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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 6 (March 15), 1998:
pp. 2108-2117
Adenovirus-Mediated Human Immunodeficiency Virus-1 Nef Expression
in Human Monocytes/Macrophages and Effect of Nef on Downmodulation
of Fc Receptors and Expression of Monokines
By
Swapan K. De,
Chettemgere N.S. Venkateshan,
Prem Seth,
D. Carleton
Gajdusek, and
Clarence J. Gibbs Jr
From the Oral Infection and Immunity Branch, National Institute of
Dental Research, the Laboratory of Central Nervous System Studies,
National Institute of Neurological Disorders and Stroke, and the
Division of Cancer Treatment, Medicine Branch, National Cancer
Institute, National Institutes of Health, Bethesda, MD.
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ABSTRACT |
To characterize the effect of human immunodeficiency virus-1 (HIV-1)
nef expression in human monocytes/macrophage (HMØ) and U937 on
the levels of Fc Rs, HLA antigens, and monokines, elutriated HMØs
and U937 cells were transfected with an adenovirus-mediated Nef
expression system. Nef-expressing cells downmodulated FcRI, FcRII, and upregulated HLA class I molecules. Nef-expressing HMØs,
treated with lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA), overexpressed tumor necrosis factor- (TNF-), interleukin-1 (IL-1), and IL-10. However, IL-6 was induced by LPS
and inhibited by PMA. Additionally, a subpopulation of Nef-expressing HMØs underwent apoptosis. Our data suggest that HIV-1 nef
downmodulated FcRs in myeloid cells in a manner similar to that
previously reported for its effect on CD4+ in T cells.
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INTRODUCTION |
THE VIRAL GENE, nef, encodes a
27- to 34-kD protein and is present only in the human immunodeficiency
virus (HIV) and in primate lentiviruses, simian immunodeficiency virus
(SIV).1-3 Although the precise function(s) of this gene is
not fully understood, it is known that Nef is required to maintain high
viral loads during persistent SIV infection and disease in
macaques.4 HIV-1 nef downregulates cell surface CD4
and blocks interleukin-2 (IL-2) gene induction in T cells as well as in
monocytic cell lines.5-12 It has been shown that nef
perturbs cell activation pathway(s), thereby influencing viral
replication in the host.13,14 HIV-infected human
monocytes/macrophages (HMØs) play an important role in the pathogenesis of disease by acting as a persistent virus
reservoir.15,16 HMØs express both CD4 and Fc receptors
(FcRs) for IgG.17 It is not known how FcR responds to
HIV gene products in HMØs. Virus-infected HMØs secrete
immunomodulators, inflammatory monokines, neurotoxins, and
neuroexcitotoxins.18-21 Cytokines/monokines generated
during the immune response regulate both immune function and viral gene expression, thereby affecting the progression to acquired
immunodeficiency syndrome (AIDS).16,22 Tumor necrosis
factor- (TNF-), interleukin-1 (IL-1), IL-6, and IL-10 can
either enhance or suppress HIV replication during the virus life
cycle.16,23-31 Overexpression of Nef during early HIV
infection promotes active virus replication and has been suggested to
increase monokine/cytokine production.4,18 However, the Nef
gene by itself has not been shown to modulate monokines directly.
Our preliminary studies have suggested that Nef expression alters
myeloid cells surface receptors.32 In the present study, we
determined whether nef expression modulates expression of
FcRI, II, and III; HLA class I and II; CD4; IL-2R molecules; and the monokines TNF-, IL-1, IL-6, and IL-10 in dividing and
differentiated primary HMØs and U937 cells. Because several studies
have shown the potential involvement of apoptotic cell death during HIV
infection,33,34 we also examined Nef-expressing HMØs for
apoptosis. To study the expression of cell surface molecules, monokines
and apoptosis, we selected adenovirus (Ad)-mediated transfection and
retrovirus-mediated transduction,35-37 because transfection
of DNA by chemical or electroporation methods were found inefficient in
macrophages.38 To increase Nef expression, we used a
replication-deficient adenovirus that had previously been shown to
enhance the delivery of cointernalized macromolecules into the
cells.37,39-41 Because HMØs express high numbers of
transferrin receptors,42-44 we conjugated nef
plasmid DNA with transferrin and used this conjugate together with
an adenovirus mutant dl312 to transfect HMØs. The present results show
that Nef expression in HMØs and U937 cells modulated cell-surface expression of FcRI and II, CD4, and HLA class I molecules.
Activation of Nef-expressing HMØs with phorbol 12-myristate 13-acetate
(PMA) or lipopolysaccharide (LPS) altered the levels of TNF-,
IL-1, IL-10, and IL-6. Furthermore, for the first time, we have
shown that HIV Nef mediates apoptosis in a subpopulation of HMØs.
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MATERIALS AND METHODS |
Human monocytes/macrophages.
Monocytes (95% to 98% pure) were prepared from peripheral blood
mononuclear cells (PBMCs) of HIV-1-seronegative donors by centrifugal
elutriation45,46; purified cells were
cultured.21
U937 cells.
