successful species, with two life stages: i) an early and briefplanktonic developmental phase (up 3–4 weeks), and ii) a benthicadult with a lifespan of 8–15 years (Ebert, 2007; Tomsic et al., 2010).Embryos from this species were used by the 19th century Europe-an biologists to perform classical studies that led to major basicdiscoveries in developmental biology (Pederson, 2006). Amongstthose, a poorly known example is the use of the sea urchin embryofor the fundamental discovery of cyclins, which are the key mol-ecules that regulate the cell cycle in all eukaryotic organisms(Minshull et al., 1989) including yeast, plants, animals and humans.This work received the 2001 Nobel Prize in Physiology or Medi-cine, which was awarded jointly to Leland H. Hartwell, Tim Huntand Sir Paul M. Nurse. Following the publication of the first echi-noderm genome (Sea Urchin Genome Sequencing Consortium et al.,2006), a similar effort has been made by a core group of Europeanlaboratories forming a consortium for the sequencing, assembly andannotation of the genome of the sea urchin P. lividus (P. lividus

Fig. 2. Immune response can be activated by Hsp70 proteins. The model represents an immune cell undergoing stress. This increases the levels of misfolded proteins, ac-tivating the Hsp70-dependent stress response resulting in the increased levels of Hsp70 proteins. In the stress-protected cell, Hsp70 proteins stabilize misfolded proteinsand activate an immune-signalling cascade that triggers cytokine production.

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genome Project). The P. lividus genome is expected to be releasedin 2015. The full understanding of the morpho-functional proper-ties of sea urchin immune cells is still controversial, but some oftheir immune mechanisms are relatively well known and includecellular recognition and cytotoxicity (Arizza et al., 2007; Bertheussen,1979), phagocytosis and ROS production (Ito et al., 1992), antibac-terial and anti-biofilm properties (Majeske et al., 2013a; Schillaciet al., 2010; Stevens et al., 2010) and a complement system that in-cludes C3 and factor B homologues, that is likely initiated by a largeset of homologues similar to mannose binding lectin and C1q, anda number of antimicrobial peptides (Li et al. 2014; Smith et al., 2010).

Three major cell types of freely circulating immune cells havebeen described in P. lividus (amoebocytes, vibratile cells, phago-cytes) (Fig. 4), which can be identified easily when cells are inspectedunder the microscope immediately after collection (Matranga et al.,2005, 2006; Pinsino et al., 2008). Under these conditions, a few cellsare capable of rapid movements, while others show a slow loco-motion (Supplementary Video S1). Of the mobile group, theamoebocytes (red and white) constitute about 13% ± 3 (mean ± SE)of the total cell population. Their locomotion is achieved by rapidchanges in the body shape, closely resembling the motions ofamoebae (Fig. 4A, Supplementary Video S1). Due to their relativelyfast movement, it seems plausible that these cells may be in-volved in the first phase of pathogen immobilization (Smith, 1981).Red amoebocytes carry natural red pigments (echinochrome) uni-formly dispersed within the cytoplasmic vesicles, and thought tobe utilized as an anti-bactericidal agent (Service and Warklaw, 1985;Smith, 1981). The homeostasis of red and white amoebocytesdepends on the healthy state of the sea urchin to which they belong.For example, there is a rapid increase in the number of red amoe-bocytes in specimens collected from polluted sea water or subjectedto accidental injury (Matranga and Bonaventura, 2002; Matrangaet al., 2000, 2005, 2006; Pinsino et al., 2008). The vibratile cells ofP. lividus sea urchin constitute about 7.45% ± 0.86 of the total cellpopulation (Matranga et al., 2006). These are round, very fast movingcells (Fig. 4B, Supplementary Video S1) that can move in a straightdirection along a helicoidal pattern based on the actions of a singlelong flagellum, which may contribute to the mixing of the coelo-mic fluid. Vibratile cells contain large cytoplasmic granules, identifiedas primary lysosomes by in vivo assay using the Neutral Red (NR)dye (Annalisa Pinsino, personal communication). Exocytosis of thesegranules may be associated with the clotting reaction (Smith et al.,2010). Immune cells from P. lividus after challenge with Escheri-chia coli show an increased number of circulating vibratile cells 3hours after injection (Pinsino, personal communication). Phago-cytes are the most abundant immune cell type in P. lividus andaccount for approximately 80% ± 1.77 of the total population(Matranga et al., 2006; Smith et al., 2010). These cells have adendritic-like phenotype that undergoes a striking morphological

