Echinoderm Coelomocytes as Immune Effector Cells Morphological Features and Recognized Functions

In all organisms the first defence against infections involves a physical barrier to invasions by pathogens or foreign substances, provided by their body coats which physically prevent the interaction between the host and the invading organism. However, when pathogens accidentally or experimentally penetrate this barrier they first encounter generic or specific molecules that restrict the infection. The second-line defence consists of cells that can engulf (phagocytose) foreign substances or pathogens or can aggregate under formation of large coagulates (clots) in the case of wounds. Other cells secrete in the medium specific anti-host molecules, which will be discussed below. Phagocytosis is a complex process which involves several steps, including: (1) chemotaxis, whereby phagocytic cells are attracted towards chemotactic chemicals like microbial products, complements, or damaged cells; (2) adhesion, whereby phagocytes stick to each other and attach to foreign agents; (3) opsonization, a process that enhances adhesion, whereby specific proteins (opsonins) are coated on the microbial surface; and (4) ingestion, whereby phagocytes extend their cellular projections, engulfing the foreign organism. Finally, the foreign organism can be digested by enzymes in the lysosome. All the steps described above have been studied using echinoderms as a model system. In the past, each of the echinoderm classes has preferentially been used for a certain type of study; in the case of classical immune/defence studies, involving self/non-self recognition, which occurs when grafting cells from one individual to another, scientists have mostly used the classes of Holothu-riodea and Echinoidea. For the purpose of our description and to take care of the amount and the heterogeneity of information present in the literature, we will focus our interest on echinoids, specifically sea urchins. In the body of adult sea urchins a cell population lives, which has been identified as the true immune effector because of many proven characteristics. Quite vast, although not so recent, literature is available which describes the morphologies present in this mixed cell population (see Smith 1981; Matranga 1996). However, possibly due to the way in which cells have been collected or preserved before their observation by optical or electron microscopy, a variety of misleading classifications are reported, which have generated confusion. The easiest way to give a nomenclature that reflects the actual morphology of the cells is the immediate observation of fresh and live cells just taken from the sea urchin without any addition of anti-coagulant solution (Fig. 1). Under these conditions it appears clear that some cells are capable of rapid movements, while others show very slow motions. Within the first group, fast-moving cells are red and colourless, each constituting 5-7% of the total cell population. Peculiar to this cell type is their characteristic locomotion (about 0.5 |m s-1) achieved by rapid changes in their body shape, where a leading edge of the cell is protruded towards their march direction (Fig. 2). Consequently, these cells are referred to as amoebocytes, from the Greek amoeba, which means change, referring to the shape of the cell (Fig. 1a,b). The red pigment, called echinochrome, is thought to be utilized by echinoderms as an anti-bactericidal agent. This notion comes from the single report present to our knowledge in the literature, describing the release of a red pigment by the sand dollar Mellita quinquiesperforata coelomocytes in response to stress (Smith and Smith 1985). In the same study the authors showed the release of histamine on challenge and suggested that the coelomocyte stress response may be an evolutionary precursor to the mammalian allergic response. Due to their fast movement, it seems conceivable to suppose that this cell type is utilized as a first defence mobilization, as in the case of wound healing or prompt attack by foreign agents. Indeed, red amoebocytes have been observed trapped in clotting and encapsulation (Smith 1981).

Another type of very fast cells, constituting 5-6 % of the total cell population, are the so-called vibratile cells (Fig. 1 c); these are round cells which, thanks to a long flagellum, can move in a straight direction along a helicoidal pattern. The function of this less studied cell type is at present unknown.

Finally, the most abundant morphology observed (80-85 %) is constituted by cells with a dendritic-like phenotype, namely "petaloid" or "philopodial." Their conversion from the first to the second morphology is easily observed under the microscope within 5-10 min, and a detailed description, as well as

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Fig. 1. Paracentrotus lividus coelomocytes: a red amoebocyte; b colourless amoebocyte; c vibratile cell; d petaloid phagocyte; e philopodial phagocyte. Bar 10 |m

Fig. 1. Paracentrotus lividus coelomocytes: a red amoebocyte; b colourless amoebocyte; c vibratile cell; d petaloid phagocyte; e philopodial phagocyte. Bar 10 |m

Vibratile Cells

Fig. 2. Red and colourless amoebocytes freshly collected and rapidly moving under the microscope. Bright fields of cells viewed on a Zeiss Axioscope 2 plus using a 63 x objective lent. Pictures taken every 8 s. Bar 10 |m

Fig. 2. Red and colourless amoebocytes freshly collected and rapidly moving under the microscope. Bright fields of cells viewed on a Zeiss Axioscope 2 plus using a 63 x objective lent. Pictures taken every 8 s. Bar 10 |m different functional states of the same cell type, can be found in several papers (Edds 1985,1993; Henson et al. 1999).

These four major cell types have been consistently seen and described for at least three different species, i.e. the Pacific Ocean Strongylocentrotus purpu-ratus (Hillier and Vacquier 2003), the North Sea Strongylocentrotus droe-bachiensis (Bertheussen and Seljelid 1978), and the Mediterranean Sea Para-centrotus lividus (Matranga and Bonaventura 2002). However, 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, as we will see below. It should be recalled that these cells, within a few minutes (5-10 min) after collection from the sea urchin body, tend to clump; therefore, for their use and/or observation, they need to be placed in an anti-coagulant solution, which, on the other hand, might affect their physiological morphology. However, much work still needs to be done on the function of all these cell types. Most of the immune responses that we expect to be carried out are performed by phagocytes (reviewed by Smith 1981). It seems that the petaloid stage is involved in migration towards the sites of injuries, while the fillopo-dial stage is involved in clotting. In fact, because of their high numeric representation and the ability to change the shape of their cytoplasmic protrusions, phagocytes are able to form large clots in which other coelomocyte types are trapped or possibly contribute to their formation. However, of course, phagocytes deserve their name from their ability to engulf (phagocyte) foreign particles, such as pathogens, as observed in the clearance of injected bacteria (Yui and Bayne 1983) or even synthetic beads (Bertheussen 1981). In addition, encapsulation, a process by which the host is surrounded by a layer of cells and eventually digested by lytic enzymes released into the cavity, is performed by phagocytes and so-called spherule cells (possibly colorless amoe-bocytes), the latter being claimed to release bactericidal substances (Johnson 1969; Gerardi et al. 1990).

However, due to the small number of controversial reports present in the literature, the real function of each of these cell types is still not acknowledged.

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