The human histiocytic lymphoma cell line U937,47 expressing
many monocytic characteristics including FcRs obtained from ATCC
(Rockville, MD), was propagated in RPMI 1640 containing 10% fetal
bovine serum (FBS), penicillin, streptomycin, and glutamine (Inovar
Biologicals, Gaithersburg, MD).
PA317 cells.
Mouse embryo fibroblasts, derived from NIH/3T3 cells,48
were purchased from ATCC. Transfection of retrovirus vectors into these
cells resulted in production of virions able to infect human cells.
PA317 cells were used to prepare nef- or fen-expressing retrovirus stocks. Cell culture conditions were the same as for the
U937 cells.
293 cells.
Primary human embryonic kidney cells transformed by human adenovirus
type 5 DNA49 were obtained from ATCC and cultured in
Eagle's minimum essential medium (MEM) with Earle's
buffered saline solution (BSS), 10% FBS, and antibiotics
to prepare an adenovirus stock of dl312.
Proliferation assay of HMØs.
HMØs were cultured and maintained in macrophage medium with
L-glutamine (GIBCO-BRL, Life Technologies, Gaithersburg, MD) with 5%
filtered FBS (Intergen, Purchase, NY) and gentamicin sulfate (GIBCO-BRL) at 37°C, 5% CO2. Twenty-six thousand cells
were plated in each well of a 96-well flat-bottom culture plate
(Costar, Cambridge, MA) in the presence or absence of 5 ng/mL IL-3 (R & D System, Minneapolis, MN) and different concentrations (1 to 20 ng/mL; Fig 1) of granulocyte-macrophage
colony-stimulating factor (GM-CSF; R & D System). Cell proliferation
assay was performed as previously described.35 In brief, at
different days (1, 2, 4, and 6), 0.1 µmol/L or 2.5 µCi/mL of
[3H] thymidine (ICN Pharmaceuticals Inc, Costa Mesa, CA)
was added and the cells were harvested after 6 hours. The cells were
dissociated in an enzyme-free EDTA solution (Speciality Media Inc,
Lavallette, NJ) before harvesting on a filtermat paper (Skatron Inc,
Sterling, VA). Filters were then counted in a Beckman LS 5800 liquid
scintillation counter (Beckman Instrument Inc, Fullerton,
CA.
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| Fig 1.
Dose-response effect of IL-3 and GM-CSF on DNA synthesis
in freshly isolated primary HMØs. Purified MØs were cultured in
serum-free macrophage medium alone as a control or in the presence of 5 ng/mL IL-3 and at concentrations of 1, 10, or 20 ng/mL GM-CSF, as
indicated. Cells (2.6 × 104) were seeded in each well of
a 96-well plate. On different days, as shown in the figure, cells were
pulsed with 0.1 µmol/L or 2.5 µCi/mL [3H] thymidine
and harvested after 6 hours, and counts were taken. The data presented
here are the average of counts from 6 individual wells ± standard
error.
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Nef, fen, and -gal retrovirus vectors.
A Moloney murine leukemia virus-based retrovirus vector encoding
nef or fen (antisense) with neo-R gene, LnefSN,
and LfenSN from HIV-1 SF-2 isolate, nucleotide position 8504 to
9573,36 was used to transduce HMØs and U937 cells. The
fragment contained the entire nef coding region as well as 289 bp of 5 and 140 bp of 3 noncoding sequences. Virus made
by PA317 amphotropic retrovirus packaging cells48 was
stored at  20°C and used to transduce cells. Retrovirus
vector containing the -gal gene50 (kindly provided by M.V. Eiden, National Institute of Mental
Health, National Institutes of Health, Bethesda, MD) was
used to transduce HMØs.
Retrovirus-mediated transduction of nef, fen, and
-gal into HMØs.
Purified HMØs were transduced with a retroviral vector containing
nef or fen, nef, and -gal together using two
separate retrovirus vectors. Briefly, HMØs, 1 to 2 × 107 cells/flask (Costar) were cultured in macrophage media
plus 5% FBS and 5 ng/mL IL-3 in the presence or absence of 20 ng/mL
GM-CSF for 4 days (Fig 1). On day 4, polybrene (5 µg/mL; Sigma
Chemical Co, St Louis, MO) was added to the cells to assist gene
transfer51 in the presence of 1 mL (1 × 105 PFU) of tissue culture supernatant containing the
nef retroviral vector. Cells were cultured for 48 hours;
transduced cells were used for -gal staining, apoptosis, Nef
immunoprecipitation, fluorescence-activated cell sorting (FACS), and
cytokine analysis.
Transduction of nef and fen into U937.
U937 cells (1 × 105/well) were plated into each well
of a 6-well flat-bottom plate (Costar) and cultured as described above. The cells were then transduced with nef or fen
containing 1 mL of tissue culture supernatant of retrovirus in the
presence of polybrene.7 Cells and virus were incubated for
48 hours. Media containing G418 (2 mg/mL; GIBCO-BRL) were added to the
culture. Nef transduction-positive cells were resistant to G418
and were selected after 10 days in culture7 for
immunoprecipitation of Nef, turnover of cytoplasmic FcRs, and
analysis of FcRs and other cell surface antigens.
