Hyperactivity and reactivity of peripheral blood neutrophils in chronic periodontitis

Abstract

SummarySome evidence exists that peripheral neutrophils from patients with chronic periodontitis generate higher levels of reactive oxygen species (ROS) after Fcg-receptor stimulation than those from healthy controls. We hypothesized that peripheral neutrophils in periodontitis also show both hyper-reactivity to plaque organisms and hyperactivity in terms of baseline, unstimulated generation and release of ROS. Peripheral neutrophils from chronic periodontitis patients and age/sex/smoking-matched healthy controls (18 pairs) were assayed for total ROS generation and extracellular ROS release, with and without stimulation (Fcg-receptor and Fusobacterium nucleatum), using luminol and isoluminol chemiluminescence. Assays were performed with and without priming with Escherichia coli lipopolysaccharide (LPS) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Phox gene expression (p22, p47, p67, gp91) was investigated using reverse transcriptionpolymerase chain reaction (RT-PCR). Neutrophils from patients produced higher mean levels of ROS in all assays. Total generation and extracellular release of ROS by patients' cells were significantly greater than those from controls after FcgR-stimulation, with (P = 0·023) and without (P Յ 0·023) priming with GM-CSF. Differences in unstimulated total ROS generation were not significant. By contrast, patients' cells demonstrated greater baseline, extracellular ROS release than those from controls (P = 0·004). This difference was maintained after priming with LPS (P = 0·028) but not GM-CSF (P = 0·217). Phox gene expression was similar in patient and control cells at baseline and stimulation with F. nucleatum (3 h) consistently reduced gp91 PHOX transcripts. Our data demonstrate that peripheral neutrophils from periodontitis patients exhibit hyper-reactivity following stimulation (Fcgreceptor and F. nucleatum) and hyperactivity in terms of excess ROS release in the absence of exogenous stimulation. This hyperactive/-reactive neutrophil phenotype is not associated with elevated phox gene expression.

Periodontitis Associates with a Type 1 IFN Signature in Peripheral Blood Neutrophils

“…Patient and control pairs were previously demonstrated to exhibit a high hyperresponsive differential (both hyperreactivity to Fc␥R-stimulation and baseline unstimulated hyperactivity) as determined by ECL (mean differential periodontitis vs health ϭ 13,897 RLU, range ϭ 8,300 -27,061 RLU), and these data have been published elsewhere (9,10). RNA samples were analyzed using human Affymetrix HG_U133A oligonucleotide arrays, as described at www.affymetrix.com/products/arrays/specific/hgu133.affx.…”

“…The resultant collateral host tissue damage to the supporting periodontal tissues leads to progressive periodontitis and ultimately culminates in tooth loss (6). Several studies have demonstrated that PBN from chronic periodontitis patients are not only hyperreactive, in response to Fc␥R stimulation by periodontal pathogens, but also hyperactive, with respect to baseline unstimulated ROS production (7)(8)(9)(10). Although a host molecular defect in intracellular lipid signaling may explain peripheral neutrophil ROS hyperreactivity in the relatively rare form of the disease, localized aggressive periodontitis (11), this mechanism does not explain the patient predisposition observed in chronic periodontitis.…”

“…All volunteers were never smokers with no history of recreational drug use and no special dietary requirements. This population has been investigated in both cross-sectional and longitudinal studies of the effects of nonsurgical periodontal therapy upon PBN ROS hyperresponsiveness (9,10).…”

“…All assays were performed as previously described (9,10). In brief, neutrophils (1 ϫ 10 5 ) were placed in preblocked (PBS with 1% BSA, overnight at 4°C) white microwells (Microlite 2; Dynex) with supplemented PBS or supplemented PBS containing 25 IU of IFN-␣, -␤, -␥ (R&D Systems), or 0.1 g of LPS (Sigma-Aldrich; serotype 026:B6; 35 l), luminol (3 mmol/L; 30 l) for detection of total ROS production, or isoluminol (3 mmol/L; 60 l) with 6 U of HRP for detection of extracellular ROS release.…”

Nuclear Factor Erythroid 2-Related Factor 2 Down-Regulation in Oral Neutrophils Is Associated with Periodontal Oxidative Damage and Severe Chronic Periodontitis

“…22,23 Bone marrow PMNs from six WT and four Nrf2 À/À mice were incubated in a mixture of 3 mmol/L isoluminol and 6 U of horseradish peroxidase (Sigma-Aldrich) on preblocked 96-well plates, and stimulated with 10 À6 mol/L phorbol myristate acetate (Sigma-Aldrich). Relative light units (RU) were measured using a fluorescence spectrophotometer (Fisher Scientific, Waltham, MA).…”