Primary human monocyte isolation and HIV-1 infection
Monocytes were obtained from leukopheresis of HIV-1, 2 and hepatitis B seronegative donors and purified by countercurrent centrifugal elutriation. Cells were cultured with 10% heat-inactivated pooled human serum, 1% glutamine (Sigma Chemical Co., St. Louis, MO), 10 μg/ml ciprofloxacin (Sigma), and 1000 U/ml highly purified recombinant human MCSF (a generous gift from Genetics Institute, Inc., Cambridge, MA) in Dulbecco’s Modified Eagle’s Medium (DMEM). After 7 days, the MDM were infected with HIV-1ADA
(a macrophage tropic viral strain) at a multiplicity of infection (MOI) of 0.01 (40
). The ADA strain was used in these experiments as a result of prior and extensive analyses of macrophage function and neurotoxicity based on strain differences (12
). We found in these systems used that the levels of viral replication not the strains per se
govern the levels of neurotoxicity. Thus we used the laboratory adapted HIV-1 ADA strain as the levels of viral growth are uniform and not dependent on host cell differences. This ensured that the data acquired was reproducible from one experiment to another regardless of macrophage donor.
HIV reverse transcription assays
HIV replication was examined by measuring viral RT activity as previously described (41
). In these assays, 90,000 monocytes were cultured in 96-well plates for seven days, then infected with HIV-1ADA
at a MOI of 0.01 and washed 24 hr later to remove virus. Media was changed every other day. To estimate HIV-1 replication, RT activity was determined by incubating 10 mL of sample with a reaction mixture consisting of 0.05% Nonidet P-40 (Sigma) and [3
H]dTTP (2 Ci/mmol; Amersham, Arlington Heights, IL) in Tris-HCl buffer (pH 7.9) for 24 hr at 37°C on days 3, 5, 7 and 10. Radiolabeled nucleotides were precipitated on paper filters in an automatic cell harvester (Skatron, Sterling, VA), and incorporated activity was measured by liquid scintillation spectroscopy.
Murine cortical neurons (MCN) cultures
Cerebral cultures, containing neurons and glia in similar proportions to that found in the brain, were derived from the cerebral hemisphere of embryonic C57Bl/6 mice (Jackson Laboratories) on day 17 of gestation and cultured as described previously (42
) following dissociation in 0.027% trypsin. Neuron-enriched cells were resuspended in neurobasal medium (Invitrogen, Grand Island, NY) with heat-inactivated fetal calf serum supplemented with B-27, 500 μM glutamine, and 25 μM glutamate then seeded at a density of 2.8 × 105
on poly-d-lysine-coated 96 and 24-well plates.
Cell viability assays
Ninety thousand human MDM were cultured in 96-well plates and treated with 0, 80, 160 or 220 nM CEP-1347 24 hr before, at, or 4 hr after viral infection. Media was changed every other day and appropriate concentrations of CEP-1347 added. Cultures were monitored for 10 days. Cell viability assays were performed on days 1, 3, 5, 7 and 10 by measuring mitochondrial activity by reduction of tetrazolium salt MTT as described previously (43
MCN cytotoxicity assays
One million human MDM were cultured in 24-well plates and treated with/without CEP-1347 as outlined above, cultured for 5 days and then washed with PBS. Media was replaced with neural basal media (Invitrogen) every 24 hr. At that time, a ratio of 1:5 (supernatant:media) fluids were placed onto murine cortical neurons for 24 hrs to assess neurotoxicity by neuronal immunohistochemistry. For the neuronal antigen measurements, cells were blocked with 5% normal goat sera for 1 hr then incubated with antibodies to microtubule-associated protein 2 (MAP-2) (neural cell bodies, axons and dendrites, 1:1000) and neuron-specific nuclear protein (NeuN) (1:100) for 1 hr (Chemicon International, Inc., Temecula, CA). Cells were then washed three times with PBS and then incubated with fluorescent antibodies (1:1000 dilutions) to rabbit and mouse Alexa Fluor® 488 (green) and 594 (red) as a secondary antibody (Molecular Probes Inc., Eugene, OR) for 1 hr. Cells were washed twice with water, and water was allowed to cover the wells. Plates were read at 617 and 519 nM for MAP-2 and NeuN using a fluorescent plate reader, and the data were quantified (4 wells/group). Data were expresses as the changes in the mean of fluorescence intensity (MFI) in treated versus non-treated MCN. Lactate dehydrogenize (LDH) levels in neuronal fluids were determined by a cytotoxicity detection kit (Roche, Indianapolis, IN). In this case, MCN culture plates were read at 490 nM. Data were expressed as percent changes in MCN LDH-release amongst conditioned media treatments as compared to 1nM staurosporin (St)-treated cells (listed as 100%). For characterization of neuronal morphology double-immunofluorescence staining was carried out using Alexa Fluor® 488 (green) and 594 (red) as a secondary antibody (Molecular Probes Inc.). Confocal laser scanning imaging was used, which contains Argon, and Argon/Krypton lasers allowing for up to 4 Acousto-Optical Tunable Filter (AOTF) modulated excitation lines of 488 nm, 514 nm, 568 nm and 633 nm (Melles Griot, Prairie Technologies, Inc., Madison, WI). The lasers are connected to SweptField scanner head (Nikon Instruments, Melville, NY) attached with Cascade 512B back illuminated 12-bit CCD digital camera (Roper Scientific, Duluth, GA), which are connected to the side port of Nikon TE2000U Inverted Microscope with a high-resolution X-Y-Z-motorized stage.