Selection of nef-transduction-positive cells.
U937 cells and HMØs were treated with G418 after retrovirus
transduction to select the nef-positive cells. U937 cells were selected under the above-described conditions; however, G418 was toxic
to HMØs. Therefore, nef-transduced HMØs in the absence of G418 were used. To determine the transduction efficiency, HMØs were
also transduced with both nef and -gal genes; they
were then stained49 for -gal, and the blue cells were
quantified.
Adenovirus vector system.
A replication-deficient mutant of AD, dl312 (kindly provided by T. Shenk, Princeton University, Princeton, NJ), was used.52 AD, dl312 was propagated in 293 cells49 and purified by
CsCl2 double-density centrifugation.39 Titrated
virus stock was stored in Tris-Cl buffer, pH 7.5, containing 20%
glycerol and stored at 70°C. AD, dl312 was used with HIV-1
nef plasmid for efficient protein expression in HMØs.
Conjugation of human apotransferrin with poly(L-lysine).
Human apotransferrin (Sigma) was conjugated with poly(L-lysine) (Sigma)
using a previously published procedure.53,54 In brief, 100 mg of apotransferrin was mixed with 10 mg sodium periodate (Sigma) at
4°C for 1 hour. Excess periodate was removed by Sephadex G-25; pH
of the protein solution was adjusted to 7.5 by sodium bicarbonate.
Apotransferrin was incubated with 10 mg poly(L-lysine) and 10 mg
cyanocarbohydride (Sigma). Conjugated apotransferrin was then separated
from unconjugated apotransferrin with a MonoS column (Pharmacia
Biotech, Piscataway, NJ) using a salt gradient (0.7 to 2.5 mol/L NaCl).
Conjugated protein was dialyzed against 20 mmol/L
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer and
stored at 70°C until used.
Coupling of apotransferrin-poly(L-lysine) with nef plasmid
DNA.
Apotransferrin (500 µg) was incubated with 5 mmol/L ferric citrate
(Sigma) solution for 5 minutes at room temperature, followed by the
addition of 100 µg of nef plasmid DNA for 4 hours.
Adenovirus and lipofectamine-mediated nef transfection into
HMØs.
To express HIV-1 Nef protein, a plasmid containing a 1.0-kb nef
gene fragment of HIV-1 SF2 clone (kindly provided by J. A. Levy,
University of California, San Francisco, CA) was used. HMØs were
transfected either with HIV-1 nef or HIV-1 fen plasmid
alone. Adenovirus- (Ad-) and lipofectamine-mediated HIV-1 nef
or HIV-1 fen were also transfected into the HMØs using a
recently described transfection protocol.37 In brief, 5 × 106 cells were cultured in 8 mL of macrophage media
with 5% FBS, IL-3 (5 ng/mL), and GM-CSF (20 ng/mL) in
25-cm2 tissue culture flasks (Costar) and incubated for 4 days at 37°C, 5% CO2 until transfection.
Nef or fen plasmid DNA conjugated to transferrin-poly(L-lysine) at 20 µg DNA/flask was mixed
with lipofectamine (20 µg/flask) in a total volume of 200 µL. The
mixture was added to the HMØs. After 10 minutes, 100 µL of AD, dl312
(100 PFU/cell) were added to the experimental flasks. Cells were
incubated at 37°C in 5% CO2 for 4 hours. Cells were
washed; 5 mL of fresh macrophage media containing IL-3, GM-CSF, and 5%
FBS with antibiotics was added. Flasks were incubated for an additional
24 hours. Cells from these flasks were used for the analysis of
-gal, Nef protein, FcRs, and other cell surface antigens.
Assessment of nef-transduced/transfected HMØ survival.
HMØs and U937 cells, transduced with retrovirus vector containing
nef and fen, were assessed for toxicity by
phase-contrast microscopy and trypan blue exclusion.55
Similarly, Ad-mediated nef-transfected HMØs were also examined
for signs of toxicity.
Determination of apoptosis in nef-transduced/transfected
HMØs.
Ad-mediated nef-transfected and retrovirus-transduced HMØs
were examined for apoptosis by an in situ nick/end-labeling technique using an in situ apoptosis detection kit; cells on the slides were
stained by peroxidase (ApopTag plus; Oncor, Gaithersburg, MD; catalogue
no. S7101-kit).
Assesment of -gal-expressing HMØs.
Nef-transduced/transfected HMØs with -gal and
nef were scraped and collected from the flasks. The cells were
centrifuged at 1,100 rpm for 10 minutes, and the pellet was resuspended
in phosphate-buffered saline (PBS). After two washes in PBS, the cells
were resuspended at a concentration of 106 cells/mL. The
cells were placed (100 µL/well) in a poly(L-lysine)-coated 96-well
plate and kept at room temperature for 30 minutes. Cells were fixed for
2 minutes with a cold mixture containing 0.8% formalin (Mallinckrodt
Chemicals Inc, Paris, KY) and 0.5% glutaraldehyde (Sigma) in PBS.