Fig. 3. Paracentrotus lividus sea urchins occurring in the Mediterranean Sea. Sixcoloured adult sea urchins are shown. The image was taken during the oceano-graphic campaigns 2003–2004 on board of the ASTREA boat, around the TremitiIslands Archipelago, Southern Adriatic Sea, Italy.

Fig. 4. Immune cells from Paracentrotus lividus sea urchin. (A, B) Live immune cells collected as a total cell population in an anticoagulant solution containing EGTA wereinspected under a Zeiss Axioskop 2 Plus microscope (Zeiss, Arese, Italy) just after collection. Cell types are indicated by captions of different colours and correspondingpointing arrows. Specifically, in panel (A): red amoebocyte (red arrows), white amoebocyte (white arrows), and filopodial phagocyte (black arrow); in panel (B): vibratilecell (blue arrow), filopodial and petaloid phagocytes (black arrows). (C–E) Sea urchin immune cells fixed in cold methanol and immuno-stained with β-Actin (C) or α-Tubulin(D) Abs, or incubated with Dihexyloxacarbocyanine iodide (DiOC6) for ER labelling (E). The ER stained by DiOC6 showed a network of flat vesicles, more dense around thenuclei. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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transition from petaloid to filopodial shape (Fig. 4A and B). Thischange in shape is induced by a calcium-dependent clotting processthat mediates the reorganization of cytoskeletal microfilaments,which in turn causes cytoplasmic retraction and filopodial elonga-tion (Henson et al., 1992). Phagocytes collected in an anticoagulantsolution containing EGTA (a calcium chelator) appear in a petaloidform where thin sheets of cytoplasm, as petals of a flower, are or-ganized around a central nuclear region. The cytoplasm containselongated actin bundles, tubulin filaments and associated organ-elles including a well developed endoplasmic reticulum (ER)(Fig. 4C–E). The two morphotypes of phagocytes exhibit two majordifferent functions; petaloid cells are actively involved in phago-cytosis, whereas filopodial cells trigger the formation of the clot(Supplementary Video S1) by the aggregation of single cells (phago-cytes, amoebocytes, and probably vibratile cells). Once placed inculture and after removal of the anticoagulant, sea urchin phago-cytes aggregate over time into large syncytia-like structures whichform on glass or plastic surfaces, as well as in response to foreignparticles, bacteria and LPS (Majeske et al., 2013a; Matranga et al.,2005). Phagocytes have also been implicated in encapsulation, ag-gregation, graft rejection, wound repair, as well as cytolytic/cytotoxic reactions and transport of materials through vesicles (Hillierand Vacquier, 2007; Matranga et al., 2000; Smith, 1981). Furtherstudies to clarify the role of each immune cell type in the above men-tioned functions are awaited.

