Stimulation Of Neutrophil Oxidative Burst By Calcium Phosphate Particles With Adsorbed Mucin

Abstract

Objective — Mucin can promote formation of gallstones via precipitation with calcium phosphate. The proinflammatory effect of mucin-coated particles is still unclear, and our aim was to study the role of mucin sorption in activation of neutrophil respiratory burst. Material and Methods — Polydisperse calcium phosphate nanowires (CP) were prepared from hot gelatin solution and according to scanning electron microscopy (SEM) had the length 1-10 μm and thickness 50-450 nm. CP were incubated in mucin or human serum albumin (HSA) giving CP-Muc and CP-HSA. Their hemolytic activity towards human erythrocytes was assayed, and neutrophil lucigenin- and luminol- chemiluminescence (Luc-CL and Lum-CL) response to CP, CP-HSA and CP-Muc was measured. Cytokine RNA was detected in neutrophils by means of reverse transcription with subsequent real-time PCR. Cytokines (IL-1β, IL-6, IL-8, IL-10) were assessed in cell medium by ELISA. Results and Conclusion — Hemolytic activity of CP was 3.0±0.5%, mucin sorption (0.019 mg/mg) reduced it to 0.24±0.04% (p<0.05) as well as HSA. CP and CP-HSA stimulated neutrophil Lum-CL and Luc-Cl by 2-3 times vs. spontaneous values while for CP-Muc the effect was 10-fold and higher. No increased cytokine gene expression or cytokine secretion was detected after 1h incubation of neutrophils with samples. Obviously, sorption of mucin but not that of HSA stimulated generation of reactive oxygen and halogen species with no increase in cytokine production. Thus, the mucin-coated CP has the potential to contribute to gallstone-associated cholecystitis via oxidative damage of mucosa and epithelium.