Cells were then washed twice with 0.15 mol/L Tris-HCl buffer, pH 7.8, and dried for 2 to 3 minutes. A freshly prepared staining solution
containing 5 mmol/L potassium ferricyanide, 5 mmol/L potassium
ferrocyanide (Aldrich Chemical Co, Milwaukee, WI), 2 mmol/L
MgCl2, and 1 mg/mL X-gal (GIBCO-BRL) was filtered and added
to the well and incubated at 37°C without CO2 for 2 hours; blue cells were quantified.
Analysis of steady-state Nef protein levels in
retrovirus-/Ad-mediated nef-transduced/transfected HMØs.
To analyze Nef protein in the transduced or transfected cells,
immunoprecipitation methods were performed as described.35 In brief, the cells were starved for 1 hour in cysteine- and
methionine-free RPMI 1640 and then labeled with 200 µCi/mL and 1,000 Ci/mmol of 35 S-cysteine and 35 S-methionine
(ICN Pharmaceuticals), respectively, for 4 hours. Cells were lysed in
buffer containing 50 mmol/L Tris-HCl, pH 7.5, 0.1% sodium dodecyl
sulfate (SDS), 300 mmol/L NaCl, 0.5% deoxycholate, 1.0% Triton X-100,
1 mg/mL leupeptin, 1 mg/mL aprotinin, and 50 mg/mL phenylmethylsulfonyl
fluoride (PMSF; Sigma). Cellular extracts were immunoprecipitated
overnight with polyclonal rabbit anti-Nef antisera (NIH AIDS and
Research Reagent Program, Rockville, MD) and fractionated on a reducing
10% SDS-polyacrylamide gel. The gels were dried and exposed to x-ray
film.
Flow cytometric analysis of FcRs, HLA, CD4, and IL-2R molecules
in Ad-mediated nef-transfected HMØs and U937 cells.
Flow cytometric analysis was performed on a Becton Dickinson FACScan
using Consort 30, and Lysis II software as previously described (Becton
Dickinson, Mountain View, CA).10 Ten million Ad-mediated fen- or nef-transfected HMØ/U937 cells
were washed twice with modified Hank's buffer (GIBCO-BRL) containing
0.1% bovine serum albumin, 0.1% sodium azide, and 25 mmol/L HEPES, pH
7.2, without phenol red, and placed on ice. Washed cells were then
filtered through a nylon monofilament cloth (53 µ opening; Small
Parts Inc, Miami, FL); filtered cells were counted. Cells (5 × 105) were taken in 0.1 mL of Hank's buffer for each cell
surface antigen analysis. Ten microliters of a 10 mg/mL solution of
nonfluorescent human IgG (Sigma Immunochemicals, St Louis, MO) was
added to all the tubes except those containing FcRs to minimize
nonspecific binding. Human -globulin (Pierce, Rockford, IL; 20 µL
of stock concentration [12 mg/mL]) was added to the FcRs tubes to
block Fc region-specific binding of monoclonal antibody according to the manufacturer's protocol (Medarex Inc, West Lebanon, NH). The following antihuman antibody-fluorescein conjugates were used: antihuman CD4, antihuman CD14, and antihuman HLA-ABC (Olympus Corporation Immunochemicals, Lake Success, NY); antihuman HLA-DR (Beckton Dickinson Immunocytometry System, San Jose, CA); antihuman IL-2R (T Cell Diagnostics, Cambridge, MA); and antihuman FcRI, II,
and III (Medarex, Inc). After the addition of IgG and -globulin, antibody-fluorescein conjugates were added according to the
manufacturer's protocol and kept on ice for 30 minutes. The cells were
then washed with 2 mL of Hank's buffer, centrifuged, and resuspended
in 1 mL of the same buffer; 5 mg/mL of propidium iodide was added and the cells were analyzed for FcRI, II, and III; HLA-ABC; HLA-DR; CD4;
CD14; and IL-2R molecules. A total of 10,000 cells was analyzed; living
cells were gated, and the relative fluorescence intensity was measured
and plotted for each of the above-mentioned cell surface antigens. The
effect of Nef expression on cell surface antigens in U937 cells and
HMØs was compared.
Turnover of FcRI and II in Nef-expressing U937 cells.
Turnover of cytoplasmic FcRs was analyzed as previously described
for CD4.36 In brief, 1 × 106
Nef-expressing exponentially growing U937 cells were starved for 30 minutes in cysteine- and methioneine-free RPMI 1640 and then
pulse-labeled for 30 minutes with Trans35S-label (ICN
Pharmaceuticals) at 0.2 mCi/mL. Pulse-labeled cells were washed,
aliquoted, and chased for 0, 4, 12, 20, and 36 hours, incubating them
in complete growth medium. At designated times, aliqouts were washed in
cold PBS, pH 7.4. Cytoplasmic proteins were extracted in RIPA buffer
consisting of PBS containing 0.1% SDS, 0.5% deoxycholate, 1% Triton
X-100, 1 µg/mL leupeptin, 1 µg/mL aprotinin, and 1 mmol/L PMSF.
