Fusion pore expansion in horse eosinophils is modulated by Ca2+ and protein kinase C via distinct mechanisms.

Using the patch-clamp technique, we studied the role of protein phosphorylation and dephosphorylation on the exocytotic fusion of secretory granules with the plasma membrane in horse eosinophils. Phorbol 12-myristate 13-acetate (PMA) had no effect on the amplitude and dynamics of degranulation, indicating that the formation of fusion pores is insensitive to activation of protein kinase C (PKC). Fusion pore expansion, however, was accelerated approximately 2-fold by PMA, and this effect was abolished by staurosporine. Elevating intracellular Ca2+ to 1.5 microM also resulted in a 2-fold acceleration of pore expansion; this effect was not prevented by staurosporine, indicating that intracellular Ca2+ and activation of PKC accelerate fusion pore expansion via distinct mechanisms. However, fusion pores can expand fully even when PKC is inhibited. In contrast, the phosphatase inhibitor alpha-naphthylphosphate inhibits exocytotic fusion and slows fusion pore expansion. These results demonstrate that, subsequent to its formation, fusion pore expansion is under control of proteins subject to functional changes based on their phosphorylation states.

Exocytotic competence and intergranular fusion in cord blood-derived eosinophils during differentiation.

We studied degranulation of single cord blood-derived mononuclear cells differentiating to eosinophils in cultures containing recombinant human interleukin-5 (rhIL-5) and rhIL-3 by whole-cell patch-clamp capacitance measurements. As in mature cells, degranulation can be stimulated by intracellular application of guanosine-5'-O-(3-thiotriphosphate) (GTP) gamma S after 10 days in culture, simultaneously with the first morphological appearance of granules. These results demonstrate that the fusion machineries for exocytotic fusion are present and functional as soon as the granules are formed, presumably at the myeloblast stage. In the third week, the total amount of granules exocytosed upon stimulation is similar to that in mature eosinophils from peripheral blood. The capacitance step size distributions in promyelocytes and myelocytes confirm that mature large specific granules are formed by homotypic fusion of unit granules with similar size. Homotypic fusion is facilitated during early stages of differentiation associated with granulogenesis. Between day 10 and day 35 in culture the plasma membrane area of resting cells decreases from approximately 700 microns2 to approximately 400 microns2, approaching the value of mature cells from peripheral blood. The most prominent decrease occurs between day 25 and day 35 and is accompanied by the appearance of an exocytotic component due to small vesicles. This suggests that a class of small secretory vesicles is formed by endocytosis during a late phase in maturation.

Regulation of granule size in human and horse eosinophils by number of fusion events among unit granules.

1. We have investigated the granule size distributions in human and horse eosinophils by time-resolved patch-clamp capacitance measurements. 2. During exocytosis of single granules the electrical capacitance of the plasma membrane increases in discrete steps. The steps in horse cells are about six times larger than those in human cells in accordance with the difference in granule size. 3. In both species a multimodal capacitance step size distribution is observed with a first peak at 6-7 fF corresponding to granules with a diameter of about 450-500 nm and a surface area of about 0.7 microns2, which we call the unit granule. The other peaks in the distributions correspond to multiples of the surface area of these units. 4. These results show that the larger granules are formed by fusion of several unit granules and the final size of mature granules is determined by the number of units allowed to fuse with each other. Whereas in human eosinophils most granules consist of one or two units, most granules of horse eosinophils are formed by fusion of seven to fifteen units. 5. The intracellular fusion events associated with vesicular traffic are believed to occur constitutively. In contrast, our results indicate that a cellular mechanism exists which regulates the size of the mature granules by determining the number of units allowed to fuse with each other. In view of our recent report that granule-granule fusion can be activated by GTP gamma S, this regulation may possibly involve GTP-binding proteins.

Compound exocytosis and cumulative degranulation by eosinophils and their role in parasite killing.

The killing of metazoan parasitic larvae by eosinophils occurs following cell adhesion and the secretion o f their cytotoxic proteins onto the surface o f these targets. In eosinophils, as in mast cells and neutrophils, stimulus-secretion coupling is mediated by GTP-binding proteins. In this article, Susanne Scepek Redwon Moqbel and Manfred Lindou summarize recent results indicating that the granule-fusion events activated by GTP-binding proteins lead to compound exocytosis and cumulative fusion. They propose that these exocytotic processes, following contact with opsonized larvae, may direct secretion to a restricted space defined by the site o f contact Such a focused release may be essential for effective targeting in parasite killing, thus preventing uncontrolled random diffusion of the secreted cytotoxic proteins with the possible undesirable consequences of damage to intact host tissue.

Focal exocytosis by eosinophils--compound exocytosis and cumulative fusion.

We have investigated the granule fusion events during exocytosis in horse eosinophils by time-resolved patch-clamp capacitance measurements. Stimulation with intracellular GTP gamma S leads to a stepwise capacitance increase by 4.0 +/- 0.9 pF. At GTP gamma S concentrations < 20 microM the step size distribution is in agreement with the granule size distribution in resting cells. Above 80 microM the number of steps is reduced and very large steps occur. The total capacitance increase, however, is unaffected. These results show that at high GTP gamma S concentrations granule--granule fusion occurs inside the cell forming large compound granules, which then fuse with the plasma membrane (compound exocytosis). The electrical equivalent circuit of the cell during degranulation indicates the formation of a degranulation sac by cumulative fusion events. Fusion of the first granule with the plasma membrane induces fusion of further granules with this granule directing the release of all the granular material to the first fusion pore. The physiological function of eosinophils is the killing of parasites. Compound exocytosis and cumulative fusion enable the cells to focus the release of cytotoxic proteins to well defined target regions and prevent uncontrolled diffusion of this material, which would damage intact host cells.