Aggregation of Sea Urchin Phagocytes Is Augmented In Vitro by Lipopolysaccharide

Abstract

Development of protocols and media for culturing immune cells from marine invertebrates has not kept pace with advancements in mammalian immune cell culture, the latter having been driven by the need to understand the causes of and develop therapies for human and animal diseases. However, expansion of the aquaculture industry and the diseases that threaten these systems creates the need to develop cell and tissue culture methods for marine invertebrates. Such methods will enable us to better understand the causes of disease outbreaks and to develop means to avoid and remedy epidemics. We report a method for the short-term culture of phagocytes from the purple sea urchin, Strongylocentrotus purpuratus, by modifying an approach previously used to culture cells from another sea urchin species. The viability of cultured phagocytes from the purple sea urchin decreases from 91.6% to 57% over six days and phagocyte morphology changes from single cells to aggregates leading to the formation of syncytia-like structures. This process is accelerated in the presence of lipopolysaccharide suggesting that phagocytes are capable of detecting this molecular pattern in culture conditions. Sea urchin immune response proteins, called Sp185/333, are expressed on the surface of a subset of phagocytes and have been associated with syncytia-like structures. We evaluated their expression in cultured phagocytes to determine their possible role in cell aggregation and in the formation of syncytia-like structures. Between 0 and 3 hr, syncytia-like structures were observed in cultures when only ∼10% of the cells were positive for Sp185/333 proteins. At 24 hr, ∼90% of the nuclei were Sp185/333-positive when all of the phagocytes had aggregated into syncytia-like structures. Consequently, we conclude that the Sp185/333 proteins do not have a major role in initiating the aggregation of cultured phagocytes, however the Sp185/333 proteins are associated with the clustered nuclei within the syncytia-like structures.

Echinochrome A Release by Red Spherule Cells Is an Iron-Withholding Strategy of Sea Urchin Innate Immunity

“…The responses of invertebrate immune cells to PAMP and DAMP are well characterised across diverse taxa; however, sea urchin RSC are an exception [9,10,23,27]. When insect and crustacean hemocytes encounter pathogens they release a battery of immune effectors (through exocytosis) to immobilise/entrap the intruders as part of their inflammatory programmes [28].…”

“…Activation of invertebrate defences generally leads to an increase in free-floating immune cell numbers and the liberation of bioactive compounds (e.g., antimicrobial peptides and lysozyme) [33]. Likewise, exposure of sea urchin phagocytes to LPS in vitro induces cellular aggregation (reminiscent of encapsulation) and de novo synthesis of SpTransformer proteins (formerly Sp185/333) [9]. Intriguingly, both phagocytes and RSC from the green sea urchin (S. droebachiensis) increase the gene expression of a defensin-like antimicrobial peptide, i.e., strongylocin 2, when exposed to E. coli in vitro [34].…”

“…The former can be subdivided into discoidal, polygonal, and small phagocytes, which express a myriad of immune effectors belonging to the (Sp)Transformer gene family [6,7]. The phagocytes are tasked with identifying, ingesting, and destroying invading pathogens, whereas the vibratile cells are said to be involved in hemostasis [8][9][10]. The immunological function of colourless spherule cells (CSC) remains unclear, although some evidence supports a cytotoxic role [11].…”

Sea urchin immune cells as sentinels of environmental stress

“…The P. lividus genome is expected to be released in 2015. The full understanding of the morpho-functional properties of sea urchin immune cells is still controversial, but some of their immune mechanisms are relatively well known and include cellular recognition and cytotoxicity (Arizza et al, 2007;Bertheussen, 1979), phagocytosis and ROS production (Ito et al, 1992), antibacterial and anti-biofilm properties (Majeske et al, 2013a;Schillaci et al, 2010;Stevens et al, 2010) and a complement system that includes C3 and factor B homologues, that is likely initiated by a large set of homologues similar to mannose binding lectin and C1q, and a number of antimicrobial peptides (Li et al 2014;Smith et al, 2010).…”

Single Sea Urchin Phagocytes Express Messages of a Single Sequence from the DiverseSp185/333Gene Family in Response to Bacterial Challenge

“…Although sea urchins have been evaluated for immune responses to a variety of PAMPs and uncharacterized marine bacteria (11,12,20,22), most reports of sea urchin activation have employed LPS (23,36,37). Consequently, a putative sea urchin pathogen, V. diazotrophicus (Vibrio), that was originally isolated from an infected green sea urchin, Strongylocentrotus droebachiensis (38), was used as the immune challenge.…”