Reference: BinI Hull 191: 159-167. (October. Acute Cytotoxic Allogeneic Histoincompatibility Reactions Involving Gray Cells in the Marine Sponge, Callyspongia diffusa CHAOQUAN YIN AND TOM HUMPHREYS Kewalo Marine Laboratory, Pacific Biomedical Research Center, University of Hawaii, 41 Ahui Street, Honolulu. Hawaii 96813 Abstract. A variety of procedures were used in a study of the histoincompatibility reactions of Callyspongia diffusa. Rejection reactions as traditionally tested be-tween laterally apposed intact fingers cut from two different sponges require about a week of contact to ex-hibit cytotoxicity. In a miniaturized assay involving re-actions 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 allo-geneic mixtures were significantly smaller than aggre-gates of cells from a single individual. This rapid slowing of aggregation is the earliest response to allogeneic con-tact that we noted and does not appear to reflect early cytotoxic processes. Apposition of an aggregate contain-ing cells from one sponge to an aggregate containing cells from a second individual leads to mutual destruction. Aggregates harvested and apposed 4 hours after initia-tion of aggregation begin to show mutual cytotoxicity at 36 hours of contact. Aggregates placed in contact 48 hours after the initiation of aggregation exhibit cyto-toxicity 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) genera-Received 16 October 1995; accepted 24 May 1996. tion of signals that suppress cell aggregation and cell movement and attract gray cells to the boundary of con-tact; (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 in-dividual cells; (4) arrival of gray cells at the boundary of allogeneic contact: and (5) initiation of cytotoxic pro-cesses. Introduction The invertebrate immune system must protect the in-dividual animal from a full range of parasitic and patho-genic threats (Ratcliffe el ai. 1985). For sessile species, individual-specific recognition may also prevent cellular parasitism between contacting individuals of the same species (Buss, 1987). Very specific, immune-like histoin-compatibility reactions occur in every invertebrate phy-lum (Cooper et a/.. 1992) including sponges, the simplest metazoans (Hildemann et ai. 1979). Because these reac-tions are usually very slow, and not well defined histolog-ically, cells functioning like vertebrate T or B cells are not evident in any invertebrate. The idea that evolutionary precedents for specific recognition events characteristic of these vertebrate immune cells cannot be found in in-vertebrates is often expressed (Klein, 1989; Janeway, 1992; Smith and Davidson, 1992). This laboratory has observed very rapid allogeneic rec-ognition reactions involving the intimate participation of a specific cell type gray cells in the marine sponge Microciona prolifera (Humphreys, 1994). The rapidity and selectivity of these reactions have led us to focus again on the possibility of specific cellular recognition in the immune reactions of invertebrates (Humphreys and 159
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