Accumulating investigations have proven that elevated levels of several inflammatory mediators among apparently healthy men and women have predictive value for future adverse events 123. In particular, prospective epidemiological studies have found increased vascular risk to be associated with increased basal levels of cytokines such as interleukin (IL) -6 and tumor necrosis factor (TNF)-α 45, and downstream acute-phase reactants such as C-reactive protein (CRP), fibrinogen, and serum amyloid A (SAA) 67. The local inflammatory response is accompanied by a systemic response known as the acute-phase response 8. The levels of those inflammatory markers increase several fold and decrease when the infection or trauma has healed. In contrast, chronic low-grade inflammation is typically characterized by mild to moderate increases in the systemic concentrations of these inflammatory markers 9. Ageing is also associated with low-grade inflammation, and increases in circulating levels of CRP, TNF-α, and IL-6 have been identified in age-related inflammatory diseases in cross-sectional studies 1011. In this regards, circulating levels of these inflammatory mediators in patients and/or elderly humans may provide clinically useful information about the state of chronic medical disorders 12.

Neutrophils have a variety of functions including chemotaxis, adhesion to the endothelium and foreign agents, phagocytosis, and microbicidal activity. Neutrophils can penetrate and migrate into infected tissues, where they internalize and destroy invading microorganisms by producing toxic agents such as reactive oxygen species (ROS), proteases (elastase), and proteins that interfere with bacterial development (lactoferrin) 13. ROS are generated during the complex process of respiratory burst, during which superoxide anion (O₂^-) is formed immediately after the reduction of molecular oxygen by single electrons through the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase system 13. O₂^- is then rapidly transformed through enzyme activity (superoxide dismutase, catalase, myeloperoxidase) into other ROS, which include hydrogen peroxide (H₂O₂), hydroxyl radicals, and hypochlorous acid 13. The importance of O₂^- and H₂O₂ production for neutrophil bactericidal activity is illustrated by patients with chronic granulomatous disease who do not produce enough O₂^.- and H₂O₂, thus becoming more susceptible to bacterial infection 14. Despite the intrinsic function of neutrophils in the innate immune response, potent cytotoxic compounds are released into the extracellular space, which damage host tissues 13. Herman's free radical theory, which has been accepted as a plausible explanation of the primary chemical reactions involved in ageing process, proposes that oxygen-derived free radicals are responsible (due to their high reactivity) for the age-associated damage at the cellular and tissue levels through the oxidative modification of biological molecules (lipids, proteins, and nucleic acids), which leads to functional impairment 15. Cells that use oxygen and consequently produce ROS have evolved complex antioxidant defense systems to neutralize ROS and protect themselves against free radical damage. The increased oxidative stress in ageing is likely to be the consequence of an imbalance between free radical production and antioxidant defenses (with a higher production of the former) 16. From a physiological perspective, endogenous ROS produced by the NADPH oxidase system regulate tyrosine phosphorylation dependent pathways that in turn modulate host defense responses such as phagocytosis and, via nuclear factor-κB, the expression of cytokines and chemokines that further modulate the inflammatory response 17. Under pathological circumstances such as inflammatory tissue injury, excess production of ROS may destroy vicinal cells such as endothelium or epithelium 18. In addition, since ROS are membrane permeable, they may influence intracellular signaling pathways in the cell as well as the adjacent cells in the inflammatory milieu 19. During a systemic inflammatory response, it is feasible that ROS act as signaling molecules leading to modulation of crucial events including phagocytosis, secretion, gene expression, and apoptosis, resulting in dysregulation of inflammation 19. In this regards, we hypothesized that the spontaneous production of ROS from neutrophils might change with ageing and associated with the conventional inflammatory markers, even though the intrinsically microbicidal task of ROS decreases with ageing. To examine the hypothesis, we compared ROS production by neutrophils and the circulating levels of inflammatory markers in older and younger people and investigated whether the elevated ROS levels were associated with inflammatory markers including cytokines. The objective of this study was to determine whether spontaneous ROS production by neutrophils is an effective inflammatory marker.