Antibody array and flow cytometric analyseis of cytokines and chemokines
One million human MDM were cultured in 24-well plates and infected with HIV-1ADA at an MOI of 0.01 for 1 day, then washed with PBS. Infection was allowed to grow for 5 days. On day 5, cells were treated with 220 nM CEP-1347 for 45 min, washed with PBS, and phenol-red free and serum free media were added. Supernatants were harvested and assayed 24 hr later by Panomic antibody array 3.0 (Panomics, Inc., Fremont, CA) for human cytokines. Panomic antibody array 3.0 measured human cytokines: ApoI/Fas, Leptin, RANTES, ICAM-1, IL-2, IL-7, CTLA, MIP1α, TGFβ, VCAM-1, IL-3, IL-8, Eotaxin, MIP1β, IFNγ, VEGF, IL-4, IL-10, GM-CSF, MIP4, TNF-α, IL-1α, IL-5, IL-12(p40), EGF, MIP-5, TNFRI, IL-1β, IL-6, IL-15, IP-10, MMP3, TNFRII, IL-IRα, IL-6R, and IL-17. For the Cytometric Bead Array (CBA) flow cytometric of secreted chemokine analysis, one million human MDM were cultured in 24-well plates and infected with HIV-1ADA at an MOI of 0.01 for 1 day, then washed with PBS. At time of infection, cells were treated with 80, 160 or 220 nM CEP-1347. Half media exchanges were carried out on day 2 and 4 with respective drug concentrations. On days 3 and 5, 50 μL of supernatant was assayed for each group by CBA Human Inflammation Kit and Chemokine Kit (BD Biosciences, San Diego, CA). Human Inflammation and Chemokine Kit measures IL-8, IL-1β, IL-6, IL-10, TNF-α, IL-12p70 and IL-8, RANTES, MIG, MCP-1, and IP-10, respectively.
Western blot analysis
MDM were infected and cultured with CEP-1347 for 5 days then lysed using RIPA lysis buffer (Fischer Scientific, Pittsburgh, PA) containing additional protease and phosphatase inhibitors (Calbiochem, San Diego, CA). Protein concentrations of whole cell lysates were estimated using BSA assay and stored at −80°C. Cell lysates were allowed to thaw on ice then processed for Western by boiling with SDS laemmli buffer (Bio-Rad Laboratories, Hercules, CA). Whole cell lysate (40 μg protein) was loaded into each well of the Bio-Rad SDS gradient PAGE ranging from 4–15 % and run at 100 V for 1 hour 30 minutes. Protein was transferred to polyvinylidene fluoride (PVDF) membrane (Millipore Billerica, MA) using Bio-Rad semidry transfer apparatus at 25 V for 1 h. The membrane was then blocked with 5% BSA in TBS. Primary antibodies used were anti-phosphorylated-MLK3 (1:1000, Cell Signaling Technology, Inc., Danvers, MA), anti-MLK3 (1:1000, Cell Signaling Technology, Inc.), anti-JNK (1:1000, Cell Signaling), anti-phosphorylated-JNK (1:2000, Cell Signaling Technology, Inc.), anti-p38 (1:1000, Cell Signaling), anti-phosphorylated-p38 (1:1000, Cell Signaling Technology, Inc.), anti-phosphorylated- nuclear factor-kappa beta (NF-κB) p65 (Ser 468) (1:1000, Cell Signaling) anti-NF-κB p65 (1:1000, Cell Signaling), anti-phosphorylated-NF-κB p105 (Ser933) (1:1000, Cell Signaling), anti- NF-κB p105/50 (1:1000, Cell Signaling) and GAPDH (1:1000, Cell Signaling Technology, Inc.). Primary antibodies were detected with HRP-linked secondary antibodies, anti-mouse or anti-rabbit as per antibody used (1:20,000, Chemicon International) followed by detection by ECL femto-detection reagent (Pierce Biotechnology, Rockford, IL) and subsequent exposure to X-ray films for 5–30 min (for phosphoproteins up to 6h). Blots were quantified by inverting scanned images of the blots using the program ImageJ (Naitonal Institutes of Health, NIH) and measuring intensity. Readings were normalized to respective GAPDH expression levels and compared to uninfected, untreated MDM.
