Human microparticles generated during sepsis in patients with critical illness are neutrophil-derived and modulate the immune response

Abstract

Background: Microparticles (MPs) are 0.3 μm to 1.0 μm vesicles generated after cell activation or apoptosis that may play a role in the pathophysiology of sepsis. We sought to elucidate the role of MPs in patients with critical illness and hypothesized that MPs are generated at the site of inflammation and can modulate the immune response.

Methods: Surgical patients with critical illness with ongoing sepsis were enrolled from the intensive care unit of an urban, Level I trauma center from March to June 2011. Abdominal washings and bronchoalveolar lavage fluid were collected from sites of inflammation. MPs were isolated using differential centrifugation, then characterized by flow cytometry. Immunologic assays were conducted by incubating neutrophil-derived MPs (NDMPs) with a human monocytic cell line (THP-1). A p value ≤0.05 was considered significant.

Results: MPs were absent in noninflamed foci in patients, whereas NDMPs were present in locations of inflammation. NDMPs were added to cultured THP-1 cells to quantify immunomodulatory effects. THP-1 cells were able to phagocytose NDMPs. Cells that ingested NDMPs demonstrated increased activation. In contrast, bystander THP-1 cells without ingested NDMPs demonstrated decreased activation.

Conclusion: NDMPs are generated at the site of inflammation in patients with critical illness during sepsis. They have a divergent effect on the immune response by activating phagocytic cells and deactivating bystander cells. NDMPs may play an important role in regulating the inflammatory response to sepsis in patients with critical illness.

Figure 1

Sizing and gating strategy for the identification of MPs. (A) Latex beads of a predetermined size were used to set the forward and side scatter voltages to best distinguish particles ranging from 0.3 μm to 3.0 μm. (B) Representative gating demonstrating sizing strategy to detect MPs.

Figure 2

NDMPs are generated in the infected foci of patients with critical illness and are the predominate type of MP identified. (A) Abdominal irrigation fluid was obtained from patients either undergoing exploratory laparotomy for peritonitis or elective surgery. BAU was obtained from patients with suspected pneumonia or lung donors without pneumonia. MPs were characterized as described in the Patients and Methods section. (B) Flow cytometric analysis demonstrates significantly increased percent NDMPs in abdominal infections. Analysis on BAU was not included in this data set. n = 3 for infected abdomens, and n = 4 for uninfected abdomens.

Figure 3

NDMPs are binding to or phagocytosed by THP-1 cells. Human NDMPs were fluorescently labeled with CFSE and then incubated for 18 hours with THP-1 cells, a human monocytic cell line. THP-1 cells were gated on CD14 and CD1 lb surface expression levels, and CFSE fluorescence was determined by flow cytometry as described in the Patients and Methods section. Five independent samples were assayed. Data are expressed as the mean (SEM). *p < 0.05 as determined by Student’s t test.

Figure 4

THP-1 cells incubated with NDMP demonstrate divergent activation. Fluorescently labeled NDMPs from patients with critical illness were incubated 18 hours with THP-1 cells. Untreated, (+)CFSE THP-1, and (−)CFSE THP-1 cells were analyzed by flow cytometry for (A) HLA-Dr, (B) CDBO, and (C) CD86 surface expression levels. Six independent samples were assayed, *p < 0.05 as compared with untreated and bystander, and ^#p < 0.05 as compared with ingested as determined by ANOVA pairwise comparison.

Figure 5

Ingestion of NDMPs increases the phagocytic ability of THP-1 cells. THP-1 cells were incubated with CFSE-labeled NDMPs. After 18 hours, the phagocytic ability of (+)CFSE THP-1 and (−)CFSE THP-1 cells was determined as describe in the Patients and Methods section. Data are expressed as the mean (SEM). *p < 0.05 as determined by Student’s t test.