Limpet haemocytes. I. Studies on aggregation and spike formation.

In this paper a promising new spontaneously aggregating system of amoebocytes from the blood of limpets is described. The system has the advantage of being readily available without cell culture and of being operational at room temperature. The blood of the limpet Patella contains 2 types of haemocytes, amoebocytes and macrophages, the latter accounting for less than 1 % of the cell population. The concentration of cells in suspension varies with the body temperature of the animal, from about 1 x io cells/ml at S °C to 9 x io cells/ml at 25 °C. Following blood withdrawal the amoebocytes are approximately spherical in shape with superficial convoluted lamellae, but over a period of 5 min develop stout marginal spikes. This development is temperature dependent and independent of divalent cations in the medium. Most interestingly, and apparently associated with the stimulus of blood withdrawal, the cells become spontaneously adhesive and commence an extremely rapid aggregation. With a shaking speed of 700 strokes/min the half time of aggregation varies from 15 s at an initial cell density of 4-7 x io cells/ml to 60 s at a density of 1-4 x io cells/ml. The aggregates so formed are very resistant to mechanical dispersion and tight junctions have been observed between aggregated cells in electron micrographs. Complete aggregation requires the presence of divalent cations and another unidentified plasma component. Aggregation will occur at o °C but the process is slower and less complete than at 20 °C. It is suggested that aggregation involves the triggering or activation of a pre-synthesized adhesive system and that the adhesiveness per unit area increases with increase in temperature.