At the beginning of this study, the granule localisation and regulation of release of human
neutrophil (PMNL) precursor collagenases, proMMP-8 and -9 (type I and type TV/V
collagenases, respectively), enzymes highly active against the extracellular matrix (ECM) and
thought to be relevant in invasion and inflammation, had been established while that of their
inhibitor, tissue inhibitor of matrix metalloproteinases-I (TIMP-1), had not.
Electron microscopy immunogold labelling of cryoultramicrotomy sections for granule marker
proteins, lysosome-associated membrane proteins (LAMPs) and endocytosed bovine serum
albumin-coated gold probes, followed by stereology, established that TIMP-1 was mainly
located in a distinct oval, electron translucent organelle, a little larger than azurophil granules.
A lack of labelling for endocytic markers and for glycosylphosphatidylinositol-anchored
proteins, established using granule fractionation and immunolabelling to be markers for the
secretory vesicles, and LAMPs-1 and -2, indicated the non-endosomal, non-secretory and nonlysosomal
nature of this organelle. Density gradient cofractionation with the least dense
secretory vesicle population and some pleiomorphism of the organelle suggested that it is a
"vesicle" rather than a "granule" population. Colocalisation with proMMP-9 in minor
subpopulations suggests that TIMP-1 vesicle biogenesis occurs between metamyelocytic and
termination differentiation, but before secretory vesicle synthesis.
Immunolabelling of phagocytosed and pulse-chased IgG-opsonised latex beads showed that
specific and azurophil granules and a small number of proMMP-8-containing granules (a
specific granule subpopulation) fuse with the phagosome whereas the TIMP-1 vesicle and
proMMP-9-containing granules do not, suggesting that the latter play no role in phagosomal
destruction of IgG-opsonised bacteria and that their phagosomal release is not calcium
regulated. However, studies using the calcium ionophore, ionomycin, and monitoring
extracellular granule marker protein release upon addition of increasing levels of extracellular
calcium, showed that all granules, except the TIMP-1 vesicle, appeared to be calcium regulated.
This suggests that the regulation of proMMP-9 release is not exclusively via calcium and that
TIMP-1 vesicle release is not calcium regulated. Whereas most granules were shown to be associated with microtubule-like structures, the
TIMP-1 vesicle and proMMP-9-containing granules were shown to associate with two
morphologically different cytoskeletal elements, neither resembling actin nor tubulin. These
elements, and the release of the TIMP-1 vesicle and proMMP-9-containing granules, need to be
studied further, but results achieved to date may explain the observed differential mode of
release of TIMP-1 relative to proMMP-9.
The proMMP-9-binding and inhibitory capacity of a 66 kDa high molecular mass form of
TIMP-1 was demonstrated in PMNL homogenates and plasma using western ligand blots and a
novel reverse zymography method. The role and relevance of this form remains unknown as
does the relevance and potential role of proMMP-9ffIMP-1 complexes seen during isolation
procedures. The proMMP-9ffIMP-1 complex may occur in vivo, as evidenced by
immunolocalisation studies, and, together with TIMP-1 released from its own discrete vesicle
population, may be responsible for the fine regulation of extracellular proteolysis. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 2002.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ukzn/oai:http://researchspace.ukzn.ac.za:10413/10014 |
| Date | 15 November 2013 |
| Creators | Price, Brendon. |
| Contributors | Elliott, Edith., Tschesche, Harald. |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
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