Five-week old male CB17/scid mice and C57Bl/6 mice were purchased from Charles River Laboratory, Wilmington, WA. Animals were administered i.p. injections either 1.5 or 15.0 mg/kg CEP-1347. The C57B1/6 mice received both drug dosages while the CB17/scid received only the lower 1.5 mg/kg dosage. Blood was extracted by cheek puncture at 0, 0.5, 1, 2, 4, 6 or 8 hr. Blood samples were centrifuged at 5000 rpm for 10 min at 4°C. Plasma was collected and stored at −20°C pending analysis. The plasma samples were prepared for bioanalysis by adding ten volumes of acetonitrile containing an internal standard (alprenolol). After the samples were vortexed and centrifuged, the supernatant was transferred to a 96-well plate for analysis by liquid chromatography/mass spectrometry. The amount of CEP-1347 in the sample was quantified using a plasma standard curve made via serial dilution in a concentration range from 10 to 5000 ng/mL.
SCID mouse model of HIVE
Four-week old male C.B-17/IcrCrl-SCIDbr (CB17/scid
) mice were purchased from Charles River Laboratory. Animals were maintained in sterile microisolator cages under pathogen-free conditions in the Laboratory of Animal Medicine at the University of Nebraska Medical Center in accordance with ethical guidelines for care of laboratory animals set forth by the National Institutes of Health. HIV-1ADA
-infected MDM (1.5 × 105
cells infected at an MOI of 0.1 in 5 μl) were stereotactically injected intracranially (i.c.) after 1 day of viral infection and referred to as HIVE mice (44
). The higher multiplicity used in the animal studies as compared to the in vitro experiments reflected the need to infect a larger proportion of cells prior to cell injections. CEP-1347 was then administered i.p. daily for 7 days at doses 0.5, 1.0, 1.5, 5.0 and 15.0 mg/kg/d (n
= 4 mice/treatment group). Vehicle only was the control. CB17/scid
mice received i.c. injections of media (sham-operated) and served as additional controls. Seventeen animals were included in each group. Animals were treated with vehicle or CEP-1347 starting 1day post-i.c. injection and for 7 days following MDM injections and CEP-1347 treatments.
Histopathology and image analysis
Brain tissue was collected at necropsy, fixed in 4% phosphate-buffered paraformaldehyde and embedded in paraffin. Paraffin blocks were cut until the injection site of the human MDM was identified. HIV-1 p24 antigen (clone Kal-1; Dako, Carpinteria, CA) was used to test for virus infected human MDM. For each mouse, 30–100 serial (5-μm-thick) sections were cut from the injection site and 3–7 sections (10 sections apart) analyzed. Antibodies to vimentin intermediate filaments (clone VIM 3B4; Boehringer Mannheim, Indianapolis, IN) were used for detection of human cells in mouse brains. Mouse microglia were detected by antibodies to Iba-1 (WAKO, Osaka, Japan), and astrocytes were detected by antibodies for glial fibrillary acidic protein (GFAP; Dako). NeuN, MAP-2 (both from Chemicon International Inc.) and heavy chain (200kDa) neurofilaments (Dako) were used for detection of neurons. Appropriate secondary antibodies and the Vectastain Elite ABC kit (Vector Laboratories, Berlingame, CA) were used to complete the immunohistochemical tests. All sections were counterstained with Mayer’s hematoxylin. The numbers of human MDM and HIV-1 p24 antigen-positive cells were counted with a Nikon Microphot-FXA microscope. All obtained images were imported into Image-Pro Plus, v. 4.0 (Media Cybernetics, Silver Spring, MD) for quantifying area (%) of GFAP, Iba-1, MAP-2 and NeuN positive staining.
Real time polymerase chain reaction (PCR)
The levels of HIV RNA in the injected hemisphere were determined by real time PCR using ABI 7000 prism (Perkin-Elmer, Applied Biosystems, Foster City, CA). Briefly, total RNA was extracted from the brain tissues. The HIV-1gag RNA
specific primers and probe were used as previously described (45
) and are: forward, 5′-ACA TCA AGC CAT GCA AAT -3′; reverse, 5′-ATC TGG CCT GGT GCA ATA GG -3′; and probe, 5′-CAT CAA TGA GGA AGC TGC AGA ATG GGA TAG A -3′. The reverse primer was used to make cDNA from RNA, which was further amplified using primers and probe at 50°C for 2 min., 95°C for 10 min., and 40 cycles at 95°C for 15 sec. and 60°C for 1 min. Separate GAPDH amplifications were used as an endogenous control to ensure that equal amounts of RNA were used. For GAPDH, Mac-1, TNF, GFAP and IL-10 Taqman gene expression assays were used (Applied Biosystems). Results were expressed as mean copy number ± SEM.
Data was analyzed using Excel (Macintosh, 1994) with Student t-test for comparisons. P<0.05 was designated statistically significant.