L. Courtney Smith

Echinoderm immunity and the evolution of the complement system

Our understanding of inflammatory responses in humans has its roots in the comparative approach to immunology. In the late 1900s, 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, cytotoxicity, and the production of antimicrobial agents. In addition, a variety of humoral factors found in the coelomic fluid, including lectins, agglutinins, and lysins, are important in host defense against pathogens and other foreign substances. Recently, a simple complement system has been identified in the purple sea urchin that is homologous to the alternative pathway in vertebrates. The sea urchin [corrected] homologue of C3, is inducible by challenge with lipopolysaccharide, which is known to activate coelomocytes. Complement components have been identified 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 ancestor of the deuterostomes.

The ancestral complement system in sea urchins.

The origin of adaptive immunity in the vertebrates can be traced to the appearance of the ancestral RAG genes in the ancestral jawed vertebrate; however, the innate immune system is more ancient. A central subsystem within innate immunity is the complement system, which has been identified throughout and seems to be restricted to the deuterostomes. The evolutionary history of complement can be traced from the sea urchins (members of the echinoderm phylum), which have a simplified system homologous to the alternative pathway, through the agnathans (hagfish and lamprey) and the elasmobranchs (sharks and rays) to the teleosts (bony fish) and tetrapods, with increases in the numbers of complement components and duplications in complement pathways. Increasing complexity in the complement system parallels increasing complexity in the deuterostome animals. This review focuses on the simplest of the complement systems that is present in the sea urchin. Two components have been identified that show significant homology to vertebrate C3 and factor B (Bf), called SpC3 and SpBf, respectively. Sequence analysis from both molecules reveals their ancestral characteristics. Immune challenge of sea urchins indicates that SpC3 is inducible and is present in coelomic fluid (the body fluids) in relatively high concentrations, while SpBf expression is constitutive and is present in much lower concentrations. Opsonization of foreign cells and particles followed by augmented uptake by phagocytic coelomocytes appears to be a central function for this simpler complement system and important for host defense in the sea urchin. These activities are similar to some of the functions of the homologous proteins in the vertebrate complement system. The selective advantage for the ancestral deuterostome may have been the amplification feedback loop that is still of central importance in the alternative pathway of complement in higher vertebrates. Feedback loop functions would quickly coat pathogens with complement leading to phagocytosis and removal of foreign cells, a system that would be significantly more effective than an opsonin that binds upon contact as a result of simple diffusion. An understanding of the immune response of the sea urchin, an animal that is a good estimator of what the ancestral deuterostome immune system was like, will aid us in understanding how adaptive immunity might have been selected for during the early evolution of the vertebrates and how it might have been integrated into the pre‐existing innate immune system that was already in place in those animals.

Complement systems in invertebrates. The ancient alternative and lectin pathways

The complement system in higher vertebrates is composed of about thirty proteins that function in three activation cascades and converge in a single terminal pathway. It is believed that these cascades, as they function in the higher vertebrates, evolved from a few ancestral genes through a combination of gene duplications and divergences plus pathway duplication (perhaps as a result of genome duplication). Evidence of this evolutionary history is based on sequence analysis of complement components from animals in the vertebrate lineage. There are fewer components and reduced or absent pathways in lower vertebrates compared to mammals. Modern examples of the putatively ancestral complement system have been identified in sea urchins and tunicates, members of the echinoderm phylum and the protochordate subphylum, which are sister groups to the vertebrates. Thus far, this simpler system is composed of homologues of C3, factor B, and mannose binding protein associated serine protease suggesting the presence of simpler alternative and lectin pathways. Additional components are predicted to be present. A complete analysis of this invertebrate defense system, which evolved before the invention of rearranging genes, will provide keys to the primitive beginnings of innate immunity in the deuterostome lineage of animals.

The echinoid immune system and the phylogenetic occurrence of immune mechanisms in deuterostomes.

In this article, Courtney Smith and Eric Davidson reinterpret the published data on immune function in lower deuterostomes and primitive chordates. It leads them to a new model of immune system phylogeny in which MHC-directed T-cell responses are the last to evolve and are not derived from subchordate self-nonself recognition systems.

SpC3, the complement homologue from the purple sea urchin, Strongylocentrotus purpuratus, is expressed in two subpopulations of the phagocytic coelomocytes.

Abstract. The lower deuterostomes, including the echinoderms, possess an innate immune system that includes a subsystem with similarities to the vertebrate complement system. A homologue of the central component of this system, C3, has recently been identified in the purple sea urchin, Strongylocentrotus purpuratus, and is called SpC3. We determined previously that coelomocytes specifically express the SpC3 gene (Sp064); however, the sea urchin has at least four different types of coelomocytes: amoeboid phagocytes, red spherule cells, colorless spherule cells, and vibratile cells. To determine which of these subpopulations expresses Sp064 and produces SpC3, coelomocytes were separated by discontinuous gradient density centrifugation. Relatively homogenous fractions were obtained consisting of the four major cell types in addition to two types of amoeboid phagocytes with different densities and distinct morphologies. Analysis of proteins from separated cell subpopulations by Western blot and analysis of gene expression by RT-PCR revealed that phagocytes express the gene and contain the protein. Immunolocalization showed that SpC3+ phagocytes are present as subsets of both the low- and high-density subpopulations of phagocytes; however, the subcellular localization of SpC3 is different in these two subpopulations.