5. P. lividus immune cells: new tools to monitor the state ofmarine environmental health

For more than a decade, sea urchin immune cells from P. lividushave been proposed as tools for toxicological testing and environ-mental monitoring (Matranga et al., 2000, 2005) and they have beenadded to the list of proposed alternative non-mammalian modelsfor assessing toxicity as presented by the European Centre for theValidation of Alternative Methods (EURL-ECVAM at JRC). P. lividuscan be considered a suitable immune-toxicology model due to themodest lifespan of the benthic adults and direct exposure to accu-mulating man-made contaminants discharged into the sea andtrapped in the sediments. Based on measurements of the test di-ameter (excluding spines), age estimates indicate that P. lividus hasa lifespan of about 8–15 years (Ebert, 2007; Tomsic et al., 2010).The fact that this sea urchin is not particularly long-lived com-pared to other echinoderms that can live for 50–100 years(S. purpuratus and S. franciscanus, respectively), supports it as a sen-sitive sentinel organism to monitor the state of marine environmentalhealth. Recent analysis of DNA damage and DNA repair capabili-ties of immune cells from four echinoderm species (L. variegatus,Echinometra lucunter, Isostichopus badionotus, and Tripneustesventricosus) indicate that species with the shortest estimated lifes-pan have a greater sensitivity to DNA damage than the longer-lived species (El-Bibany et al., 2014). As suggested by these authors,longevity may be an important determinant for species vulnera-bility to environmental genotoxicity.

According to the World Health Organization, more than 100,000chemical compounds are released in the marine environment everyyear as a consequence of their production, use and disposal. Thecapability of sea urchin immune cells to sense rapid and/or slowenvironmental changes and to activate a specific immune defencehas lately been shown in both field and laboratory studies. Oceantemperatures are rising throughout the world, the seawater pH isdecreasing, the ions trapped in the sediments are released intothe water column, and the emerging contaminants are notregulated. Of concern is the fact that the rapid anthropogenicallyinduced changes that are occurring in the environment are beyondthe range of the protective mechanisms of the sea urchin to allowit to survive.

6. Testing different environmental hazards at cellular andmolecular levels

6.1. Controlled studies

In pioneering studies, the capability of P. lividus immune cellsto respond to adverse external conditions was assessed at the mo-lecular level by evaluating the impact of temperature changes underlaboratory controlled conditions (Matranga et al., 2000). The stressproteins belonging to the Hsp70 family are known to serve as crit-ical indicators of changes in the steady state homeostasis of cells,tissues and organs (Lindquist and Craig, 1988). The highly con-served Hsp70 family includes Hsc70, which is constitutivelysynthesized and shows moderate modulation upon mild stress, andHsp70, which is usually not present in cells, but is highly inducedby heavy stress (Deane and Woo, 2006; Franzelletti and Fabbri, 2005;Pinsino et al., 2008, 2010, 2011). Both Hsc70 and Hsp70 have chap-erone functions, participating in i) protein synthesis and maturation,ii) folding, assembly, and disassembly of nascent proteins, iii) re-folding of mature proteins, and iv) proteolysis and intracellulartrafficking (Lindquist and Craig, 1988). As a consequence, becausethey might influence the activity of intracellular signalling mol-ecules, Hsc70/Hsp70 have crucial activities in determining stressresistance, immune resistance and apoptosis, thus being recog-nized as ubiquitous biomarkers of environmental stress (Gupta et al.,2010).

As described by Matranga et al. (2000), immune cells from adultsea urchins that are placed in warm (35 °C) or cold (4 °C) water for4 hours, immediately followed by a recovery for an hour at 16 °C(control temperature) show an increase in the Hsc70 levels, two-and five-fold higher than controls, respectively (Matranga et al.,2000). The Hsc70 levels were evaluated by immunoblotting withan anti-Hsp70 antibody that recognized both Hsc70 and Hsp70 formsof the stress proteins, but also exhibits a strong reactivity with theconstitutive form present in both sea urchin embryonic and immunecells (Geraci et al., 2004; Matranga et al., 2006; Pinsino et al., 2008,2010, 2011). The increased Hsc70 levels have been explained as anactivated thermo-tolerance capability of the sea urchins, in agree-ment with the Hsp70-mediated thermo-tolerance described instudies on P. lividus sea urchin embryos (Giudice et al., 1999; Roccheriet al., 1995) and embryos or adult organs of other echinoderm species(Dong et al., 2011; Hammond and Hofmann, 2010).

