Developmental and Comparative Immunology 23 (1999) 429±442 Echinoderm immunity and the evolution of the complement system Paul S. Gross a,c, Walid Z. Al-Sharif b, Lori A. Clow b, L. Courtney Smith a,b,* aDepartment of Biological Sciences, George Washington University, 2023 G St. NW, Washington, DC 20052 USA bInstitute of Biomedical Sciences Program in Genetics, George Washington University, Washington, DC 20052 USA cLife Sciences, University of Maryland, College Park, MD 20742 USA Accepted 1 October 1998 Abstract Our understanding of in¯ammatory responses in humans has it roots in the comparative approach to immunology. In the late 1900's, research on echinoderms provided the initial evidence for the importance of phagocytic cells in reactions to foreign material. Studies of allograft rejection kinetics have shown that echinoderms have a non-adaptive, activation type of immune response. Coelomocytes mediate the cellular responses to immune challenges through phagocytosis, encapsulation, cytoxicity, and the production of antimicrobial agents. In addition, a variety of humoral factors found in the coelomic ¯uid, including lectins, agglutinins, and lysins, are important in host defense against pathogens and other foreign substances. Recently, a simple complement system has been identi®ed in the purple sea urchin that is homologous to the alternative pathway in vertebrates. The sea machine homologue of C3, is inducible by challenge with lipopolysaccharide, which is known to activate coelomocytes. Complement components have been identi®ed in all vertebrate classes, and now have been characterized in protochordates and echinoderms indicating the primordial nature of the complement system. Because it is thought that the complement system evolved from a few primordial genes by gene duplication and divergence, the origin of this system appears to have occurred within the common ancester of the deuterostomes. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Echinodermata; Complement; Alternative pathway; Evolution; Coelomocytes; Immunology 1. Historical background had its origin in early studies of the causes and mechanisms of in¯ammation. Elie The comparative approach to immunology Metchniko, the chief proponent of the cellu- lar theory of immunity, asserted that in¯am- mation was the most important phenomenon in pathology and proposed that the primary * Corresponding author. Tel.: +1-202-994-9211; fax: +1- 202-994-6100. eectors of the immune response were circulat- E-mail address: [email protected] (L.C. Smith) ing, amoeboid-like, phagocytic cells [1]. In sup- 0145-305X/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S0145-305X(99)00022-1 430 P.S. Gross et al. / Developmental and Comparative Immunology 23 (1999) 429±442 port of his cellular theory of in¯ammation, These studies were the ®rst to demonstrate that Metchniko observed that under natural con- echinoderms could respond to allogeneic tissue ditions, the bipinnaria larvae of the sea star, although the speci®city of the response was Astropecten pentacanthus, would respond to unclear. both physical injury and to infection by mar- To clarify the non-self recognition capabilities ine algae. These initial observations led of echinoderms and to characterize their rejection Metchniko to perform the benchmark exper- kinetics, primary, secondary and third party allo- iments in cellular immunology which included grafting experiments were performed on the sea introducing rose prickles and glass rods into a urchin, Lytechinus pictus [5±7] and to a lesser bipinnaria larva and ®nding that mesodermal extent on the sea urchin, Strongylocentrotus pur- cells migrated to the injury site and encapsu- puratus [6]. Results indicated that sea urchins lated the prickle (for review see Ref. [1]). lacked speci®c non-self recognition and true im- Furthermore, he demonstrated that this munological memory since 2nd set and 3rd party phenomenon also occurred when bacteria were tissues were rejected at the same rate [6,8]. introduced into the larva, noting that the bac- teria were `devoured alive' by the phagocytic cells. In 1908, Metchniko, along with Paul 3. Coelomocyte functions Ehrlich, was awarded the Nobel Prize for this groundbreaking work using echinoderms as a The defense capabilities of echinoderms were model, thus beginning the ®eld of comparative de®ned from allograft rejection experiments, but cellular immunology. research has also concentrated on characterizing and understanding the functions of the coelomo- cytes, the cells that mediate these responses. 