Cell Types Involved in Allogeneic Contact Reactions of the Solitary Ascidian, Halocynthia roretzi

Abstract

ABSTRACT-The blood cells of a solitary ascidian, Halocynthia roretzi exhibit the allogeneic cellular reaction in vitro denoted as the "contact reaction" (Fuke, 1980). Nine cell types have been recognized in the blood and body fluid of H. roretzi (Fuke and Fukumoto, 1993). In the present study, the precise role of each cell type in allogeneic reactions is investigated in vitro. The vacuolated cells show devacuolation after contact with almost all other cell types from different reactive animals. These cells include hyaline amoebocytes, granular amoebocytes, macrogranular cells, small amoebocytes and giant cells (large basophilic cells), as well as vacuolated cells. The hyaline amoebocytes and small amoebocytes, which belong to the phagocyte series exhibit contact reactions with the cells of phagocyte series from other specimens of H. roretzi. They also show a contact reaction when in contact with granular amoebocytes. Therefore, almost all cell types have the materials responsible for individuality on their cell surface and can directly show the allogeneic cellular reactivity when they contact each other. To determine whether the contact reaction is involved in cell death, the loss of plasma membrane integrity was examined using fluorescent dyes. The number of cells showing uptake of ethidium bromide increased immediately after mixing of allogeneic cells. Almost the same cell types as those showing allogeneic behavior by light microscopy, as described above, exhibited loss of cell membrane integrity.These results are discussed in the context of immune systems in invertebrates and vertebrates.

Mutual and Directional Allogeneic Cytotoxic Reaction of Hemocytes in the Solitary Ascidian Halocynthia roretzi Revealed by One-step Quantitative Fluorimetric Assay

“…Ethidium homodimer is excluded by the intact plasma membrane of live cells. Loss of plasma membrane integrity of almost all types of hemocytes after cytotoxic reactions is recently observed by this assay with the fluorescence microscope (Fuke, 2001). A two-color fluorescence cell viability assay based on the simultaneous determination of live and dead cells with the two fluorescent dyes, calcein AM and ethidium homodimer, is possible and may improve the ACR assay developed in this report.…”

“…Since contact reactions occur in almost all types of hemocytes (Fuke, 1980;Fuke, 2001), impartial staining of freshly isolated hemocytes with fluorogenic dyes and decrease in fluorescence of all the stained cells are essential for quantitative measurements of contact reactions with fluorimetric assay. In the ACR assay, loading of the hemocytes with calcein AM resulted in strong and impartial staining of almost all types of hemocytes, and decrease in fluorescence from almost all of the cells after contact reactions.…”

Comparison of Evolutionary Relationships between Branchiostoma floridae, Ciona intestinalis, and Homo sapiens Globins Provide Evidence of Gene Co-Option and Convergent Evolution

“…The heart also beats continuously, even though the pumping direction is flipped periodically because the ascidian has an open circulation system. Ascidian hemocytes are a heterogeneous population consisting of at least nine cell types, most of which can crawl [25]. Thus, Gb-1 and -2 are thought to be expressed mainly in tissues with continuous movement and high energy consumption.…”