Concepto y descripción de la Comunicación Referencial

Eosinophil granules contain a number of basic cationic proteins such as major basic protein (MBP), eosinophil peroxidase (EPO), eosinophil derived neurotoxin (EDN) and eosinophil cationic protein (ECP) [145]. The

mechanism by which these are released is important because the release of the granule components is central to the toxic activities of these cells.

Degranulation is thought to take four routes, necrosis, apoptosis, piece-meal degranulation (PMD) and secretion [164]. Eosinophils at sites of

inflammation have centralised granules that have been disrupted, the plasma membranes and nuclei of these cells tend to be lysed. This is a state of cell death termed necrosis [44]. In culture, eosinophils can undergo programmed cell death but unlike necrosis, the integrity of the cell is kept intact [169]. This process of cell death can be decelerated in the presence of cytokines such IL-5, IL3 and granulocyte macrophage colony stimulating factor (GM-CSF) [146]. PMD is a process by which the contents of the granule are emptied either partially or completely into another vesicle. This vesicle then fuses with the plasma membrane to release their contents. The constituents of the granule core (MBP) or part of the granule matrix (for example EPO) or all of the granule contents may also be released.

Intact eosinophils cultured in the presence of IL-5 release some of their granule contents such as MBP and EDN over time and these cells also remain viable for longer periods [84].

Secretion from eosinophils has been studied by the use of other techniques such as patch-damp and by permeabilisation. Both techniques have

demonstrated that secretion from eosinophils of at least three animal species, namely guinea pig [39], horse [139] and human [2] occurs by an exocytotic mechanism.

Here I have studied the requirements for secretion from S L -0 permeabilised eosinophils by the application of Ca^"" and a non-hydrolysable analogue of

GTP (GTPyS). As mentioned earlier, exocytosis requires the presence of both Ca^^ and GTPyS [39]. If NaCI based buffers are substituted by isotonic buffered glutamate, then^Ca^'^independent component of secretion occurs in response to GTPyS alone [121]. In my study of exocytosis from permeabilised eosinophils, I discovered that in simple NaCI based buffers, exocytosis can be induced by GTPyS alone but this requires the presence of

ATP. I have also studied the effect of permeabilising the cells under

different conditions (e.g. in the presence of Ca^'" or ATP or both) and leaving the cells for a period of time before the application of the stimulus. This process allows the leakage of cytosolic factors from the permeabilised cells. Following a long time delay after permeabilisation, the cells are unable to respond to stimuli . The lack of responsiveness to stimulation after leakage has occurred is referred to as “Run Down”. Understanding the requirements for this process is important. This is because one of the aims of my work has been the application of exogenous proteins to permeabilised

eosinophils to test for their possible roles in the regulation of exocytosis.

I have successfully measured the release of eosinophil peroxidase (EPO) from permeabilised eosinophils for the first time (as far as I know). The release of other granule markers such as hexosaminidase and aryl

sulphatase were also measured. I have also observed that when the cells are 'run-down' in the presence of Ca^^ and ATP they can maintain

RESULTS

4.1 Dependence of exocytosis from permeabilised eosinophils on both and GTPS.

This experiment was carried out at an early stage in the work, before I had realised that impurities present in commercial preparations of SL-0 could affect the activation characteristics of secretion (see Chapter 3). It was therefore performed in the presence of excess SL-0 and its purpose was to confirm that secretion from eosinophils, like mast cells requires the two effectors, Ca^"" and GTPyS. The effect of adding ATP was also tested. The observation that Ca^^ and GTPyS are required for eosinophil secretion had been made previously but only with respect to the release of

hexosaminidase [39].

In this experiment (fig4.1), I stimulated the permeabilised eosinophils with a range of Ca^^ (pCa9-pCa5) concentrations and a range of concentrations of GTPyS (0-100}aM). This style of experiment is called a Ca^VGTPyS matrix. Secretion of eosinophil peroxidase (EPO), hexosaminidase, aryl sulphatase were measured from the supernatants after the cells had been allowed to secrete for 1 0minutes.

As shown in fig 4.1, when the cells are stimulated by lOOpM GTPyS and in the effective absence of Ca^^ (<pCa8), no secretion is achieved (<10%).

Also, when the cells are stimulated by Ca^'" (pCa5) in the absence of GTPyS, no secretion is achieved. In other words secretion requires both Ca^^ and GTPyS. In the absence of ATP, secretion commences at lOpM GTPyS and at concentrations of Ca^"" > pCa5.5. Similarly, very little Ca independent secretion occurs if ATP is provided, but in its presence, the ECso for both Ca^"" and GTPyS are enhanced and the maximum release of EPO increases from about 35% (no ATP) to over 80% (plus ATP). When ATP is present the EC50 for both Ca^^ and GTPyS are reduced and

Fig 4.1

The requirement for both and GTPyS for exocytosis from permeabilised eosinophils.

The cells were permeabilised with SL-0 in excess (not prebound) and stimulated for lOmins with a range of Ca^^ (pCa9-pCa5) and GTPyS (O-lOOpM) concentrations and in the presence of ATP (zero or ImM). Secretion was quenched with ice-cold HEPES buffered saline (pH 7).

no ATP