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Blood, Vol. 95 No. 9 (May 1), 2000:
pp. 2760-2769
CHEMOKINES
HTLV-II down-regulates HIV-1 replication in IL-2-stimulated
primary PBMC of coinfected individuals through expression of
MIP-1
Claudio Casoli,
Elisa Vicenzi,
Andrea Cimarelli,
Giacomo Magnani,
Paolo Ciancianaini,
Ercole Cattaneo,
PierPaolo Dall'Aglio,
Guido Poli, and
Umberto Bertazzoni
From the Istituto di Patologia Medica, Università di Parma,
Parma; Unità di Immunopatogenesi dell'AIDS, Istituto Scientifico
San Raffaele, Milano; Istituto di Genetica Biochimica ed
Evoluzionistica del CNR, Pavia; Divisione di Malattie Infettive,
Ospedale di Parma, Parma; Laboratorio Retrovirus, IRCCS Policlinico S. Matteo, Pavia; and Sezione di Biologia e Genetica DMIBG,
Università di Verona, Italy.
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Abstract |
The influence of human T-cell leukemia/lymphoma virus type II
(HTLV-II) in individuals also infected with HIV-1 is poorly understood.
To evaluate the reciprocal influence of HTLV-II and HIV-1 infection,
primary peripheral blood mononuclear cell (PBMC) cultures from
coinfected individuals were established in the presence of interleukin
2 (IL-2). In these cultures, the kinetics of HTLV-II replication
always preceded those of HIV-1. Noteworthy, the kinetics of HIV-1
production were inversely correlated to the HTLV-II proviral load in
vivo and its replication ex vivo. These observations
suggested a potential interaction between the 2 retroviruses. In
this regard, the levels of IL-2, IL-6, and tumor necrosis factor-
(TNF-) were measured in the same coinfected PBMC cultures.
Endogenous IL-2 was not produced, whereas IL-6 and TNF- were
secreted at levels compatible with their known ability to up-regulate
HIV-1 expression. The HIV-suppressive CC-chemokines RANTES, macrophage inflammatory protein-1 (MIP-1), and MIP-1 were also determined in IL-2-stimulated PBMC cultures. Of interest, their kinetics and
concentrations were inversely related to those of HIV-1 replication. Experiments were performed in which CD8+ T cells or PBMCs
from HTLV-II monoinfected individuals were cocultivated with
CD4+ T cells from HIV-1 monoinfected individuals
separated by a semipermeable membrane in the presence or absence of
antichemokine neutralizing antibodies. The results indicate that
HTLV-II can interfere with the replicative potential of HIV-1 by
up-regulating viral suppressive CC-chemokines and, in particular,
MIP-1. This study is the first report indicating that HTLV-II can
influence HIV replication, at least in vitro, via up-regulation of
HIV-suppressive chemokines.
(Blood. 2000;95:2760-2769)
© 2000 by The American Society of Hematology.
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Introduction |
Human T-cell leukemia/lymphoma types I and II (HTLV-I
and -II) are T-lymphotropic retroviruses. HTLV-I mostly infects
CD4+ T cells, whereas HTLV-II shows a preferential tropism
for CD8+ T lymphocytes.1 A strong association
of HTLV-I infection with the subsequent development of adult T-cell
leukemia,2 as well as of HTLV-I-associated
myelopathy-tropical spastic paraparesis (HAM/TSP)3 has been
described. In contrast, no definitive relationship between HTLV-II and
human diseases has been yet established.4
Both HTLV-I and HTLV-II are potential viral cofactors in individuals
also infected by HIV-1. In Europe, HTLV-II is predominant among
intravenous drug users (IVDUs),5 and it is frequently found
in individuals coinfected with HIV-1.6 Some studies in homosexual men concomitantly infected by HTLV-I and HIV-1 have suggested that they have a significantly accelerated HIV disease progression toward AIDS, compared with individuals infected with HIV-1
alone.7 Similarly, it has been suggested that a concomitant HTLV-I/HIV-1 infection causes a higher mortality in IVDUs with AIDS.8 This potentiation of HIV pathogenicity is supported by in vitro studies on HTLV-I tax gene products, showing that the production of infectious HIV-1 is enhanced in cell lines
transformed by HTLV-I.9 This hypothesis has not been
confirmed thus far in individuals with concomitant HIV-1 and HTLV-II
infections, likely as a result of the different cellular tropism of
these 2 viruses.10 Unlike HTLV-I, HTLV-II coinfection was
not statistically associated with progression to AIDS in subjects from
some cohorts of IVDUs.11,12
Some Italian individuals coinfected with HIV-1 and HTLV-II have shown a
less steep decline of CD4+ T cells and a very long interval
before the development of AIDS, in association with a high HTLV-II
proviral load, even in the presence of high levels of HIV-1
replication, as measured by plasma viremia.13,14 These
findings suggest that HTLV-II may play a protective rather than
potentiating role on HIV disease progression.