Radiolabeled FcRs were immunoprecipitated using protein
A-Sepharose CL-4B (GIBCO-BRL). In brief, cell lysates were
incubated with protein-A for 30 minutes and centrifuged, and the
supernatants were incubated with 1 µg of antihuman FcRI or
FcRII antibodies (Medarex, Inc) at 4°C for 2 hours. The immune complexes were washed in RIPA buffer and analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. The radioactivity incorporated into protein is quantified by exposing the dried gel to a
storage phosphor screen and scanning the screen with a phosphor imaging
instrument (Molecular Dynamics, Sunnyvale, CA).
Analysis of TNF-, IL-1, IL-6, and IL-10 by enzyme-linked
immunosorbent assay (ELISA) in Ad-mediated nef-transfected
HMØs.
HMØs were cultured for 4 days and nef or fen
transfection was performed in an Ad expression system as described
above. Two days after transfection, cells were washed and media were
changed to remove growth factors and transferrin. Fresh media
containing 5% FBS were replaced and treated with LPS (100 ng/mL;
Sigma) or PMA (1 or 20 ng/mL; Calbiochem, San Diego, CA) for 72 hours.
The tissue culture fluid was centrifuged at 1,100 rpm for 10 minutes, and the supernatant was assayed for TNF-, IL-1, IL-6, and IL-10 by ELISA. An enzyme immunoassay kit (Endogen Inc, Cambridge, MA) to
quantitatively determine cytokines was used according to the manufacturer's protocol.
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RESULTS |
Purified HMØs treated with IL-3 and GM-CSF proliferated on days 1, 2, 4, and 6 (Fig 1). This proliferation was dose-dependent and was maximal
on day 4 of culture in the presence of 5 ng/mL IL-3 and 20 ng/mL
GM-CSF.
Retrovirus-mediated transduction produced very low levels of Nef
expression. The adenovirus-enhanced delivery of the nef plasmid conjugated with transferrin resulted in expression of Nef protein (Fig 2), allowing us to
investigate Nef activity in HMØs. Only 1% to 2% of the cells were
positive for -gal expression in retrovirus-mediated nef gene
transfer in HMØs. However, Nef protein could not be immunoprecipitated from such low levels of transduction in HMØs. Retrovirus
vector-mediated Nef-expressing U937 cells (Figs 3 and
4) were selected with G418 treatment, and
Nef protein was precipitable in the selected U937 cells (data not
shown). Ad-mediated transfection of HMØs yielded better expression of
the Nef and -gal genes (Figs 2 and 5b) than retrovirus
vector-mediated gene expression (data not shown). About 10% of
Ad-mediated transfected HMØs were -gal positive; these cells were
identified as intense or light blue by histochemical analysis (Fig
5b) Ad-mediated transfected
cells were analyzed for Nef protein by immunoprecipitation (Fig 2).
HMØs transfected with nef, in the presence of AD, dl312 and
GM-CSF had the highest Nef expression (Fig 2, lane 1). The absence of
GM-CSF markedly reduced Nef expression (Fig 2, lane 2). The absence of
Ad-d1312 from the transfection resulted in minimum Nef expression in
the presence or absence of GM-CSF (Fig 2, lane 3 and 4).
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| Fig 2.
Detection of HIV-1 Nef expression in HMØs
after Ad-d1312-mediated transfection of nef cDNA. HMØs were
transfected either with nef cDNA alone or cDNA conjugated with
transferrin-poly(L-lysine) in the presence of a replication-deficient
AD, dl312. HMØs were cultured in macrophage media in the presence of
IL-3 (5 ng/mL) and GM-CSF (20 ng/mL). After 4 days in culture, cells
were transfected and cultured in macrophage media containing FBS, IL-3,
with/without GM-CSF for 24 hours. The cells were then labeled with 200 µCi/mL of 35 S-labeled cysteine/methionine for 4 hours
after starving them with cysteine/methionine-free media for 1 hour.
Cells were then harvested, lysed, and immunoprecipitated with Nef
antibody followed by SDS-PAGE and autoradiography. HMØs transfected
with nef, AD, dl312, and lipofectamine in the presence of
GM-CSF had the highest levels of Nef expression (lane 1). The absence
of GM-CSF markedly reduced Nef expression (lane 2). The absence of Ad
from the transfection resulted in minimal Nef expression in the
presence of GM-CSF (lane 3) or in the absence of GM-CSF (lane 4).
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| Fig 3.
Cell surface expression of human FcRI, II, and III;
HLA class I and II; CD4; CD14; and IL-2R molecules in U937 cells
expressing HIV-1 Nef or HIV-1 fen. U937 cells were
transduced with retroviral vector containing nef or fen
and selected after treatment with G418. Selected cells expressing Nef
and Fen were cultured and analyzed for cell surface molecules as
described in Fig 6. Downregulation of FcRI and II and CD4 and
upregulation of HLA class I molecules and no change in HLA class II,
CD14, and IL-2R were observed in the Nef-expressing population of U937
cells.
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| Fig 4.
Determination of FcRI and II turnover in retrovirus
vector-mediated Nef and Fen-expressing U937 cells. Cells were starved for 1 hour and pulse-labeled with 35S-labeled
cysteine/methionine for 30 minutes; cytoplasmic extracts were prepared
at the indicated time points and analyzed by immunoprecipitation with
anti-FcRI and II antibodies. As shown, Nef-expressing U937 cells had
significantly lower half-lives of FcRI and II than those of the
fen-transduced U937 cells.
