The cases at the First Affiliated Hospital of Zhejiang University (Hangzhou, China) and the First and Fifth Affiliated Hospitals of Suzhou University (Suzhou, China) between February 2011 and December 2013 were retrospectively reviewed. This study was approved by the Ethics Committee of the The First Affiliated Hospital of Soochow University (Suzhou, China). One hundred and four HIV-1-infected individuals and 37 healthy donors were enrolled in this study. Consent of the blood donors or their guardians was obtained in a manner consistent with the policies of the appropriate local institutions. HIV-1 infection was confirmed by a positive immunoblot and acquired immune deficiency syndrome (AIDS) was diagnosed based on the CDC classification (26). Of the 104 HIV-1 positive patients, 82 met WHO criteria (27) for highly active anti-retroviral therapy (HAART) initiation and received a stable antiretroviral regimen. In total, 22 were seropositive, but did not meet WHO criteria for HAART initiation. Healthy control participants (n=37) were also recruited and were age-, gender-, and ethnicity-matched. A short medical history was obtained from all healthy control donors to ensure that they did not have an infectious disease in the past 3 months. Peripheral blood samples (5 ml) from healthy, HIV-negative individuals and HIV-1-positive patients were drawn into a syringe containing EDTA and stored at −80°C.

Whole blood was treated with the red blood cell lysis buffer to lyse the red blood cells, and then centrifuged at 1,500 × g for 5 min. The supernatant was discarded, and pellets were re-suspended in 200 μl phosphate-buffered saline. The resultant cells were incubated with mouse fluorescein isothiocyanate (FITC)-conjugated CD4 monoclonal antibody (cat. no. 6603850; 1:10; Beckman Coulter, Brea, CA, USA) at room temperature for 1 h, and analyzed using a flow cytometer.

Isolated lymphocytes from whole blood cells were stained with a PC5-conjugated CD4-directed monoclonal antibody (cat. no. A07752; 1:10; CD4-PC5; Beckman Coulter) and staining was analyzed on a FACS Calibur cell analyzer (Becton Dickinson, USA). Flow cytometry data were analyzed using WINMDI software version 2.8 (The Scripps Institute, San Diego, CA, USA).

The peripheral level of RON and MSP in blood samples was measured with a dual antibody switch enzyme-linked immunosorbent assay (ELISA) using the RON-directed mouse anti-Zt/G4 and 2F2 monoclonal antibodies (1:200; provided by Professor Wang, Texas Tech University Health Sciences Center, Amarillo, TX, USA) as described previously (28–31) and human MSP/MST1 α Chain MAb (Clone 45904), mouse IgG1 (R&D Systems, Inc., Minneapolis, MN, USA).

The JLTRG cell line was a gift from the National Institutes of Health, (Baltimore, MD, USA), and the H9/HTLV-IIIB (human T cell line infected with HIV III) cell line was purchased from the American Type Culture Collection (Mannassas, VA, USA). The HeLa, L02, MRC, 293T, Huvee, Wish and Sup T1 cell lines were provided by First Affiliated Hospital, Zhejiang University School of Medicine (Hangzhou, China). All cell lines were cultured in RPMI-1640 (Invitrogen, Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal calf serum (Gibco, Thermo Fisher Scientific, Inc.), 100 U/ml penicillin, 100 mg/ml streptomycin and 0.2 M L-glutamine at 37°C and 5% CO₂.

JLTRG cells (1×10⁶) were cultured in a 10 cm culture dish, and co-cultured with H9/HTLV-IIIB cells to achieve a ratio of 10:1. After 24, 48, 72 and 96 h, infection was assessed by fluorescence microscopy (Olympus IX81, Tokyo, Japan). RON, MSP and NF-κB content was assessed by western blotting, as described below.

JLTRG cells were cultured on slides (Lab-Tek Chamber Slide system, Thermo Fisher Scientific, Inc.) and fixed with 4% paraformaldehyde at 4°C for 20 min and incubated with RON directed monoclonal antibody (2F2; 1:200; provided by Professor Wang) at room temperature for 1 h. The cells were then further incubated with anti-mouse FITC-conjugated secondary antibody (cat. no. sc-3699; 1:10; Santa Cruz Biotechnology Inc., CA, USA) for 30 min. Fluorescence was observed and photographed by a fluorescence microscope (Olympus IX81).

