Ariffin, Nur Azrah Fazera Mohd
(2018)
Phenotypic and functional characterisation of monocytic microparticles (mMP) derived from U937 and THP-1 cell lines.
Masters thesis, Universiti Sains Malaysia.
Abstract
Monocytic microparticles (mMP) are 0.1-1.0 μm membrane particle, shed from
human monocytes under physiological condition such as cellular activation and apoptosis
or during certain pathological conditions. It has been reported in the literatures that mMP
play important roles in inflammation, endothelial cell function and blood coagulation,
however, the mechanism involved is still unclear. Monocytic MP can be characterised by
the expression of their parental cell antigen, CD14 along with phosphatidylserine (PS) on
mMP surface which can be detected by flow cytometry. This study was conducted using
U937 and THP-1 monocytic cell lines. To induce mMP production in vitro, 1 x 106 cells
were stimulated with 1 μg/mL lipopolysaccharide (LPS) or 10 ng/mL recombinant human
tumor necrosis factor-α (TNF-α) for 18 hours in 5% CO2 humidified incubator at 37°C.
Then, mMP were isolated through three differential centrifugations: (i) 500 xg to remove
cell pellet, (ii) 1,500 xg to remove cell debris and (iii) 20,000 xg ultra-centrifugation to
pellet mMP. This study was aimed to identify mMP surface marker expression by flow
cytometry, measure mMP-induced proinflammatory cytokine secretions and assess
coagulation potential of mMP. Theoretically, mMP populations are defined as positive for
Annexin-V in combination with monocytic marker, CD14. Our finding has shown that
U937 mMP and THP-1 mMP does not necessarily follow the same surface antigen
expression as their parent cells. We then investigated the ability of mMP to secrete
proinflammatory cytokines; TNF-α and interleukin 1 beta (IL-1β) and also tested mMP
potential to modulate cytokine secretions in U937 and THP-1 cells. We found that IL-1β
was secreted from both mMP but was down regulated when co-cultured with U937 and
THP-1 cells. TNF-α was secreted from all TNF-α-stimulated samples and only mMPderived
U937 upregulated TNF-α secretion almost up to 400 pg/mL from TNF-α-
stimulated U937 cells. Besides that, mMP may have a procoagulant potential as shown by
high expression of CD142 on both mMP surface and shorter prothrombin time (PT) over
an increasing number of mMP. In conclusion, different monocytic cells may give rise to
different properties of mMP. Further study should be carried out to establish a detail
characterisation of mMP isolated from human blood and discover the clinical implications
of mMP especially in inflammation-related diseases.
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