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| Fig 5.
Analysis of the -gal-positive cells after transfection
of HMØs with -gal and nef cDNA conjugated with
transferrin-poly (L-lysine). A replication-deficient AD,
dl312 was added to mediate HIV-1 nef and -gal transfection.
The transfected MØs were washed and resuspended in PBS; the
resuspended cells were then placed in a poly(L-lysine)-coated 96-well
plate, fixed, and stained for -gal. The experiment was repeated four
times and a representative field is shown. (a) shows the nontransfected
HMØs that are negative in -gal staining. (b) shows HMØs
transfected with AD, dl312 and the -gal plasmid. Fields were chosen
to show distinct -gal staining. Positive cells are blue and are
shown with white arrows. A dividing MØ with positive -gal staining
is indicated with a red arrow. Microscopic examination of apoptotic
cells identified by condensation of cytoplasm and chromatin in HMØs
transfected with HIV-1 nef (c and d). MØs were cultured on
slide chambers after transfection; cells were fixed in 10%
neutral-buffered formalin and stained with the ApopTag peroxidase kit.
A red arrow shows unstained apoptotic cells with blebbing (c). A single
stained apoptotic cell (red arrow) and many unstained nonapoptotic
cells (white arrows) are shown in (d).
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Initial examination of the HMØs by trypan blue exclusion showed 6% to
10% more cell death in nef-transfected/-transduced cells than
in control cells. This observation led us to investigate the
possibility that Nef expression results in apoptotic cell death. Our
studies using in situ nick-end labeling show that the Nef-expressing
population of HMØs contained more apoptotic cells (Fig 5d) than did
their normal counterparts. We observed Nef-mediated apoptosis in about
2% to 5% of HMØs.
To investigate whether expression of cell surface molecules were
modulated by Nef, Ad-d1312-mediated nef- and
fen-transfected HMØs were analyzed by flow cytometry. We found
that FcRI and FcRII were downmodulated in the Nef-expressing
primary HMØs (Fig 6). Downmodulation of
FcRIII was minimal in HMØs (Fig 6). Retrovirus-mediated transduction downregulated FcRI and II in U937 cells. We could not
detect FcRIII in the U937 cells (Fig 3); CD4 was also downregulated in U937 cells and minimally downregulated in HMØs. HLA-DR expression on the cell surface was unchanged in both HMØs and U937 cells (Figs 3
and 6). Among the other cell surface markers examined, HLA class I
molecules were upregulated in both Nef-expressing HMØs and U937 cells
(Figs 3 and 6). Nef-mediated downmodulation of cell surface FcRs and
CD4 on HMØs is specific, because other molecules, such as IL-2R, HLA
II, and CD14, had no effect on the nef-transfected HMØs and
Nef-expressing U937 cells (Figs 3 and 6).
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| Fig 6.
Cell surface expression of human FcRI, II, and III;
HLA class I and II; CD4; and IL-2R molecules in an HMØ population
transfected with HIV-1 Nef or HIV-1 fen plasmid. After
AD, dl312-mediated transfection of nef cDNA, HMØs were
cultured and analyzed for expression of cell surface molecules. Cells
in separate tubes were suspended in modified Hank's buffer.
FITC-labeled specific monoclonal antibody was added; after incubation,
cells were washed and analyzed by fluorescence histograms. FITC-labeled
similar isotype antibody was added as control in each case.
Downregulation of FcRI, II, and III and CD4 and upregulation of HLA
class I molecules and no change in IL-2R and HLA class II were observed in a Nef-expressing population of HMØs.
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The action of nef in U937 cells and steady-state levels of
total FcRI and FcRII were compared with retrovirus-mediated
control fen- and Nef-expressing cells. FcRI expression in
both nef- and fen-tranduced cells was higher than
FcRII levels. Phosphor-imaging analysis of these gels confirmed that
both fen- and nef-transduced cells synthesized
equivalent levels of the FcRI proteins (Fig 4, time 0); the FcRI
protein in Nef-expressing cells had a significantly abbreviated
lifespan, going from the normal t1/2 of approximately 150 hours (Fig 4, U937fen) to approximately 36 hours in the U937 (Nef) cell. Similarly, FcRII was also synthesized by cells
expressing both Nef and Fen. As shown in Fig 4, FcRII had a
t1/2 of 185 hours in cells transduced with the control
vector (fen). In contrast, FcRII in Nef-expressing cells was
rapidly degraded; the t1/2 was approximately 24 hours.
