Morphological characterization of para- and proinflammatory neutrophil phenotypes using transmission electron microscopy.

Abstract

Bacterial challenge is constant in the oral cavity. To contain the commensal biofilm, partly activated neutrophils are continuously recruited as part of a normal physiologic process, without exposing the host to the harmful effect of a fully active neutrophil response. This intermediate immune state has been termed para-inflammation, as opposed to the fully activated proinflammatory state in oral disease. Directly visualizing these cells and their components via transmission electron microscopy (TEM) enhances our understanding of neutrophil activation state differences in oral health and disease, as obtained from molecular studies. The aim of this study was to describe the morphology of the para-inflammatory phenotype displayed by oral neutrophils in health, and compare it to the morphology of the naïve blood neutrophil, and the proinflammatory oral neutrophils in chronic periodontitis. This morphology was characterized by differences in granule content, phagosome content and cytoplasm and nuclear changes. We also examined the morphological changes induced in naïve neutrophils, which were stimulated in vitro by bacteria, and in oral neutrophils in full tissue samples in vivo. Neutrophils were isolated from blood and saliva samples of patients with chronic periodontitis and healthy individuals. The cells were viewed under TEM and analyzed in imaging software examining granularity, cytoplasm density, euchromatin amount in the nucleus and phagosome content. A separate cohort of blood neutrophils was incubated with Streptococcus oralis and analyzed under TEM in the same manner. Gingival tissue samples were obtained from patients with chronic periodontitis and viewed under TEM, with the neutrophils present analyzed in the same manner. The proinflammatory cells showed less granulation, lighter cytoplasm and higher amount of nuclear euchromatin. These changes were accentuated in the proinflammatory oral chronic periodontitis neutrophils compared to the para-inflammatory oral health neutrophils. The oral chronic periodontitis neutrophils also contained more phagosomes and had more phagosomes containing undigested bacteria. These changes were partially reproduced in the naïve blood cells after exposing them to S.oralis. The neutrophils in the gingival tissues displayed naïve morphology when viewed in the blood vessels and gradually showed proinflammatory morphological changes as they traveled through the connective tissue into the epithelium. Oral neutrophils display morphological changes consistent with partial or full activation, corresponding to their para- or proinflammatory states. These changes can also be induced in naïve cells by incubating them with commensal bacteria. Neutrophils change their morphology towards an activated state as they travel through the gingival tissue.