Infection by HTLVs leads to several immune dysfunctions, including
spontaneous proliferation of T cells, which have been attributed to the
effect of the viral regulatory protein Tax, encoded by the px
gene.15 In addition to acting on the long terminal repeat (LTR) region to enhance viral RNA transcription, this protein is also
capable of transactivating both eukaryotic promoters involved in T-cell
activation and proliferation16 and the promoter of HIV-1
via activation of NF- B.4 In this regard, in vitro
experiments have shown that reciprocal virus activation occurs in cell
lines coinfected with members of these virus families.17,18
Recently, it has been defined that expression of some chemokine
receptors is required for fusion and entry of HIV-1 into
CD4+ cells. In particular, CCR5, a cell surface receptor
for the CC-chemokines RANTES, MIP-1 , and MIP-1, acts as a
coreceptor for nonsyncytium-inducing (NSI) macrophage-tropic strains of
HIV-1, which dominate after transmission and during early disease. In
addition, CXCR4, which binds stromal cell-derived factor-1 (SDF-1), is
the coreceptor of SI viruses19 emerging with
disease progression in approximately 50% of the
individuals.20 The acquisition of CXCR4 usage as coreceptor
corresponds to the switch from an NSI to an SI phenotype, the loss of
sensitivity to the suppressive effects of CC-chemokines, and
a steeper decrease in CD4+ T-cell counts.20,21
Of interest, the 3 CCR5 binding chemokines, RANTES, MIP-1, and
MIP-1, were characterized as the major HIV-1-suppressive factor
released by HTLV-I immortalized T cells, as well as by primary CD8+ T
cells.22 In this regard, constitutive expression of various
chemokines has been described in HTLV-I positive T-cell lines as a
consequence of the effect of the viral transactivator Tax.23 In addition, HTLV-I-specific CD8+
cytotoxic T lymphocyte (CTL) clones derived from patients with HAM/TSP are an important source of MIP-1 and
MIP-1,24 suggesting the possibility that HTLV-I may
profoundly influence HIV replication via chemokine expression and
release. In contrast, little information is available on whether
HTLV-II can modulate the expression of CC chemokines.22
To address this issue and to investigate the potential relationship
between HIV and HTLV-II replication, we have established peripheral
blood mononuclear cell (PBMC) cultures from 6 coinfected individuals in
whom the proviral load of both retroviruses was previously determined.
We observed that both retroviruses were able to replicate in PBMC
cultures in the presence of exogenous IL-2.
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Materials and methods |
Patients
The patient population included 12 IVDUs (6 men and 6 women, mean
age 37.8 ± 7.4 years) belonging to the Parma
cohort.14 Dual seropositivity for HTLV-I/II and HIV-1 was
documented for patients PR-7, PR-8, PR-9, PR-10, PR-11, and PR-12 since
1986-87 (Table 1). Three individuals were
infected exclusively with HTLV-II (PR-1, PR-2, and PR-3), and 3 additional patients exclusively with HIV-1 (PR-4, PR-5, and PR-6). The
clinical history and immunologic features of all 12 patients were
available through medical records, including the determination of
CD4+ and CD8+ T-cell counts at entry and every
3 to 4 months thereafter. At the time of inclusion in the study,
monoinfected and coinfected individuals presented the same pattern of
seroprevalence for herpes simplex types 1 and 2; cytomegalovirus (CMV);
hepatitis B, C, and D viruses; and Toxoplasma gondii.