Because different HIV proteins have been shown to alter cytokine
levels, we studied the effect of Nef expression on the proinflammatory cytokines. As shown in Fig 7A and B, both
PMA and LPS induced basal levels of TNF-, IL-1, IL-6, and IL-10
in HMØs. PMA-treated Nef-expressing HMØs induced more TNF-,
IL-1, and IL-10 (Fig 7Aa, b, and d), whereas HMØs express less IL-6
compared with control cells (Fig 7Ac). Four different donors' HMØs
were studied and the amount of monokine levels varied from
donor-to-donor (Fig 7A). After induction with PMA or LPS,
Nef-expressing cells produced twofold to fourfold and twofold to
20-fold more TNF- and IL-1, respectively, than control cells (Fig
7B). PMA and LPS induced twofold to eightfold more IL-10 than the
control cells (data not shown). LPS induced twofold to fourfold more
IL-6 secretion in the Nef-expressing HMØs when compared with control
cells (Fig 7B).
View larger version (69K):
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| Fig 7.
(A) Analysis of TNF-, IL-1, IL-6, and IL-10 by
ELISA from HIV-1 nef-transfected HMØs. Purified HMØs were
cultured in macrophage media in the presence of IL-3 (5 ng/mL) and
GM-CSF (20 ng/mL). After 4 days in culture, cells were transfected with
HIV-1 nef cDNA conjugated with transferrin-poly(L-lysine) in
the presence of AD, dl312 and incubated for 48 hours. Cells were washed
to remove growth factors and transferrin. HMØs were cultured in
macrophage media containing 5% FBS and treated with 20 ng/mL PMA for
donors no. l, 2, and 4 and 1 ng/mL for donor no. 3 for 72 hours.
Conditioned media were analyzed for TNF- (a), IL-1 (b), IL-6 (c),
and IL-10 (d) by ELISA. HMØs from 4 donors were analyzed and
represented individually. (B) Comparative analysis of cytokines
produced by Ad-d1312-mediated nef-transfected HMØ treated with
LPS and PMA. Cells were transfected and maintained as described in (A).
HMØs were treated with LPS (100 ng/mL) or PMA (20 ng/mL) for 72 hours; conditioned media were analyzed for the presence of cytokines by ELISA.
Values presented are the average of four different experiments.
|
|
| |
DISCUSSION |
Our results of proliferation assays of HMØs are comparable to those of
previous findings.56,57 We selected optimally dividing and
differentiated cells for transfection/transduction of nef and
-galactosidase (-gal). As judged by -gal staining, about 10%
of the cell population was found positive using AD, d312-mediated transfection system. Only a subpopulation of HMØs proliferate when
treated with IL-3 and GM-CSF. However, this method of transfection proved to be an effective system to study the function of nef gene in this heterogenous population of HMØs.
HIV proteins are toxic to cells,58-60 and Nef protein is
toxic and impairs induced cell proliferation.61,62
Therefore, both nef-transduced and nef-transfected
HMØs were monitored for cell viability. HIV proteins have been shown
to be toxic to different human cells and induce apoptosis in T cells
and nonmyeloid cells.63-66 Our results indicate for the
first time that Nef expression in an HMØ subpopulation in the presence
of IL-3 and GM-CSF induce apoptosis. Apoptotic MØs have also been
identified in PBMC populations in HIV patients (A.D. Bradley, Immunex
Corp, Seattle, WA; D.R. Clark, University of Arizona, Tucson, AZ;
personal communications, July, 1996). However, in other
studies, there was no apoptosis in HIV-infected HMØs.67
Because HMØs account for approximately 1% of the PBML population, the
number of cells examined in the above-mentioned study67 may
be insufficient to conclude whether HMØs are apoptotic. In our
experiments, a small percentage of the diversified population of
HMØs68 undergoes apoptosis, possibly due to their great
susceptibility. It has been previously reported that Nef expression in
HMØs induces the neurotoxic monokines, IL-6 and
TNF-.57,69 Furthermore, Nef is toxic to certain cells,
impairs induced cell proliferation, shares some properties with
scorpion peptides,61,70 and induces apoptosis in a
subpopulation of HMØs. These facts suggest that Nef is directly toxic
to certain cells and may indirectly contribute to neuropathology in
AIDS patients.
FcRI and FcRII were downmodulated in the Nef-expressing HMØs and
U937 cells (Figs 3 and 6). Downmodulation of FcRIII was minimal in
HMØs and FcRIII was not detected in U937 cells. This was not
surprising, because FcRIII is not usually expressed in U937
cells.71 CD4 downmodulation was previously noted in
Nef-expressing lymphoid and nonlymphoid cells of different species and
is considered to be a posttranslational process.7,8,36,72
Furthermore, Nef acts on CD4 molecules that are released from the
endoplasmic reticulum and migrate to the cell surface, which triggers
their endocytosis and degradation in lysosomes with a decreased
t1/2.8,36 Downmodulation of FcRs and CD4 may
have several effects on the host immune function. It has been shown
that downmodulation of CD4 in T cells results in resistance for new
viral infection, thereby promoting virus persistence.8 In
HMØs, downmodulation of CD4 by Nef might produce similar results. In
macrophages, FcRs are responsible for multiple functions such as (1)
endocytosis of antigen-antibody complexes via clathrin-coated pits; (2)
signalling exocytosis; (3) release of proteins and inflammatory
monokines; (4) antigen-dependent cellular cytotoxicity, triggering
lysis of tumor cells; and (5) generating and releasing superoxide
radicals.71,73-78 FcRs can bind to the Fc portion of the
antibody molecule, facilitate cytoplasmic uptake of the virus-antibody
complex, and mediate immunopathogenesis in many virus
infections.79-81 Downregulation of FcRI and II affects
the above-mentioned functions of macrophages and may promote persistent
viral infection.
