Nano-sized and micro-sized polystyrene particles affect phagocyte function

Abstract

Adverse effect of nanoparticles may include impairment of phagocyte function. To identify the effect of nanoparticle size on uptake, cytotoxicity, chemotaxis, cytokine secretion, phagocytosis, oxidative burst, nitric oxide production and myeloperoxidase release, leukocytes isolated from human peripheral blood, monocytes and macrophages were studied. Carboxyl polystyrene (CPS) particles in sizes between 20 and 1,000 nm served as model particles. Twenty nanometers CPS particles were taken up passively, while larger CPS particles entered cells actively and passively. Twenty nanometers CPS were cytotoxic to all phagocytes, ≥500 nm CPS particles only to macrophages. Twenty nanometers CPS particles stimulated IL-8 secretion in human monocytes and induced oxidative burst in monocytes. Five hundred nanometers and 1,000 nm CPS particles stimulated IL-6 and IL-8 secretion in monocytes and macrophages, chemotaxis towards a chemotactic stimulus of monocytes and phagocytosis of bacteria by macrophages and provoked an oxidative burst of granulocytes. At very high concentrations, CPS particles of 20 and 500 nm stimulated myeloperoxidase release of granulocytes and nitric oxide generation in macrophages. Cytotoxic effect could contribute to some of the observed effects. In the absence of cytotoxicity, 500 and 1,000 nm CPS particles appear to influence phagocyte function to a greater extent than particles in other sizes.

Figures

Fig. 1

Uptake of fluorescently labelled carboxyl polystyrene particles (FluoSpheres®, FS) in PBMCs exposed in DMEM. After short incubation at 37 °C, lymphocytes (CD3-immunoreactive) take up 20 nm FS but not larger particles. Granulocytes (CD13-immunoreactive) and monocytes (CD14-immunoreactive) also ingest 200 and 500 nm FS after this time. Uptake of ≥200 nm FS is inhibited by low temperature. After 24 h incubation, lymphocytes also contain 500 nm FS. Asterisks indicate FS taken up by platelets. Scale bar 10 μm

Fig. 2

Viability of human monocytic U937 and THP-1 cells, differentiated THP-1 cells (THPdiff) and murine DMBM-2 macrophages after incubation with carboxyl polystyrene particles (CPS particles) of ≤200 nm (a) and ≥500 nm (b) for 24 h exposed in DMEM (n=4)

Fig. 3

Viability of DMBM-2 macrophages (a) and THP-1 cells (b) dependent on particles/cell (n=4). CPS particles decrease viability of monocytic THP-1 cells at lower numbers than viability of DMBM-2 macrophages

Fig. 4

Chemotaxis of U937 cells towards a positive chemotactic stimulus (PMA) after incubation for 30 min with plasma-coated CPS particles (n=3). Significant changes to the solvent-treated controls (p<0.05) and between different particles linked by brackets are indicated by asterisk. A hatch indicates significant difference of one particle to the others

Fig. 5

Release of cytokines IL-6 (a) and IL-8 (b) from monocytic THP-1 cells upon stimulation with 20 μg/ml CPS particles suspended in DMEM for 24 h (n=6). THP-1 cells were also assessed after differentiation with PMA and releases were normalised to solvent-treated controls as 1. a IL-6 secretion was increased by incubations with CPS ≥500 nm. b 20 nm CPS stimulated IL-8 secretion in differentiated and monocytic THP-1; 100 ng/ml LPS served as positive control. Significant changes to the solvent-treated controls (p<0.05) and between different particles linked by brackets are indicated by asterisk

Fig. 6

Phagocytosis of bacteria by DMBM-2 macrophages after pre-incubation with CPS particles suspended in DMEM (n=3). Twenty nanometers CPS particles inhibited phagocytosis, while 1,000 and 500 nm CPS particles stimulated the uptake. Data are normalised (100 %) to the uptake of indicator particles in medium. Significant changes to the solvent-treated controls (p<0.05) are indicated by asterisk

Fig. 7

Generation of superoxide in monocytes (a) and granulocytes (b) in human blood samples exposed to 25 μg/ml CPS particles suspended in PBS (n=4). E. coli provided in the assay kit served as positive control (PC) and PBS as negative control (NC). Data are normalised to the generation of superoxide in cells exposed to buffer. a Uncoated and plasma-coated 20 nm CPS particles increase the production of superoxide by monocytes while 1,000 nm CPS particles display only a marginal effect. b In granulocytes, plasma-coated 1,000 nm CPS particles increase the production of superoxide while 20 nm CPS particles display only a marginal effect. Significant changes to the solvent-treated controls (p<0.05) are indicated by asterisk. u uncoated, c plasma coated