The stage of HIV-1 infection was classified according to Centers for
Disease Control and Prevention criteria.25 The HTLV-II
subtype was identified by sequencing the LTR region of the viral
isolates.26 At regular intervals, each patient was tested
for full hematology and chemistry panels and a complete physical
examination was performed. The clinical and virologic features of the
patients included in this study are summarized in Table 1. A
competitive polymerase chain reaction (PCR) method was used to quantify
HIV-1 and HTLV-II proviral DNA in PBMC obtained from infected
subjects.13,27 Among the 6 coinfected patients, PR-11
(before therapy) and PR-12 presented with a high HTLV-II proviral load,
compared with HIV-1 (ratio HIV-1/HTLV-II, < 0.1); PR-9 and PR-10 had
equivalent proviral loads (ratio 1.1 and 1.3, respectively); and PR-7
and PR-8 had very high HIV-1 and very low HTLV-II proviral loads (ratio
40.6 and >800). PR-7 and PR-8 were under antiretroviral therapy at
study entry, whereas PR-9, PR-10, and PR-12 were and remained untreated
throughout the study. PR-11 underwent antiviral therapy after a sensory
neuropathy concomitant developed with a drop in circulating
CD4+ T cells below 400 cells/µL. Three HIV-1 monoinfected
patients were chosen among antiretroviral naive individuals belonging
to the same cohort of the coinfected patients. Coinfected individuals had an average followup of about 10 years. HIV-1 staging at entry before therapy was between A1 and B3 for those with an HIV-1/HTLV-II ratio ranging from 0.1 to <2. Patient PR-7 progressed to AIDS and an
autonomic sensory neuropathy developed; he was treated with
antiretroviral agents from 1993 until his death in 1996. The monthly
rate of decline of CD4+ T lymphocytes in all coinfected
individuals, expect PR-12, and in HIV-1 monoinfected patients was 1.3 up to 3.4 × 106 cells, compatible with the
definition of slow progressors.28 PR-12 was characterized
by a high HTLV-II load and an extremely low HIV-1 load, very high
CD4+ T-cell number since 1987, and no sign of HIV disease
progression. Nevertheless, he was able to transmit both retroviruses to
his partner.
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Table 1.
Virologic and clinical features of patients infected
with HIV-1 and HTLV-II in relationship to HIV pathogenesis
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Patients mono-infected with HTLV-II maintained CD4 counts of more than
or equal to 800 cells/µL since enrollment. Persistent HTLV-II antigen
(Ag) expression was detected in the plasma of PR-1 (64 pg/mL) and PR-11
(40 pg/mL). No correlations between HTLV-II Ag expression and proviral
load were observed. The HTLV-II strain of PR-7 and PR-11 belonged to
the subtype IIa, whereas the others were of the subtype IIb.
IL-2 stimulated PBMC cultures
PBMCs were cultured in 96-well plates (Costar Corning, Cambridge,
MA) at the concentration of 1 × 106 cells/mL in
RPMI 1640 (Gibco BRL, Life Technologies Italia, Milano) medium,
supplemented with 20% fetal calf serum (FCS) (Hyclone Europe,
Cramlington, UK) in the presence or absence of 20 U/mL of rIL-2
(Genzyme, Cambridge, MA). Half (50%) of the culture medium was changed
every 8 days with fresh medium containing 20% FCS. Supernatants were
harvested for analysis of HIV-1 or HTLV-II p24 Ag, cytokine, and
chemokine contents. Exogenous rIL-2 was added every 3 to 4 days.
p24gag HIV-1 and HTLV-II Ag levels were tested on collected
culture supernatants. HIV-1 expression was determined using an HIV-1
p24 Ag capture enzyme-immunoassay (EIA) (Abbott Laboratories, North
Chicago, IL), whereas HTLV-II production was determined by using a
p24gag Ag capture assay (Retrovirology Coulter Corp,
Hialeah, FL), according to the manufacturers' instructions. At various
time points, aliquots of cells were removed for proliferation assays,
phenotypic analyses, and detection of HIV-1 and HTLV-II proviral DNA.