Another acknowledged marker of cell stress is acetylcholines-terase (AChE) that is involved in acetylcholine (Ach) metabolism(Michelson and Zeimal, 1973). In humans, ACh receptors and AChEare present in membranes of both lymphocytes and erythrocytesand are responsive to different kinds of stress (Carvalho et al., 2004;Kawashima and Fujii, 2000). Due to the similarity between sea urchinand human immune cells, in addition to the Hsc70, AChE was pro-posed as biomarker of cold-stress in P. lividus immune cells (Angeliniet al., 2003).

Recently, P. lividus immune cells have also been used as a cel-lular model to study the in vivo potential toxicity of a few selectedmetal dioxide nanoparticles (NPs), i.e. stannum oxide (SnO2), ceriumoxide (CeO2), and iron oxide (Fe3O4) (Falugi et al., 2012; Matrangaand Corsi, 2012; Corsi et al., 2014). After 5 days of exposure, nano-aggregates/agglomerates were found inside sea urchin immune cells,causing subcellular modifications of the trans-Golgi and the endo-plasmic reticulum (ER) compartments. At the molecular level, authorsshowed that NPs inhibited the activity of AChE and other two cho-linesterase isoforms (BChE and PrChE) and reduced the basal levelsof Hsc70 and glucose-regulated protein 78 (GRP78) (Falugi et al.,2012). It is noteworthy that GRP78, also known as binding immu-noglobulin protein (BiP or heat shock 70 kDa protein 5 (HspA5), isa major Hsp70 molecular chaperone located in the lumen of the ERthat assists in protein folding and assembly, protein quality control,

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Ca2+ binding, and regulating ER stress signalling (Ni et al., 2011).Taken together, results reinforce the notion that Hsp70 familymembers are sensitive markers of stress, in association with the cho-linesterase family components.

As an alternative to whole animal tests that require a largenumber of animals, several in vitro studies have been performedby exposing immune cells from P. lividus to cold temperatures, acidicpH, cadmium and UV-B radiation in short-term cultures (Matrangaand Bonaventura, 2002; Matranga et al., 2000, 2005, 2006). In general,an increase in the Hsc70 levels was noted in all these analyses afterexposure to i) 1 hour at 4 °C, ii) UV-B radiation (500, 1000, and2000 J/m2), iii) 2 hours at 4.7 ± 0.2 pH, iv) 4 hours of cadmium(10−3,10−4, 10−5 M), v) 2 hours of cadmium (10−4 M) followed by UV-Bradiation (1000 J/m2). When the cells were exposed to the combi-nation of cadmium and UV-B radiation, it caused an increase in theHsc70 levels; however, the effect was not additive perhaps becausethe cadmium exposure protected the cells from the UV-B, therebyincreasing the levels of tolerance and resistance of the immune cells(Matranga et al., 2005).

6.2. From controlled conditions to field studies

During the last fifteen years, the use of P. lividus immune cellsas cellular model for the assessment of long-term exposure to con-ventional and emerging pollutants in the environment has beenestablished. In 1995, an original field study was performed duringan EU-sponsored Summer School at the Ruder Boskovic MarineStation (Rovinj, Croatia) and focused on the use of new biotechno-logical approaches in environmental monitoring programmes. Forthe first time P. lividus immune cells were used to assess pollutionin marine coastal areas (Northern Adriatic Sea) (Matranga et al.,2000). The first difference observed between immune cells iso-lated from specimens collected from polluted (urban runoff andindustrial wastewater) and unpolluted sites (Limski Canal, north ofRovinj) was found at the cellular level, consisting in an evident in-crease in the number of the red amoebocytes in those specimenscoming from polluted seawater (Matranga et al., 2000). Similar resultswere obtained from studies performed during the oceanographiccampaigns on board of the ASTREA boat (July 2003; June/July 2004)around the Tremiti Islands (Southern Adriatic Sea, Italy). In agree-ment, an elevated level of red amoebocytes was found in P. lividussamples collected from contaminated areas surrounding the islandof Pianosa, the location of a persistent source of contamination fromWorld War II conventional ammunitions (TNT) plus a merchant boatwreck (metals) (Pinsino et al., 2008). There has been no functionalexplanation to date to account for the observed increase in red amoe-bocytes. However, a few hypotheses can be put forward: i) aconversion or pre-existing cell phenotypes such as a differentia-tion from white to red amoebocytes; ii) rapid cell division to generatemore red amoebocytes from a few circulating stem cells; iii) re-cruitment of additional red amoebocytes from the haematopoieticareas/tissues (niches). Although future studies in these directionsare needed to clarify at least one of these intriguing hypotheses, afew reviews on echinoderm adult stem cell occurrence have ap-peared in the literature (Candia-Carnevali et al., 2009; Rinkevich andMatranga, 2009).

