The study was designed to characterize monocytes, which were recovered from stimulated peripheral blood mononuclear cells (PBMC) from non-HIV-infected volunteers. In separate experiments, activated and non-activated monocytes were then placed in culture and exposed to virus.
Cells and Cell Cultures
The study was reviewed and approved by the University of Hawaii Institutional Review Board. Heparinized blood from two non-HIV-infected donors were processed as previously described with Dubeco’s phosphate buffered saline (PBS) and Ficoll-paque (Amersham Biosciences Inc, Piscataway, NJ) to isolate peripheral blood mononuclear cells (PBMC) [
6]. The cells were re-suspended in PBS, 2% fetal bovine serum (FBS) (Mediatech Inc., Herndon, VA); washed and re-suspended in RPMI (Mediatech Inc., Herndon, VA) supplemented with antibiotics (100IU/mL Penicillin, 100ng/mL Streptomycin) and 0.2% normal human serum. Priming of cells with IL-10 (10ng/mL rhIL-10) at 37°C, 5% CO
2 for 20 hours was performed as per a previously-established protocol because plasma IL-10 levels are elevated in HIV-infected patients [
7–
10]. The protocol was established from published work and in our laboratory provided consistent results with recovery of cells [
10]. The primed cells were resuspended in RPMI supplemented with antibiotics, 0.2% normal human serum and 1μg/mL indomethacin (Sigma Aldrich, St. Louis, Missouri), at 6 × 10
6 cells/mL; and incubated in Teflon flasks at 37°C, 5% CO
2 for 20 hours by stimulating with 20ng/mL
Escherichia coli LPS O111:B4 (Sigma Aldrich, St. Louis, Missouri) [
10].
Stimulated PBMC recovered from Ficoll centrifugation were suspended in 100μL PBS, 2% FBS, 1mM EDTA. Monocyte isolation was achieved using the Human Monocyte Enrichment Kit ‘without CD16 depletion’ (Stem Cell Technologies, Vancouver, Canada). Briefly 10μL of αCD32 blocking antibody was added to prevent non-specific binding to monocytes by blocking monocyte Fc receptors. The cells were kept at 4°C for 5 minutes; followed by the addition of 5μL of the antibody cocktail (CD2, CD3, CD19, CD20, CD56, CD66b, CD123, glycophorin A) and dextran; and kept at 4°C for 5 minutes. Magnetic beads were then added to the cells followed by PBS, 2% FBS, 1mM EDTA buffer; incubated at 4°C; and the cells were magnetically separated. The unbound monocytes were poured off; and the cells re-suspended in 2.5mL PBS, 2% FBS, 1mM EDTA. The recovered activated monocytes (CD14
+/CD16
+ phenotype) were washed twice with 2% FBS/PBS. An aliquot of the activated monocytes was analyzed by flow cytometry. The remaining cells were used for the virus culture experiments described below. Non-activated monocytes were obtained from PBMC isolated from fresh whole blood from the same donors by Ficoll centrifugation followed by Easysep Monocyte Enrichment Kit ‘with CD16 depletion’ as described above (Stem Cell Technologies, Vancouver, Canada). The antibody cocktail consisted of CD2, CD3, CD19, CD20, CD56, CD66b, CD123, glycophorin A; and anti-CD16 to deplete CD16
+ monocytes. An aliquot of the non-activated monocytes were stained and analyzed by flow cytometry; with the remainder of the non-activated monocytes placed in culture for the virus experiments. As described by Adib-Conquy et al, similar experiments were set up by stimulating whole blood and monocytes to determine if differences in stimulating in the presence of other cells is noted [
10].
Cell Staining and Phenotypic Characterization
Monocytes, both activated and non-activated, were washed twice and re-suspended in 100μL PBS, 2% FBS. Fluorochrome-conjugated antibodies (anti-CD14-FITC, anti-CD16-Alexa 674, anti-CCR5-APC Cys7, and anti-CD69-Cy7; BD Biosciences Pharmingen, San Diego, CA.) were added as per manufacturer’s specifications. The cells were incubated for 20 minutes at room temperature; washed with PBS 2% FBS twice and then fixed in 200μL 1% Para formaldehyde, 2% FBS; and analyzed using FACSAria (BD Biosciences, San Jose, CA) and FACSDiva software (BD Biosciences, San Jose, CA).
Intracellular staining for TNF-α was carried out following LPS stimulation by adding brefeldin A (Sigma Aldrich, St. Louis, Missouri) to the cells three hours before harvesting. The cells were washed twice in PBS, 2% FBS and stained for cell surface markers as above. The cells were then permeabilized with 200μL fixation and permeabilization buffer (BD Biosciences, San Jose, CA); and incubated for 20 minutes. After permeabilization, the cells were washed with the wash buffer; centrifuged; re-suspended in 100μL PBS, 2% FBS with anti-TNF-α-PE; and incubated for 20 minutes at room temperature. The cells were washed with PBS, 2% FBS ; fixed in PBS, 1% Para formaldehyde, 2% FBS; and analyzed as noted above.
HIV-1 Exposure on Activated Monocytes and Non-Activated Monocytes
Monocytes (activated and non-activated monocytes) were isolated and purified from fresh whole blood from the same donors noted above using Ficoll and magnetic bead selection as outlined above. To expose cells to virus, activated and non-activated monocytes were placed separately in 96-well plates (5×105 cells/well) with the addition of 2ng p24 units of LAI (X4-tropic strain; NIH AIDS Research and Reference Reagent Program, Bethesda, MD), BaL (R5-tropic strain; NIH AIDS Research and Reference Reagent Program, Bethesda, MD); or p89.6 (dual-X4/R5-tropic strain) HIV-1 strains for 1 hour in triplicate. Following three washes, the cells and virus were lysed; and RNA isolated. The final supernatants from the third washes were recovered to verify that no virus was present in the last wash. The supernatants were also assessed for HIV p24 (XpressBio, Thurmont, MD) to verify that no active virus was present that could suggest that the cells were infected.
The amount of virus that was recovered from the remaining cells was assayed from the isolated RNA by RT-PCR using HIV Gag and β-actin primers with appropriate positive and negative control RNA. Amplified fragments were resolved on 2.5% agarose gels and analyzed by densitometry. From the scanned gels, the ratio of HIV gag light units/β-actin light units was compared between the activated and non-activated monocytes and between the viruses.
Statistical Analyses
Results from the experiments carried out in triplicate were expressed as percentage of cells stained for the selected marker before and after stimulation; and the data were analyzed for statistical significance using the Student’s t test for paired values. Differences were considered significant at p < 0.05. Experiments, carried out in triplicate, showing differences in the amount of virus detected between cell groups were considered significant by Student’s t-test if p<0.05.
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