Cells were incubated with lysis buffer (cat. no. 9803, Cell Signaling Technology Inc., Beverly, MA, USA) for 30 min at 4°C. Insoluble material was then removed by centrifugation at 8,000 x g for 20 min at 4°C, and the concentration of protein in each lysate was determined using a bicinchoninic protein assay kit (Pierce Biotechnology, Rockford, IL, USA) with bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO, USA) as the standard. Immunoprecipitation and western immunoblotting was conducted as previously described (29). In brief, 20 μg of protein lysate was mixed with 2X sodium dodecyl sulfate (SDS) loading buffer containing DTT and incubated at 100°C for 10 min before resolving by SDS-polyacrylamide gel electrophoresis. Proteins were transferred to a polyvinylidene difluoride membrane and blocked with 5% non-fat dry milk in phosphate-buffered saline (PBS) with 0.02% v/v Tween-20. The membrane was incubated with RON-directed monoclonal antibodies Zt/G4 and 2F2 and anti-p65 (cat. nos. ab16502; 1:1,000; Abcam, Cambridge, UK) or phospho-p65 polyclonal antibodies (cat. no ab86299; 1:2,000; Abcam), and antibody binding was detected with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (cat. no. ab85760; 1:500; Abcam). For detection of β-actin, membranes were stripped with 100 mM 2-ME, 62.5 mM Tris-HCl (pH 6.7) and 2% SDS for 30 min at 55°C, and re-probed with β-actin directed mouse antibody for 1 h at 25°C. Samples were washed four times with PBS and resuspended in 1 × SDS buffer [50 mM Tris-HCl (pH 6.8), 2% SDS, 0.1% bromphenol blue, 10% glycerol, and 100 mM DTT] Staining was detected with HRP-conjugated goat anti-mouse secondary antibody (cat. no. ab19195; 1:1,000; Abcam). Blots were scanned and analyzed using Image J version 1.45 software (National Institutes of Health, Bethesda, MD, USA) with protein band densities normalized to β-actin.

Quantitative data is expressed as the mean ± standard deviation. Single-factor analysis of variance and linear correlation analysis was conducted using SPSS 17.0 statistical software (SPSS Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

In total, 104 HIV-1-infected individuals and 37 age- and gender-matched healthy control participants were enrolled. The mean age of the patients was 47.2±11.3 (25–64) years. There were 62 male and 42 female HIV-1-infected patients; and 10 male and 18 female control participants. Expression levels of RON and MSP in the peripheral blood of HIV-1 positive patients receiving HAART (n=22), or not (n=82) and 37 healthy control participants were determined by ELISA. The level of RON in the peripheral blood was considerably higher in the plasma of HIV-1 positive patients undergoing HAART treatment (0.77±0.202 ng/μl) than in infected patients not undergoing HAART treatment (0.49±0.135 ng/μl) (P<0.01). The levels in these groups were higher than the level in the healthy control group (0.26±0.036 ng/μl, P<0.05) (Table I).

The MSP level was considerably lower in the plasma of HIV-1 positive patients undergoing HAART treatment (0.64±0.39 ng/μl) than in infected patients not undergoing HAART treatment (0.97±0.29 ng/μl) (P<0.05). The levels in these groups were lower than the levels in the healthy control group (2.08±1.49 ng/μl, P<0.05) (Table I).

This study aimed to investigate the expression of RON and MSP in a range of human cell lines, and found RON to be strongly expressed in only JLTRG cells (Fig. 1). JLTRG cells are a Jurkat T cell-based cell line, which express CD4 and CXCR4, and have been stably transfected with an LTR-green fluorescence protein (GFP) construct in order to report HIV-1 infection. These cells can be infected with X4-tropic HIV-1 and in the presence of HIV-1 tat, expression of enhanced GFP acts as a quantitative marker of HIV-1 infection (32) The subcellular location of RON in JLTRG cells was assessed by immunofluourescent staining, and found it to be localized to the cell membrane (Fig. 2).

To assess whether HIV-1 can affect the expression of RON and MSP JLTRG cells were infected by co-culture with H9/HTLV-IIIB cells (Fig. 3). The RON content of JLTRG cells cultured alone or co-cultured with H9/HTLV-IIIB cells was assessed by western blot analysis (Fig. 4). RON expression was demonstrated to be downregulated by HIV-1 infection in JLTRG cells. NF-κB content of JLTRG cells cultured alone or co-cultured with H9/HTLV-IIIB cells (10:1) was assessed by western blot analysis. HIV-1 infection of JLTRG cells also reduced NF-κB phosphorylation (Fig. 5).