2. Allograft rejection Coelomocytes are found in the ¯uid ®lled coelom and, depending on the species, can be a mixture For nearly half a century after Metchniko, of several morphologically dierent types. Sea the comparative approach was largely ignored urchins of the genus Strongylocentrotus have four until William Hildemann became interested in types of coelomocytes which include phagocytes how sessile animals on a crowded coral reef (present in all echinoderms, also called bladder, defended themselves against overgrowth and petal or ®liform amoebocytes), red spherule cells fusion. He and his colleagues investigated echino- (also called eleocytes, morula cells and pigment derm immunity by employing allograft rejection cells), colorless spherule cells (also called white to assess immune capabilities in these organisms. morula cells) and vibratile cells [9±12]. Sea stars (Protoreaster nodosus and Dermasterias imbricata ) and a sea cucumber (Cucumaria trico- 3.1. Coelomocyte in®ltrates in injury and infection lor ) were used to demonstrate the capability of these animals to dierentiate between self and Coelomocytes have long been considered to be allogeneic tissues [2,3]. Results indicated that mediators of the immune response, in part, they would reject ®rst set skin allografts in a because of their presence surrounding injuries chronic manner, that repeated allografts (2nd set and infections in several echinoderm species and and 3rd set) were rejected more quickly, and that their appearance in grafted tissues. Skin infec- autografts healed in and remained intact. tions become ringed with black or dark red tis- Unfortunately, third party allografts were either sues that are accumulations of red spherule cells not done or were inconclusive. Histological ana- in the sea urchin, Paracentrotus lividus [13] and lyses of rejecting allografts had a signi®cant cellu- in S. purpuratus (Smith, unpublished obser- lar in®ltrate that increased with time [2±4] and vation). In®ltrates of red spherule cells have also appeared to correspond to the phagocytes in the been documented as red spots on gonadal tissue sea star larva that were observed by Metchniko. of the sea urchin Strongylocentrotus intermedius P.S. Gross et al. / Developmental and Comparative Immunology 23 (1999) 429±442 431 that surround metacercaria of a parasitic worm cytosed and encapsulated injected red blood cells [14]. Cellular in®ltrates consisting of phagocytes (RBCs) [22], as did the sea urchin S. droebachien- and red spherule cells have also been noted sis [23]. The sea urchin, L. pictus, eciently around broken, infected, and regenerating spines cleared T4 bacteriophage [24] and carmine par- of S. purpuratus [15]. Red pigmented cells accu- ticles injected into the coelomic cavity of P. cali- mulated at sites of surgical injuries during the in- fornicus resulted in agglutination, encapsulation itial phase of tissue transplantation in L. pictus and brown body formation followed by the ex- [5] and histological analysis of allografts in the cretion of the brown bodies into the cloaca [20]. sea star, D. imbricata revealed mixed cellular Even latex beads were quickly cleared from the in®ltrates in which cell densities increased during coelomic cavity of the sea urchin, S. droebachien- the chronic rejection process [3,4]. sis [23]. In addition to using repeated allografting to 3.2. Coelomocyte responses to foreign substances assess the speci®city of immune memory in echi- noderms, repeated injections of foreign sub- The survival of echinoderms in the microbe- stances have also been performed. However, no rich marine environment is dependent on their dierences have been noted in clearance rates of ability to defend themselves against microbial bacteria injected multiple times into the coelomic invasion. Combating infections must include cavity of S. purpuratus, regardless of the interval rapid, ecient, and sometimes selective clearance between inoculations [18]. Similarly, accelerated of foreign invaders in order for an animal to sur- clearance rates were not demonstrated when vive. The capability of echinoderms to eliminate xenogeneic cells were injected a second time into injected pathogens, foreign cells, and other types A. vulgaris [21], nor after repeated injections of of particles has been well documented. Injections T4 bacteriophage into L. pictus [24]. These results of bacteria into the coelomic cavity of the sea corresponded with those obtained from allograft- star D. imbricata [16] or into the sea urchins, ing experiments, which had demonstrated the Echinus esculentus [17], S. purpuratus, [18] or absence of speci®c immunological memory in Strongylocentrotus droebachiensis [19] were e- echinoderms. ciently cleared in a few hours to a few days. In The activities and functions of coelomocytes contrast to the sterile coelomic ¯uid of sea urch- have also been studied in vitro, where the coelo- ins, pre-injection analysis of coelomic ¯uid from