Cell viability was determined by trypan blue dye exclusion. For
calculation of spontaneous proliferation, triplicate samples of
1 × 106 cells/mL were pulsed with 0.037 MBq (1 µCi) [3H]-thymidine, cultured for 6 hours, and
acid-insoluble precipitates were counted in a liquid scintillation
-counter. For phenotype determination, cells were analyzed by flow
cytometry after staining with the mAbs directed to the following Ags:
CD3, CD4, CD8, CD14, CD20, CD25, and HLA-DR (Becton Dickinson, San
Jose, CA) as previously described.10
Cultures of cells separated by a semipermeable membrane
CD4+ or CD8+ T cells from PBMC were
negatively selected by column exclusion (CD4+ or
CD8+ Subset Enrichment Columns; R & D Systems, Minneapolis,
MN). Purity of CD4+ or CD8+ T cells was 96% or
more, determined by flow cytometric analysis. CD4+ T cells,
1.5 × 106, and CD8+ T cells,
1.5 × 106, (autologous or allogeneic) were cultured
in the same well, but separated by a semipermeable polyester membrane
(0.4-µm Costar Transwell; Costar Europe, Badhoevedorp, The
Netherlands), as described for PBMC primary cultures. Supernatants from
both the upper and the lower compartments of the well were removed
separately once a week, frozen, and subsequently tested for the
presence of HIV-1 p24 Ag. In some experiments, a mixture of
neutralizing anti-RANTES, anti-MIP-1, and anti-MIP-1
polyclonal Ab (R & D Systems) was added at 2 µg/mL each to the
cultures, both at the beginning of the experiments and at every medium change.
Characterization of HIV-1 isolates
Primary viral stocks of mono-HIV-1 or coinfected patients were
serially 4-fold diluted in sestuplicates, added to wells containing 3-day-old PHA-stimulated T-cell blasts from seronegative donors, and
maintained in RPMI 1640, 10% FCS (Hyclone Europe) plus rIL-2 (20 U/mL). The culture supernatants were tested every 48 to 72 hours by a
Mg++-dependent reverse transcriptase (RT) activity
assay.29
To assess the coreceptor usage of the HIV-1 isolates, U87 cells stably
expressing either CD4 or CD4 plus 1 of the following chemokine R,
CCR2b, CCR3, CCR5, and CXCR-4,30 were inoculated with the
different primary isolates (1/100 vol/vol of the viral stock). Viral
stocks of 100 µL were diluted in 1 mL of D-MEM (Bio Whittaker,
Verviers, Belgium), containing 15% FCS (Sigma Chemical Corp, St Louis,
MO), were added to 1 × 105/mL cells in a 48-well
plate (Falcon; Becton Dickinson Labware, Lincoln Park, NJ). Standard
laboratory HIV-1 strains, such as HIV-1 IIIB and BaL, using CXCR4 (X4
virus) and CCR5 (R5 virus), respectively, were used as positive
controls.19 Twenty-four hours later, the medium was
replaced with fresh medium. Cultures were examined daily for syncytia
formation with an inverted microscope, whereas culture supernatants
were collected every 2 to 3 days over a 3-week period and stored
at  80°C until tested for Mg++-dependent RT
activity contents.29
Cytokine and chemokine determinations
Levels of cytokines present in the PBMC culture supernatants were
determined by commercially available enzyme-linking immunosorbent assay
(ELISA) kits, recognizing only bioactive cytokines: IL-2, IL-3, IL-6,
and TNF- (Biosource International, Camarillo, CA). Chemokine
(RANTES, MIP-1, MIP-1) concentrations were measured by ELISA
according to the manufacturer's instructions (R & D Systems).
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Results |
Establishment of PBMC cultures from HTLV-II infected individuals
After PBMC isolation, the majority of PBMCs of HTLV-II monoinfected
PR-1 and coinfected PR-11 cultures were T lymphocytes, as shown by the
expression of CD3 Ag (Figure 1). Similar
levels of CD4+ T cells were present in these cultures,
whereas the number of CD8+ T cells was lower in PR-1 (25%)
than in PR-11 (40%). Approximately, 14% to 16% of CD3+
cells expressed the IL-2R -chain (CD25) and class II HLA-DR Ag on
their cell surface. The proportion of B lymphocytes (CD19+)
and monocytes (CD14+) was similar to those of uninfected
donors. The level of spontaneous cell proliferation in PBMC primary
cultures varied from patient to patient (range, 3050 to 4654 cpm) but
it was 2- to 3-fold greater than that seen in the cultured cells from
HTLV-seronegative individuals (mean 1452 cpm), with the exception of
the PBMC culture obtained from patient PR-7. This individual was
characterized by a very low HTLV-II proviral load (< 240 copies per
105 cells). All primary PBMC cultures remained viable for
at least 45 days, as determined by trypan blue dye exclusion, and
expressed CD25 and HLA-DR activation markers on their cell surface that increased slightly over time, with respect to starting conditions (with
18%-22% vs 14%-16% positive cells at day 7 vs day 0, respectively). In the absence of IL-2, these primary cultures did not support HTLV-II
or, in the case of dual infections, HIV-1 p24 Ag production (data not
shown).