Vulnerability to infection in the elderly may result from reduced neutrophil numbers at the site of infection, or an intrinsic reduction in bactericidal activity 20. Nevertheless circulating neutrophil numbers are not altered with age 21. Our result also showed that the neutrophil counts did not changed with age. The neutrophil counts are known as a predictor of the incidence of the low-grade inflammation-associated diseases like atherosclerosis 2223. Consistent with previous studies, our results have found that neutrophil counts in the aged group were weakly correlated with inflammatory markers, IL-6, SAA, TNF-α, and CRP (r = 0.48, 0.47, 0.37, and 0.44, respectively), and that the plasma concentration of the conventional inflammatory markers and cytokines including IL-8, TNF-α, IL-6, CRP, and HSP70 were higher in the aged group than in the younger group. In addition, there was a strong correlation between neutrophil counts and IL-8 in the young group. IL-8 is a potent neutrophil chemoattractants that gathers neutrophils, produces excess ROS, and may indicate the higher intrinsic function of neutrophils in the young group. On the other hand, we found that spontaneous ROS production by neutrophils was significantly higher in the aged group than in the young group (P < 0.01), showing that higher levels of spontaneous ROS production from neutrophils could increase with age. However, we did not find a correlation between spontaneous ROS production and any of the inflammatory markers except HSP70. Since the comparison of the magnitude of spontaneous ROS production and inflammatory markers between the aged and young groups was less than 2-fold, the lower magnitude of inflammatory markers and ROS production in this group may be insufficient to examine the low-grade inflammation.

The spontaneous ROS production negatively correlated with HSP70 (r = -0.41, P < 0.05) only in the aged group, but not in the young group. HSP70 is a very conservative family of cytoprotective proteins that are specifically induced in response to several environmental stresses at the cellular level including heat shock, cellular energy depletion, oxidative stress or inflammation. Although the role of HSP70 in the blood (i.e. extracellular HSPs) is yet not known, HSP70 (intracellular HSP70) plays an important role in cytoprotection by preventing abnormal folding of newly synthesized polypeptides, or by assisting in the repair of damaged proteins 24. The elderly people seem to be characterized by a diminished basal expression of HSP70 and a blunted induction in response to different stress conditions 25. Therefore, the negative correlation between spontaneous ROS production and HSP70 may indicate that the higher concentration of HSP70 in plasma protectively responds to the oxidative stress 26, which may cause lower levels of spontaneous ROS production, or vice versa.

The response of fMLP- or LPS-stimulated ROS production was similar in the two groups. In general, the priming action of fMLP and LPS on neutrophil activity is seen as the body providing a global defense to support a primary immune response against exposure to antigens. Age-related neutrophil alterations show impaired ROS production and phagocytosis with a subsequently decreased capability to destroy bacteria 2021. The results may be the limitation in ROS detection. To determine the level of ROS production by neutrophils, we used a flow cytometric assay with whole blood samples. Hydroethidine (HE) is one of the most commonly used fluorescent probes to measure intracellular oxidant production. Oxidation of HE by O₂^- produces ethidium, which can emit red fluorescence after intercalation with cellular DNA 27. In this study, the elevated baseline level, in part, cause the difficulties to detect the response to the stimulation between the aged and young groups. Further study is needed to improve this point.

The main limitation of this study is that it did not involve aged healthy subjects without any condition known to be associated with low-grade inflammation. More specifically, the young group consisted of healthy individuals, whereas most subjects in the aged group had one or more conditions that could be associated with low-grade inflammation (hypertension [n = 13], dyslipidemia [n = 12], and diabetes mellitus [n = 2], which might have influenced the level of ROS production.

We compared the ROS production by neutrophils and the circulating levels of inflammatory markers in older people and younger people, and investigated whether the elevated ROS levels are associated with inflammatory markers including cytokines. The results suggest that the spontaneous ROS production from neutrophils may increase with age and represent the different aspect of age-associated immune dysregulation. Further investigations on biological significance of ROS from neutrophils in ageing process are warranted

The authors declare that they have no competing interests.

KO had the overall responsibilities of the experiment design and statistical analysis, and wrote the manuscript. KS had the overall responsibilities of fund management and the experimental design. MO and KY conducted the experiments in the manuscript. SS had shared the concept and supported the manuscript. All the authors have read and approved the final manuscript.