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115 Qunantitative and functional evaluation of plasma microparticles in systemic lupus erythematosus
  1. A Lateef1,2,
  2. L Shaffiee1,
  3. GK Gill3,
  4. P Cheung1 and
  5. YC Lim4
  1. 1National University Hospital, Medicine, Singapore, Singapore
  2. 2Yong Loo Lin School of Medicine- National University of Singapore, Medicine, Singapore, Singapore
  3. 3National University of Singapore, Physiology, Singapore, Singapore
  4. 4National University of Singapore, Pathology, Singapore, Singapore

Abstract

Background and Aims Plasma microparticles (PMPs) are small (0.1 to 1.0 µm) membrane bound vesicles, released from plasma membrane during cell activation and apoptosis. The cytoplasmic and surface contents of PMPs vary according to the parental cell’s lineage and physiological state. Altered numbers and profiles have been associated with thrombotic and inflammatory disorders including rheumatic diseases.

There have been variable reports on PMPs levels and profiles in Systemic Lupus Erythematosus (SLE). Some studies have reported higher levels while others showed similar or lower numbers with altered profiles. We compared PMPs in a multi-ethnic SLE cohort with a healthy control group, and explored their role in pathophysiology of SLE

Methods Clinical data and blood samples were collected from 56 consented SLE patients (median 45 years, 95% females) who fulfilled the ACR criteria, and 39 healthy adults. PMP pellet was obtained by high speed centrifugation. Flow cytometry was used to enumerate and profile the PMPs, utilising fluorescent polystyrene nanobeads, lipophilic dyes and fluorescent-labelled antibodies. HL60, a promyelocytic cell line, was utilised to study functional effects of PMPs in a smaller subgroup of samples (8 from each arm).

Results SLE patients have lower PMP numbers compared to healthy controls (median: 12 925 vs 26 490 respectively, p=0.01, Figure 1). SLE PMPs also exhibited significantly higher proportions of leukocyte, and endothelial markers (Figure 2). SLE PMPs induced significantly higher p38 phosphorylation in HL60 cells compared to control PMPs (p=0.04, Figure 3)

Figure 1

SLE samples have lower PMPs than control samples. (A) PMP gate setup using 0.22, 0.45, 0.88 and 1.34μm fluorescent polystyrene beads. (B) A representative example of control PMP (left) and patient PMP (right) diluted 1:10.Events falling outside the gate were not considered as PMP.Thresold foe PMP collection set according to SSC. (C) Time-matched backgrounds of the diluents binding buffer (BB, left) and 1% bovine serum albumin dilutedin phosphate buffered solution (BSA/PBS, right). (D)PMP counts for control samples (n=39), patient samples (n=56), BB background (n=16) and BSA/PBS background (n=13). Controls had significantly higher PMP counts than patients (p=0.01).Each data point shown represents 1 unique control or patient sample. Bars represent median with interquartile range.

Figure 2

Highewr percentage of leukocyte and edothelial derived PMP, But not platelet-derived PMP, are detected in SLE patients. (A) Isotype control for PE and PerCP-conjugated antibodies in representative control (left) and patient (right) samples. (B) Representative examples of CD45-PE positive staining in control (left) and patient (right) samples. (C) CD31-PE and CD144-PerCP double staining in a control (left) and a patient (right) Sample. (D) CD61-PE and CD41-PerCP double staining in a control (left)and a patient (right) sample. (E) Each data point shown represents 1 control or patient sample.Bars represent median and interquartile range. Significant differences between control (n=34) and patient (n=42) groups in the population of leukocyte (p=0.02) and endothelial (p=0.01) derived PMP using Mann-Whitney test.

Figure 3

Patients PMPs induced significantly higher phosphorylated 38 expression in HL60 cells. HL60 cells were treated with control (CTL) or patient (PT) PMPs prior to cell lysis and western blot analysis for protein p38 phosphorylation.Untreated HL60 and PMA-activated HL60 were used as baseline and activated controls respectively. Phosphorylated p38 bands were quantified using using Image J software and normalised to that of the untreated condition.n=8 for each treatment condition.The intensity of phosphorylated p38 bands in PT PMP-treated-HL60 cells was significant higher than that of CTL PMP-treated (p=0.4) and PMA-activated (p=0.04) HL60 cells using Mann-Whitney test.

Conclusions Our data suggest that there are quantitative and functional differences in PMPs between SLE patients and healthy controls

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