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A variety of procedures were used in a study of the histoincompatibility reactions of Callyspongia diffusa. Rejection reactions as traditionally tested between laterally apposed intact fingers cut from two different sponges require about a week of contact to exhibit cytotoxicity. In a miniaturized assay involving reactions between small pieces of tissue snipped from sponges with scissors and pushed together on an insect pin, cytotoxicity is evident within 48 hours of contact. Reactions of cells dissociated by divalent cation removal and allowed to reaggregate in seawater were also studied. Aggregates produced from allogeneic mixtures of cells from two individuals were killed by internal cytotoxic reactions within 36 hours of the initiation of aggregation. After only one hour of aggregation, aggregates from allogeneic mixtures were significantly smaller than aggregates of cells from a single individual. This rapid slowing of aggregation is the earliest response to allogeneic contact that we noted and does not appear to reflect early cytotoxic processes. Apposition of an aggregate containing cells from one sponge to an aggregate containing cells from a second individual leads to mutual destruction. Aggregates harvested and apposed 4 hours after initiation of aggregation begin to show mutual cytotoxicity at 36 hours of contact. Aggregates placed in contact 48 hours after the initiation of aggregation exhibit cytotoxicity within 8 hours. These rapidly reacting 48-hour aggregates exhibit a pronounced accumulation of gray cells at the boundary of allogeneic contact by 8 hours. These results are interpreted as indicating at least five steps in the histoincompatibility reactions of C. diffusa: (1) recognition soon after allogeneic contact; (2) generation of signals that suppress cell aggregation and cell movement and attract gray cells to the boundary of contact; (3) acceleration of the sponge immune response--including the responsiveness of gray cells to accumulate at the boundary of allogeneic contact--by tissue trauma produced when the tissue is cut or dissociated it into individual cells; (4) arrival of gray cells at the boundary of allogeneic contact; and (5) initiation of cytotoxic processes.

Acute Cytotoxic Allogeneic Histoincompatibility Reactions Involving Gray Cells in the Marine Sponge, Callyspongia diffusa