The sea urchin complement homologue, SpC3, functions as an opsonin

SUMMARY The purple sea urchin Strongylocentrotus purpuratus expresses a homologue of complement component C3 (SpC3), which acts as a humoral opsonin. Significantly increased phagocytic activity was evident when yeast target cells were opsonized after incubation with coelomic fluid containing SpC3. SpC3 could be detected on the surface of yeast, and phagocytic activity could be inhibited by an anti-SpC3 antibody. This indicates that SpC3 promotes phagocytosis by physically tagging target cells for ingestion. Confocal microscopy showed that opsonized yeast were phagocytosed by a single coelomocyte type (polygonal phagocytes), presumably because these cells express SpC3 receptors. Overall, these data indicate that SpC3 is a major humoral opsonin in S. purpuratus coelomic fluid.

Distinctive expression patterns of 185/333 genes in the purple sea urchin, Strongylocentrotus purpuratus : an unexpectedly diverse family of transcripts in response to LPS, β-1,3-glucan, and dsRNA

BackgroundA diverse set of transcripts called 185/333 is strongly expressed in sea urchins responding to immune challenge. Optimal alignments of full-length 185/333 cDNAs requires the insertion of large gaps that define 25 blocks of sequence called elements. The presence or absence of individual elements also defines a specific element pattern for each message. Individual sea urchins were challenged with pathogen associated molecular patterns (PAMPs) (lipopolysaccharide, β-1,3-glucan, or double stranded RNA), and changes in the 185/333 message repertoire were followed over time.ResultsEach animal expressed a diverse set of 185/333 messages prior to challenge and a 0.96 kb message was the predominant size after challenge. Sequence analysis of the cloned messages indicated that the major element pattern expressed in immunoquiescent sea urchins was either C1 or E2.1. In contrast, most animals responding to lipopolysaccharide, β-1,3-glucan or injury, predominantly expressed messages of the E2 pattern. In addition to the major patterns, extensive element pattern diversity was observed among the different animals before and after challenge. Nucleotide sequence diversity of the transcripts increased in response to β-1,3-glucan, double stranded RNA and injury, whereas diversity decreased in response to LPS.ConclusionThese results illustrate that sea urchins appear to be able to differentiate among different PAMPs by inducing the transcription of different sets of 185/333 genes. Furthermore, animals may share a suite of 185/333 genes that are expressed in response to common pathogens, while also maintaining a large number of unique genes within the population.

The echinoderm immune system. Characters shared with vertebrate immune systems and characters arising later in deuterostome phylogeny.

In summary, the characters of the echinoderm immune system that we review here can be considered to illuminate the baseline nonadaptive immune systems that were our original deuterostome heritage. We still retain--and greatly rely upon--similarly functioning, nonadaptive cellular defense systems. It is worth stressing that sea urchins are long lived, normally healthy animals that display remarkable abilities to heal wounds and combat major infections. From an external point of view, their immune systems obviously work very well. Thus, their cellular defense systems are extremely sensitive, and they respond rapidly to minor perturbations, all without any specific adaptive capabilities. These systems probably function through the transduction of signals conveying information on injury and infection, just as do the equivalent systems that underlie and back up our own adaptive immune systems, and that provide the initial series of defenses against pathogenic invasions. Many extremely interesting questions remain regarding the evolution of the deuterostome immune response. Are the echinoderm and tunicate systems the same, or have the protochordates augmented the basic phagocyte system with an as yet unidentified chordate-like character? Do the jawless fishes produce Igs that would make them similar to the sharks, or are they vertebrates without an Ig system that essentially rely on an invertebrate-like, nonspecific, activated phagocyte type of immune system? How do sharks regulate their immune system without T cells and MHC class I? How do they avoid producing autoantibodies? Future research will not only answer these questions, but those answers will also be enlightening with regard to the origins of the mammalian immune system in which ancient functions and subsystems remain.

Extraordinary diversity among members of the large gene family, 185/333, from the purple sea urchin, Strongylocentrotus purpuratus

BackgroundRecent analysis of immune-related genes within the sea urchin genome revealed a number of large gene families with vertebrate homologues, such as the Toll-like and NOD/NALP-like receptor families and C-type lectins in addition to a rudimentary complement system. Therefore, the immune response of the purple sea urchin appears to be more complex than previously believed. Another component of the sea urchin immune response is an unusual family of mRNAs, known as 185/333, which is strongly upregulated in response to pathogen challenge. The work presented here indicates that this family of transcripts is derived from an unexpectedly diverse gene family.ResultsThe 185/333 genes are small (< 2 kb) with only two exons. Their extraordinary diversity was exemplified by 121 unique sequences identified from 171 cloned genes. Sequences from the second exons were aligned optimally by introducing large gaps, which defined blocks of sequence known as elements. Genes were defined by the presence or absence of elements. Phylogenetic analysis defined five intron types which, when combined with the exon element patterns resulted in 31 gene patterns, 14 of which were not described previously. Sequence diversity was present in all elements, and was higher in the intron than the exons. Repeats within the sequence facilitated multiple alignments, of which two were analyzed in detail. Although the two alignments differed in length, number of elements, and number of patterns, both were about equally accurate at describing the 185/333 sequences. The genes were closely linked and flanked by short repeats. The repeats within and between the genes may promote their diversification through gene conversion, recombination, and meiotic mispairing.ConclusionThe diversity of the 185/333 gene family represents an intriguing addition to what is known about the S. purpuratus immune response, and provides further evidence that invertebrate immune systems are neither simple nor static.

Lipopolysaccharide activates the sea urchin immune system

Profilin is a small, actin-binding protein that functions at the intersection of signal transduction and cytoskeletal modifications. Increases in the number of profilin messages per cell correlate with sea urchin coelomocytes activation in response to injury. Here we show that coelomocytes respond to immune challenge from lipopolysaccharide with significant elevations in profilin transcripts per coelomyocyte.