Effect of Lipopolysaccharide Administration on the Number, Phenotype and Content of Nuclear Molecules in Blood Microparticles of Normal Human Subjects

Abstract

Microparticles (MPs) are small membrane-bound vesicles that arise from activated and dying cells and promote inflammation and thrombosis. To characterize the in vivo release of MPs, we used flow cytometry to measure MPs in the blood of 15 healthy volunteers administered bacterial endotoxin (lipopolysaccharide or LPS) in the presence of a low dose of hydrocortisone with or without inhaled nitric oxide. MPs, defined as particles less than 1.0 lm in size, were assessed following labelling for CD42a, CD14 and CD62E or CD144 antibodies to identify MPs from platelets (PMPs), monocytes (MMPs) and endothelial cells (EMPs). In addition, PMPs and MMPs were labelled with anti-HMGB1 and stained with SYTO13 to assess nuclear acid content. Administration of LPS led to an increase in the numbers of PMPs, MMPs and EMPs as defined by CD62E, as well as the number of MMPs and PMPs staining with anti-HMGB1 and SYTO13. Inhalation of NO did not influence these findings. Together, these studies show that LPS can increase levels of blood MPs and influence phenotype, including nuclear content. As such, particles may be a source of HMGB1 and other nuclear molecules in the blood during inflammation.

CD40L expression in plasma of volunteers following LPS administration: A comparison between assay of CD40L on platelet microvesicles and soluble CD40L

“…G3E069, United States Pharmacopeia, Rockville, MD) at both occasions whereas iNO or placebo (inhaled N 2 ) were randomized. Notably, iNO had no effect on either inflammation or circulating MVs [11,12]. Written informed consent was obtained from all subjects prior to the study.…”

“…Conjugate isotype matched immunoglobulin (IgG1-FITC, IgG1-PE and IgG1-APC) with no reactivity against human antigens was used as a negative control to define the background noise of the cytometric analysis. The percentage of debris/fragments in the MV gate positive for phalloidin (cellfragment marker) was 10.6 ± 6.9 % (mean ± SD) which indicates acceptable sample handling [12]. The concentration of MVs was calculated by means of the following formula: (MV counted  standard beads/l)/standard beads counted, (FlowCount, Beckman Coulter).…”

High-Mobility Group Box-1 (HMGB1) participates in the pathogenesis of alcoholic liver disease (ALD)

“…Upon stimulation, HMGB1 undergoes translocation from the nucleus to the cytoplasm, and it is then secreted via the lysosomal pathway in most cells (2,3), by exosomes in enterocytes (4,5), or by the inflammasome in immune cells (6 -10). HMGB1 binds the receptor for advanced glycation end products, toll-like receptors-2/4/9, Mac-1, syndecan-1, phosphacan protein-tyrosine phosphatase-/␤, and CD24 (11)(12)(13)(14)(15).…”

The Expression of HMGB1 on Microparticles Released during Cell Activation and Cell Death In Vitro and In Vivo

“…The expression ion of HMGB1 on MPs was confirmed in a study of the response of healthy human volunteers receiving LPS (50). This study had originally been performed to assess possible immunomodulatory effects of inhaled nitric oxide (NO) and involved relatively low doses of LPS; furthermore, to attenuate possible adverse effects of the LPS, the volunteers were treated with glucocorticoids (51).…”