At the molecular level, in both the field studies described above,an increase in the Hsc70 levels was observed in immune cells ofsea urchins collected from polluted seawater as compared to con-trols (Matranga et al., 2000; Pinsino et al., 2008). Interestingly, resultsrecapitulated that observed in laboratory controlled conditions,confirming that Hsc70 is an excellent biomarker to test for envi-ronmental hazards using P. lividus immune cells as a sensitive cellularmodel. To the best of our knowledge, no investigation on the effectsof pollutants in the field combining the use of echinoderm immune

cells and molecular tools has been reported to date, with the onlyexception of the Asterias rubens sea star (Matranga et al., 2012).

7. Future perspective and concluding remarks

The sea urchin occupies a strategic phylogenetic position becausethe echinoderms represent an evolutionary link between inverte-brates and vertebrates. The fully sequenced genome of S. purpuratushas shown that the sea urchins are closer to humans than to othermodel invertebrate organisms. Despite immune cellular behaviourand self/non-self recognition were first established in echino-derms during the last century and the availability of the full seaurchin genome in recent years, little effort has been made to makeuse of P. lividus immune cells as a sensitive cellular model, partic-ularly suitable in immuno-toxicological studies.

Adult P. lividus immune cells have been introduced as a valid toolto uncover basic molecular and regulatory mechanisms of immuneresponse and immuno-toxicity, having many strengths for immuneresearch including: i) resistance and plasticity to environmentalchanges, ii) ease and responsiveness to experimental manipula-tion, iii) no ethical animal use restrictions when respecting the 3Rscriteria (reduction, refinement, and replacement of animal experi-ments) of EU Agency for Alternative Approaches for Animal Testing(EPAA).

Reproductive, developmental and immunological functions arecentral to the life of any organism. Thus, to know whether anthro-pogenic compounds released in the oceans have noxious effects onmarine animals can be very useful in predicting and mitigating thepotential risk related to their increase and spread in the environ-ment. The understanding of molecular pathways involved in sensingand coping with classical or emerging pollutants in a defined cel-lular model, such as the sea urchin immune cell, could be very helpfulfor developing predictive diagnostic tools to evaluate the risk tomarine organisms. In addition, the sea urchin immune cell can beconsidered a proxy to human immune cells and used as a cellularmodel for studies on immuno-toxicology. Studies on sea urchin re-sistance to immune and age-related diseases may contribute tohighlighting the key protective molecules, which could be used ininnovative applications at the cutting edge of biomedicine.

Acknowledgements

The work described has been partially supported by the Euro-pean Regional Development Fund 2007–2013 – Regione Sicilia –DeCroMed Project, CUP: G93F12000190004, to VM. The authors wishto thank one of the anonymous reviewers for suggestions and criti-cisms that improved the quality of this review article. M. Biondois acknowledged for his technical assistance in the video mount-ing and E. Amato for photographic recording. VM is grateful to R.Emlet, who helped record immune cells while both were teachingat the EU-sponsored Course “The Sea Urchin: from Basic Biology toAquaculture”, held at the International Marine Centre of Oristano(Italy) in 2000.

Appendix: Supplementary material

Supplementary data to this article can be found online atdoi:10.1016/j.dci.2014.11.013.

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