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| Fig 1.
Analysis of cell surface markers in primary cultures
established from a mono-HTLV-II-infected patient (PR-1) and a patient
(PR-11) coinfected with HIV-1 and HTLV-II.
Results are representative of 3 phenotypic analyses performed at the
indicated time, and are expressed as percentages of positive cells.
 , day 0;  , day 30;  , day 60.
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In the presence of exogenous IL-2 (20 U/mL), the majority of
CD3+ T cells expressed CD25 and HLA-DR cell surface
activation markers after 30 days of culture, at which time HTLV-II
expression was observed. Approximately 50% to 60% CD8+ T
cells and 30% to 35% CD4+ T cells were more than 80% of
the cells in culture. B cells were either stable or diminished
overtime. A substantial fraction of cells (ranging from 10% to 20% of
the total cell numbers) coexpressing a double
CD4+/CD8+ phenotype emerged in cultures of
HTLV-II-infected individuals (Figure 1).
After 30 days of culture, HIV-1-associated CD4+ T-cell
depletion was observed in primary cultures in coinfected patients, and the percentage of CD4+ cells among viable CD3+
T cells decreased rapidly to 10% to 15%. A moderate decrease (5%-10%) of CD8+ T cells was also observed that was,
however, compensated by a rise in T cells coexpressing CD4+
and CD8+.
HIV-1 and HTLV-II expression in PBMC cultures from
coinfected patients
Primary cultures were established from the PBMC of all 12 subjects
in the presence of IL-2 without additional mitogenic stimulation. This
condition has been described as an optimal system to allow replication
of both SI and NSI strains of HIV-1.31 HIV-1 and HTLV-II
replication were measured by testing the levels of
supernatant-associated p24 Gag proteins at different time points of
PBMC cultures. The kinetics of viral Ag production in
mono-HIV-1-infected PR-4, PR-5, and PR-6 and mono-HTLV-II-infected
PR-1, PR-2, and PR-3 are shown in Figure 2A
and 2B, respectively. Consistent with their low HTLV-II proviral load,
HTLV-II expression was not detected in cultures of cells from
coinfected individuals PR-7 and PR-8 (Figure
3A). In PBMC cultures of coinfected
individuals PR-9, PR-10, and PR-11, characterized by an HIV-1/HTLV-II
ratio of less than or equal to 1, the kinetics of HTLV-II production
always preceded those of HIV-1 (Figure 3B and 3C). HIV replication in
these PBMC cultures was considerably delayed (3 to 4 weeks) in
comparison with that observed in cultures established from
mono-HIV-infected individuals (Figure 2A). Strikingly, the absence of
HIV expression was observed up to 5 weeks of culture in PBMC of patient
PR-11 (Figure 3C), although his HIV-1 proviral load in PBMC was similar
to that of the mono-HIV-1-infected individual PR-4 (Table 1).
Finally, in PBMC cultures of PR-12, characterized by a very low
HIV-1/HTLV-II ratio, no replication of HIV-1 was observed (Figure 3D).
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| Fig 2.
Kinetics of virus production in IL-2-stimulated PBMCs
obtained from mono-HIV-1-infected (A), or mono-HTLV-II-infected (B)
individuals.
Supernatants from PBMC cultures were harvested every 3 to 5 days.
Supernatants were tested for concentrations of HIV-1 and HTLV-II p24
Gag Ags; the results are representative of 3 independent experiments
performed at 6-month intervals.
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| Fig 3.
Kinetics of virus production by primary cultures obtained
from HIV-1/HTLV-II-coinfected patients. (A) Cultures from PR-7 and
PR-8, characterized by a high HIV-I/HTLV-II proviral load ratio. (B)
Cultures from PR-9 and PR-10, with an HIV-I/HTLV-II ratio equal to 1. (C) Culture from PR-II before therapy, with a low HIV-I/HTLV-II ratio.
(D) Culture from PR-12 with a very low HIV-I/HTLV-II ratio. See Figure
2 for procedures.
