Subject characteristics
The 22 young subjects consisted of 12 men (age range, 21–37 years) and 10 women (age range, 22–37 years), and the 37 aged subjects who live in a nursing home consisted of 5 men (age range, 76–86 years) and 32 women (age range, 70–95 years). All subjects underwent a medical examination and blood biochemistry analysis. They were also interviewed by a physician. Moreover, the subjects were examined for any signs of infection and inflammation. None of the young subjects had any underlying acute or chronic disease that would affect the immune system nor did they have any cancers or cardiac, liver, brain, or kidney diseases. On the other hand, 13 of the aged subjects used anti-hypertension drugs, 2 had diabetes mellitus, 13 had dyslipidemia, and 12 had prior bone fractures (some had more than one of these conditions). None had suffered from bone fracture within 3 years. All participants were informed of the purpose and risks of the study, and written informed consent was obtained from them. The Research Ethics Committee at Waseda University approved the study.
Biochemical and hematological characteristics
At between 7:00 and 8:00 am after an overnight fast, blood was collected from a forearm vein into a sterile tube containing heparin (Termo, Tokyo, Japan). WBC, lymphocyte, and neutrophil cell counts were obtained with an automatic cell counter pocH-100i (Sysmex, Kobe, Japan). The collected blood was kept at room temperature for 30 min and then centrifuged for 10 min at 1000 × g to separate serum. Samples were stored at -20°C until analysis. The levels of albumin, T-BIL, T-CHO, HDL-CHO, LDL-CHO, TG, FFA, lipid peroxide, and glucose were determined at a hematology laboratory (BML Corporation, Tokyo, Japan),.
Inflammatory markers and cytokine measurement
The serum concentrations of CRP, SAA (Diagnostic Systems Laboratories Inc., Webster, TX, USA), HSP70 (Stressgen Biotechnologies Co., Victoria, BC, Canada), TNF-α (R&D Systems Inc., Minneapolis, MN, USA), IL-6 (R&D Systems), IL-8 (Becton Dickinson), and MCP-1 (R&D Systems) were measured by using commercially available enzyme-linked immunosorbent assay (ELISA) kits, according to the manufacturers' instructions. The absorbance was measured spectrophotometrically with a microplate reader (VersaMax Molecular Devices, Sunnyvale, CA, USA). The plasma concentration of each marker was calculated by comparison to a standard curve established in the same measurement.
ROS from neutrophils
HE (Polysciences Warrington, PA, USA) was dissolved in N, N-dimethylformamide (Sigma-Aldrich, St. Louis, Missouri, USA) at a concentration of 400 mM. Before the flow cytometry assay, the stock solution was further diluted with phosphate buffered saline (PBS, from Wako Pure Chemical, Tokyo, Japan) to a final concentration of 20 mM. fMLP and LPS (Sigma) were used to stimulate the cells. The respiratory burst activity was adopted the method of Rothe et al. [27] using a flow cytometric assay. In the pilot experiments, we have confirmed that not catalase but superoxide dismutases (SOD) inhibits ROS production measured by HE in isolated neutrophils, and that sodium azide, which inhibits myeloperoxidase and SOD, enhances HE responses similar to another superoxide anion detection system [28]. Our method is considered to specifically measure superoxide anion produced by neutrophils.
Heparinized whole blood (100 μl) was placed in separate polypropylene tubes used for the stimulated tests and reagent blank tests. The HE solution was added to the samples, and the same volume of PBS was added to the reagent blank tube. All tubes were loaded with HE at 37°C for 5 min. Either fMLP (final concentration; 10-5 mM) or LPS (final concentration; 1 μg/ml) was then added to the stimulated tubes. After a 35-min incubation at 37°C, 1.5 ml of FACS lysing solution (Becton Dickinson, San Jose, CA, USA) was added to all tubes. The tubes were left at room temperature for 10 min and then centrifuged at 1000 × g for 10 min. The supernatant was discarded, and the cells were resuspended in PBS before being centrifuged again. The washing procedure was repeated twice. After the third centrifugation, the supernatant was discarded and replaced with 400 μl of PBS.
All samples were then analyzed with a FACScan and CellQuest software (Becton Dickinson). Before acquiring data, the instrument was set up by using a reagent blank sample. The forward and side light scatter profiles were adjusted to ensure that the neutrophil population was clearly displayed. Fluorescence was measured on the FL2 red channel (wavelength, 480 nm), and the photomultiplier gain was adjusted so that the fluorescence of the reagent blank was confirmed to the first decade of the FL2 histogram display. Data were then collected from the reagent blank and stimulated sample tubes. A total of 10,000 events were collected for each sample. At analysis, the scattergram of forward and side scatter was displayed, and the neutrophil population was displayed by its location and selected by gating. A scattergram and a histogram of FL2 were then obtained for the neutrophil-gated region, and the median channel fluorescence was recorded from the display statistics. In order to make comparisons of fluorescence intensity between no-situmilation and stimulated ROS poroduction, we adopted the method of Shinkai et al. [29]; the median channel. The median fluorescence intensity (MFI) due to response in stimulation was calculated by subtracting the MFI for an unstimulated neutrophil population (MFI-) from the MFI on a stimulated neutrophil population (MFI+), the difference being expressed as a channel. Thus, the median channel for each assay was calculated from the following equation: median channel = 255.75 × log (MFI+ - MFI-). Fig. 1 shows an example.
Statistical analysis
The statistical analysis was performed with SPSS 15.0J (SPSS Japan Inc. Tokyo, Japan). P values less than 0.05 were considered statistically significant. Data were presented as median, minimum and maximum. The Mann-Whitney U test was used to compare the values between the aged and the younger groups. Associations between parameters were evaluated using Spearman's correlation coefficient.