Reagents

The endotoxin-free Hanks’ balanced salt solution (HBSS), phosphate-buffered saline (PBS), and deionized water were purchased from Life Technology Japan (Tokyo, Japan). All reagents were checked and confirmed to be endotoxin-free, DNase-free, RNase-free, and pyrogen-free by Life Technology Japan. Furthermore, all reagents and solutions included in the commercial kits in this study were confirmed to be free from contamination using endotoxin testing, which was performed in compliance with the following guidelines: ANSI/AAMI ST72: 2011; Bacterial Endotoxins-Test methods, routine monitoring, and alternative to batch testing; USP < 85 > Bacterial Endotoxin Test (F.D.A. n.d.).

Chemicals

All chemicals used are of pro analysis grade. Takeda Pharmaceutical (Japan) kindly provided lansoprazole (2-[[[3-methyl-4-(2,2,2 -trifluoroethoxy)-2-pyridyl]methyl]sulfinyl]-1H-benzimidazole) and AG2000 (4-methyl-3-(2,2,2-trifluoroethoxy)-5H-pyrido[1′,2′: 4, 5][1, 2, 4]thiadiazino [2, 3-a]benzimidazole-13-ium tetrafluoroborate) (Schmarda et al. 2000) (Fig. 1).

Lipopolysaccharides (LPSs)

LPSs isolated from Escherichia coli O111:B4 (B4) and Helicobacter pylori (Hp) were purchased from Wako Pure Chemical Industries (Tokyo, Japan). LPSs were suspended in 100% dimethyl sulfoxide (DMSO) (endotoxin-free) to prepare a 100 μg/mL stock solution, which was adjusted to various concentrations by dilution with HBSS (Ubagai et al. 2021).

Human blood samples

We obtained blood from 9 volunteer donors (6 men and 3 women; age 29 − 58 years: mean age, 39). Each donor donated blood more than once, and the mRNA expression levels of the 7 genes (one was the reference gene, ACTB) were averaged. Healthy individuals were confirmed, at the start of the study, through consent forms and interviews to be physically healthy, non-smokers who were not taking any medication were informed about the study’s purpose, and consent was obtained from all participants. The protocol was approved by the Ethical Review Committee at the Teikyo University School of Medicine (No. 07–104).

PMN preparation

Human PMNs were isolated from healthy volunteers’ peripheral blood (Ubagai et al. 2008). Briefly, 20 mL of heparinized whole blood was mixed with 4.5% dextran solution and allowed to stand for 40 min at room temperature. The leukocyte-rich plasma was centrifuged at 400 × g on an endotoxin-free Ficoll-Paque Plus gradient (Amersham Bioscience, WI, USA) for 20 min. Hypotonic (0.2%) saline was used to lyse erythrocytes, and osmolality was restored by hypertonic (1.6%) saline. PMNs were only used in experiments if the viability was 99% or more (as assessed by trypan blue exclusion) and the purity was at least 95% (as assessed by morphological analysis). PMNs were adjusted to a final concentration of 1 × 107 cells/mL in endotoxin-free HBSS.

PMN chemiluminescence assay

Human PMNs (5 × 105 cells/mL) were treated with several concentrations of LPS (final concentration, 0–1000 ng/mL), LPZ, or AG-2000 (final concentration, 0–25 µg/mL). Then, 20 µL of luminol solution (40 µg/mL) was added. The mixture was incubated at 37 °C for 60 min. The reaction mixture was stimulated with 10 µL fMLP (10−5 M), 20 µL zymosan A (500 µg/mL), or 5 µL PMA (500 ng/mL) for ROS production. Finally, the CL intensity was continuously measured for 10–20 min with a six-channel Biolumat LB 9505 device (Berthold, Wildbad, Germany).

PMN stimulation with LPS

LPS, resuspended in 100% DMSO (endotoxin-free), was inoculated into PMN suspension (5 × 106 cells/mL) at 10, 100, or 1000 ng/mL. Control PMNs (5 × 106 cells/mL) with serum (0.2%) that were unstimulated (LPS −) were also prepared, with a final DMSO concentration of 1%. The mixtures were incubated with controls for 10 and 30 min in an environment containing 5% CO2. Following stimulation, PMNs were collected and washed with cold endotoxin-free PBS.

RNA preparation

Human PMNs (5 × 106 cells/mL) were washed with 1 mL of cold endotoxin-free PBS. Total RNA was extracted using the RNeasy Plus Mini Kit (QIAGEN, Germany) following the manufacturer’s instructions. The quantity and quality of total RNA samples were determined using the Agilent 2100 Bioanalyzer (Agilent Technologies, Germany).

Complementary DNA synthesis

Total RNA was reverse-transcribed to cDNA using the SuperScript VILO cDNA synthesis kit (Invitrogen Life Technologies, CA, USA). Briefly, 1 µg of total RNA was incubated with 2.5 µM oligo (dT)20, 50 ng of random hexamers, and 200 U SuperScript III RT enzyme in a 20 µL reaction volume at 42 °C for 60 min, followed by 50 °C for 20 min. The reactions were terminated by heating the solution at 85 °C for 5 min.

Quantitative real-time PCR (qPCR) analysis

The mRNA expression levels of all genes were quantified in PMNs after LPS treatment for 10 and 30 min (Ubagai et al. 2009). In gene expression analysis, the control group was untreated PMNs, i.e., intact PMNs resuspended in endotoxin-free HBSS. The TLR gene expression levels (GenBank accession no. MN_018643.2) in PMNs were quantified using the StepOne Real-Time PCR System (Applied Biosystems, CA, USA). Complementary DNA was amplified with SYBR Green by using the Power SYBR Green PCR Master Mix (Invitrogen, CA, USA). Briefly, qPCR was performed for TRLs and the housekeeping gene ACTB (GenBank accession no. MN_001101.1). The PCR primer sets are described in Table 1. The following cDNA amplification program was used: 95 °C for 10 min and then 40 cycles of 95 °C for 15 s and 60 °C for 1 min. All PCR reactions were performed in 20 µL reaction volumes comprising the following components: 5 µL of cDNA solution, 0.9 U Ampli Taq Gold DNA polymerase, 1 × reaction buffer (20 mM Tris–HCl pH 8.4, 3 mM MgCl2, 200 µM dVTPs (mixture of dATP, dCTP, and dGTP), 400 µM dUTP, 500 nM ROX reference dye, and 0.6 U Uralic glycosylase), and 200 nM primers. TLR mRNA expression levels in PMNs were normalized to the ACTB gene expression levels. Fold changes of PMN TLR mRNA levels between LPS-stimulated samples and untreated controls were determined using the Sequence Detection System (SDS) software (Applied Biosystems). Briefly, in this study, we employed the absolute quantification method for qPCR analysis. The copy numbers of each target gene were divided by the copy number of the same gene in untreated PMNs. All target gene copy numbers were adjusted relative to the number of copies of the housekeeping gene ACTB.

Table 1 Primer sets for quantitative polymerase chain reactionStatistical analysis

All P values were determined by the nonparametric unpaired or paired t-tests (two-tailed) using Excel 2011 (Microsoft Corporation, Tokyo, Japan). We considered P < 0.05 to be significant, and the degree of significance was indicated as **P < 0.01.