LIQUIDEZ I SOLVENCIA

Eosinophils can be purified from human blood by taking advantage o f the

absence o f the marker CD 16 on their cell surface, which is present on the surface o f

neutrophils. Neutrophils can therefore be absorbed away from the eosinophils after

incubation with an anti-CD 16 monoclonal antibody, using magnetic beads coated with

sheep anti-mouse IgG (Fattah et a l, 1996). The pure eosinophils can then be disrupted in a ball bearing homogeniser and the subcellular fractions separated on a Nycodenz

density gradient. The generation o f fractions enriched with specific subcellular

organelles would allow a detailed study into the presence in granule fractions o f

components o f the vesicle docking and fusion machinery.

3.2.2 Results

In order to obtain a pure preparation o f human eosinophils, 250ml o f blood

was taken from donors with a high eosinophil count. These donors were not

hypereosinophilic with high numbers o f hypodense eosinophils, but had higher than

average, normodense eosinophils (Spry, 1988).

For the subcellular fractionation o f eosinophils for western blot analysis o f

specific subcellular compartments, cells were disrupted in a ball bearing homogeniser.

A post-nuclear supernatant prepared from the disrupted cells was separated by

equilibration centrifugation on a linear density gradient o f Nycodenz (Lacy et a/., 1995). Figure 3.1a demonstrates protein content, density and marker enzyme content

o f each subcellular fraction. These profiles are representative o f at least 20 similar

subfractionations. Two clear peaks o f protein concentration were detected, one at the

top o f the tube that did not penetrate the Nycodenz gradient, probably corresponding

to the cytoplasmic fraction and the second at the bottom o f the gradient, co-

sedimenting with the granule fractions at a density o f 1.23g/ml. Figure 3.1b,

demonstrates the marker activity profiles for specific subcellular compartments. These

granules could be detected in the gradient by measurement o f the granule marker

eosinophil peroxidase (EPO). EPO activity was concentrated in a sharp peak at the

bottom o f the gradient. The hexosaminidase activity, an enzyme marker o f the

E O ) (/) c Q) Q -O- Density protein 1.3 400 D) 1.2 200 1.1 1 0 2 4 6 8 10 12 14 16 18 20 22 24 Fraction number >

I

26 24 22 20 18 16 14 12 10 8 6 4 2 0 0 2 4 6 8 10 12 14 16 18 20 22 24 o EPO # AP - o Hex Fraction number

pcop

Calnexin kDa <105 <90

Figure 3.1: M arker enzyme assays and protein concentration from subcellular fractions of human eosinophils. Protein concentration and densities of fractions (measured by their relative refractive indices) (a). Profiles of secretory granules (eosinophil peroxidase activity (EPO)), lysosomes (hexosaminidase activity (Hex)) and plasma membrane (alkaline phosphatase activity (AP)) (b). Immunodetection of protein markers: pCOP (cytosol and Golgi membranes) and calnexin (ER) (c). For immunoblotting, an equal volume of each fraction equivalent to 2.5-40pg of protein was loaded into each well and subjected to SDS-PAGE and transferred onto nitrocellulose membranes.

e

A

#

Figure 3.2. Electron microscope photographs of hum an eosinophils and pellets from pooled granule fractions produced by subcellular fractionation, (a) Purified human eosinophils (x6500 magnification); (b) secretory granules (equivalent to fractions 19-21 in figure 3.1) (x25000 magnification).

the most dense o f which co-sedimented with EPO. The second peak was always

smaller and sedimented just above the first peak in the gradient. The peak o f plasma

membrane activity sedimented at a density o f approximately 1.12g/ml, well separated

from the granule population. Figure 3.1c, illustrates the distribution o f p-COP, a

component o f Golgi coat complex that is both cytosolic and bound to budding Golgi

membranes and demonstrates the lack o f separation between plasma membrane and

Golgi membranes. Western blotting across eosinophil subfi*actions demonstrated clear

immunoreactivity at the top o f the gradient demonstrating the presence o f the

coatomer subunit in cytoplasmic fractions and fi'actions co-sedimenting with the

plasma membrane marker. Calnexin was used as a marker o f the endoplasmic

reticulum and Western blotting with an antibody to calnexin demonstrated that the

peak o f ER immunoreactivity sedimented at a density between the peaks o f plasma

membrane and granule activity at a density o f 1.15g/ml (Fig. 3.1c).

To confirm the findings o f the enzyme marker analysis, electron micrographs

were taken o f the pooled granule fractions as determined by the peak o f EPO activity.

Figure 3.2a shows electron micrographs o f purified human eosinophils. The bilobed

nucleus is clearly visible, as are the electron dense crystalloid granules. Figure 3.2b

demonstrated that these fractions were highly enriched in electron-dense crystalloid

secretory granules, with little damage to their surrounding membranes.

3.2.3 Discussion

The aim o f this study was to develop a fi*actionation protocol for human

peripheral blood eosinophils. It was o f primary importance to be able to resolve the

granule fraction to sufficient purity to allow further biochemical characterisation. The

eosinophil was the haematopoietic cell o f choice for these studies. As with other cells

o f this lineage, the eosinophil undergoes degranulation in response to a variety o f

extracellular stimuli and unlike mast cells, can be isolated from peripheral blood. The

advantage o f using the eosinophil for these studies over the neutrophil, which is

available in much higher numbers, is that eosinophils have a simpler profile o f

granules, making subsequent isolation and biochemical analysis more straightforward.

iodonated material, which exhibits low tonicity and low viscosity throughout the

gradient (Rickwood, 1993).

The tracking o f subcellular fractions by marker enzymes after centrifugation

demonstrated that this gradient was not suitable for the resolution o f plasma and Golgi

membranes (Fig. 3.1). The Nycodenz gradient was however, particularly effective at

resolving the dense, crystalloid secretory granules, characteristic o f mature

eosinophils from other intracellular compartments. It was also clear that the peaks for

the granule marker EPO and the second peak o f activity o f the lysosomal enzyme,

P-hexosaminidase overlap exactly. Hexosaminidase is released upon degranulation o f

cells o f this lineage and this observation demonstrated the proposed lysosomal nature

o f this organelle (Griffiths, 1996b). The upper peak o f hexosaminidase activity, which

overlapped with the minor peak o f EPO activity could be immature, or small

eosinophil granules. This peak was not analysed further because o f its expected

impurity. The purity o f the denser granule fraction was demonstrated by the electron

micrographs (Fig. 3.2).

In summary, a reproducible subfractionation protocol has been developed that

results in the production o f gradient fractions substantially enriched in eosinophil

granules compared to other subcellular components. This should allow further

3.3 Western blotting across eosinophil subfractions

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