Involvement of apoptosis-like cell death in coelomocytes of Themiste petricola (Sipuncula) in the formation of a cellular clot with haemostatic and immune functions

BLANCO G.A.

New Cell Apoptosis Research

Nova Science Publisher Inc

Lugar: New York; Año: 2007; p. 121 - 160

  Involvement of apoptosis-like cell death in coelomocytes of Themiste petricola (Sipuncula) in the formation of a cellular clot with haemostatic and immune functions   ABSTRACT Coagulation in vertebrates and invertebrates with open circulatory system involves humoral and cellular mechanisms that lead to the transformation of hemolymph or blood into a gelatinous insoluble mass designated as clot. Activation of cells within the clot leads to several changes such as discharge of granular content, cell spread, extrusion of filopodia, exposure of phosphatidylserine (PS) in the outer leaflet of cell membrane and shedding of procoagulant microvesicles. In insect coagulocytes and vertebrate platelets, these changes have been related to apoptosis. Sipunculans are non-segmented coelomate marine worms that do not have a true circulatory system. Cell-free coelomic fluid of sipunculans does not transform itself into a gelatinous mass under any stimulus, but we hypothesized that worms could bear the capacity to form an exclusively cellular clot. We evaluated clot formation by exposing coelomic fluid of the sipunculan Themiste petricola to paramagnetic beads both in vivo and ex vivo and isolating the cell clot with a magnet. Cells involved in clot formation were observed under phase contrast microscopy, Giemsa stain and supravital staining with acridine orange (AO), propidium iodide (PI), Annexin V-FITC and JC-1, and were evaluated for the presence of changes indicative of apoptosis. A cellular clot was formed ex vivo in the presence of sea water (SW) and served to entrap paramagnetic beads. Beads were entrapped in vivo into small cell aggregates when injected into the coelomic cavity. In contrast large cell aggregates, macroscopically visible as a small clot, were seen to entrap beads ex vivo in the presence of SW. Morphological changes in the coelomocyte subtype involved in clotting and entrapping beads ex vivo were reproduced by exposing cells to contact with glass surface in the presence of SW. These changes resembled apoptotic changes both in nucleus and cytoplasm and included as follows: early loss of mitochondrial membrane potential as detected by JC-1 dye, exposure of PS at the external leaflet of cell membrane as indicated by Annexin V-FITC binding, rippled nuclear contour, chromatin condensation and fragmentation, as indicated by staining with AO and PI, membrane blebbing and cytoplasm fragmentation with formation of microvesicles. Other coelomocyte subtypes not directly involved in cell aggregates and clot formation did not show apoptotic changes, maintain viability, and were found surrounding cell aggregates and phagocytosing microvesicles shed from apoptotic cells. We conclude that clotting may take place without transformation of cell-free coelomic fluid into a gelatinous mass as occurs in arthropods. This cellular clot may aid in haemostasis precluding loss of coelomic fluid upon injury of the body wall and may serve to entrap microbes that may attempt to invade the worm through the wound. The peculiar haemostatic and immune strategy of these marine worms required a fast series of morphological and biochemical changes that were related to apoptosis in a specific coelomocyte subtype and was elicited upon contact with external environment epitomized by sea water. Our findings underscore the usefulness of tools derived from apoptosis research in aiding to unravel the peculiar biological strategies of this small phylum of marine worms.