HLA class I molecules were upregulated in both Nef-expressing HMØs and
U937 cells (Figs 3 and 6). Macrophages use major histocompatibility complex (MHC) class I molecules to present endogenous peptides and
exogenous antigens, whereas the class II pathway presents exogenous
antigen only.82-84 This allows macrophages to monitor tissues for the presence of viral infections and tumor
cells.85 In Nef-expressing macrophages, downregulation of
FcRI and II prevents efficient uptake, processing, and presentation
of exogenous antigens. Thus, overexpression of HLA class I in
Nef-expressing macrophages may partially compensate the
antigen-processing and presentation functions that have been altered
due to FcRI and II downregulation. In U937 cells chronically
infected with HIV-1, MHC class I molecules were downmodulated by the
virus.86 Viral infection and monokines, including IL-10,
interferon (IFN), and TNF- upregulate MHC class I
molecules.87-89 In our study, the combination of Nef,
TNF-, and IL-10 might upregulate HLA class I. Overexpression of HLA
class I is also associated with autoimmune diseases.90
Nef-expressing macrophages lack some of the important HMØ functions,
but not antigen presentation, because they overexpress HLA class I. Late-stage HIV infection is associated with autoimmune diseases,90 which may be due in part to HLA class I
overexpression by HIV-infected macrophages.
To examine the mechanism of FcRs downmodulation, U937 cells were
used, because cells that stably expressed Nef could be selected by
culturing in the presence of cells with G418.7,91 Reduction of t1/2 has also been shown to downmodulate
CD4.8,36 Here, for the first time, we have shown that Nef
downmodulates surface FcRI and FcRII.
Monokines (TNF-, IL-1, IL-6, and IL-10) play a very important
role in AIDS pathogenesis and can be induced by different stimuli
including LPS,92-95 PMA,96 and UV
light97 in myeloid cells. Therefore, we analyzed whether
Nef-expressing HMØs in the presence of LPS or PMA can alter secretion
of TNF-, IL-1, IL-6, and IL-10. Induction of IL-6 by LPS but not
by PMA in Nef-expressing cells showed that LPS-mediated IL-6 induction
differed from PMA. Cytokines/monokines regulate both immune function
and viral replication, and contribute to immunopathogenesis during HIV
infection.16 In HIV-infected individuals, secretion of the
proinflammatory monokines TNF-, IL-1, and IL-6 is increased in
HMØs, lymphoid tissues, plasma, and cerebrospinal fluid; these
monokines can also enhance viral expression when added to acutely or
chronically infected cell cultures.18 In contrast, IL-10
can either induce or suppress HIV expression, depending on the culture
system used.16 IL-10 can inhibit HIV replication by
blocking secretion of TNF- and IL-6.30 TNF- has been
shown to be the most potent inducer of HIV
replication.24,27 In Nef-expressing human T cells, signal transduction is altered.6,98,99 Nef can upregulate
TNF-, IL-4, IL-8, and IL-13 and downregulate IL-2 and IFN- after
induction by a mitogenic combination of PMA and
ionomycin.100 Furthermore, IL-10 mRNA increased during the
acute phase of infection of Cynomolgus macaques with a pathogenic
primary isolate of SIVmac consisting of a full-length
nef.101 Among other regulatory proteins, HIV-1 Tat
induces overexpression of TNF- and IL-6.102,103
Moreover, gp120 enhances expression of TNF-, IL-1, IL-10,
IFN-, complement proteins, nitric oxide, and endothelin-1 in many
cell types, including macrophages.104,105 Our results
suggest that HIV-1 Nef upregulates TNF-, IL-1, and IL-10 in LPS-
or PMA- stimulated HMØs. Although the mechanism of induction of
monokines by HIV-1 Nef is unknown, upregulation of these monokines in
an activated state may regulate cell-mediated and humoral immune
responses as well as control viral gene transcription and virus
replication, thereby contributing to viral pathogenesis.
In summary, our present study shows that Nef downmodulates FcRI and
II and CD4; upregulates HLA class I molecules in HMØs; induces
apoptosis in a subpopulation of HMØs; and modulates monokine expression in the presence of LPS and PMA. Moreover, the Ad vector system described here has potentially attractive therapeutic
applications for gene delivery into HMØs.
| |
FOOTNOTES |
Submitted June 4, 1997;
accepted October 31, 1997.
S.K.D and C.N.S.V. contributed equally to this paper.
Address reprint requests to Swapan K. De, PhD, Bldg 30, Room 232, Oral Infection and Immunity Branch, National Institute of Dental Research, NIH, Bethesda, MD 20892.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors acknowledge the editorial guidance of Devera Schoenberg,
MS. C.N.S.V. dedicates this paper to the memory of Dr Somnath Ghosh and
Ashwatha Shetty. We are grateful to Charles Carter for providing the
monocytes.
| |
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Abstract
Introduction
Abstract