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In these PBMC cultures, HTLV-II expression was associated with cellular
proliferation and moderate (approximately 10%) cell death, whereas a
dramatic cytopathic effect was observed during the peak of HIV-1
replication (data not shown).
HIV-1 coreceptor usage
All HIV-1 primary isolates were NSI by the MT-2 cells32
and used CCR5 as entry coreceptor (Table 1). The primary PR-11-derived HIV did not score positive in a coreceptor usage assay and was further
passaged onto allogeneic PHA-activated PBMC of a seronegative donor.
This second passage isolate showed usage of both CCR5 and CXCR4 in the
U87 assay coreceptors assay (Table 1). Finally, in the face of several
attempts, the PR-10-derived HIV-1 could not be characterized for
coreceptor usage.
Cytokine secretion in primary cultures of PBMCs from
coinfected patients
It has been reported that T-cell lines infected by HTLVs
constitutively express high levels of different cytokines, including IL-2, IL-6, and TNF-,33,34 which in turn can
profoundly influence the state of activation of T lymphocytes and
monocytes. Expression of cytokines in IL-2-stimulated PBMC infected in
vitro with HIV-1 has been previously shown to significantly sustain
virus replication.31,35 Therefore, the cytokine content in
the supernatants of primary PBMC cultures from mono- and coinfected
patients with high HTLV-II proviral loads was investigated both in the
presence or absence of exogenous IL-2.
Unstimulated PBMC cultures obtained from HTLV-II monoinfected PR-1 and
from coinfected PR-11 initially secreted very low levels of IL-6 and
TNF-, whereas production progressively increased after 15 days and
peaked after approximately 40 days of culture (Figure
4A). A modest (up to 2-fold) increase of
cytokine production was noted in PBMC cultures from the coinfected
patients in comparison with those of monoinfected patients. Of interest
is the fact that in both cases the cytokine levels that were reached
during cultivation were in the range of concentrations capable of
sustaining HIV replication after in vitro infection.31
Secretion of endogenous IL-2 was not detected during the entire period
of both in monoinfected PR-1 and coinfected PR-11 cultures (Figure 4A).
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| Fig 4.
Cytokine production in primary cultures obtained from
mono-HTLV-II-infected patient PR-1 and from HIV-1/HTLV-II-coinfected
patient PR-11.
Results are representative of 3 separate experiments. Patients' PBMCs
were cultivated in the absence (A) or presence (B) of IL-2.
Supernatants were harvested at various times and tested for cytokine
content.
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No significant differences in the levels or kinetics of cytokine
production in monoinfected or coinfected cell cultures were observed
between unstimulated and IL-2-stimulated cultures (Figure 4B).
Worthy of note, cytokine production peaked simultaneously or close
to maximal HIV-1 replication, suggesting their positive role on HIV
production, as reported in vitro- infected PBMC.31
Chemokine secretion and HIV-1 replication in
IL-2-stimulated PBMC cultures
The ability of HTLV-I/-II to up-regulate expression of chemokines
has been reported.22,23 Because some CC-chemokines, such as
MIP-1, MIP-1, and RANTES, have shown inhibitory effects on NSI
HIV-1 replication,19,22 their concentrations were tested in
the supernatants of primary cultures established from
HTLV-II-monoinfected PR-1, PR-2, and PR-3 and
HIV-1/HTLV-II-coinfected PR-7, PR-8, PR-10, PR-9, PR-11, and PR-12.
All chemokines were indeed secreted on a 6-day time course by primary
cultures from PR-1 (Figure 5A) in the
presence of IL-2; similar levels were found in PBMC cultures established from PR-2 and PR-3. Substantial variability in terms of
chemokine secretion was observed in cultures from coinfected individuals. The PBMC cultures of PR-7 showed early peaks of MIP-1, MIP-1, and RANTES secretion within 6 to 14 days of culture, which then decreased to undetectable levels (Figure 5B); a similar pattern was observed in PBMC cultures of PR-8, who, like PR-7, was
characterized by a very low HTLV-II load (Table 1). MIP-1 was
produced early at low levels in the cultures of PR-10 (Figure 5C) and
PR-9, whereas MIP-1 and RANTES peaked at higher levels than MIP-1
later during the culture (Figure 5C). In the case of PR-12,
characterized by a particularly low HIV-1/HTLV-II ratio, a substantial
production of all 3 chemokines occurs and was maintained during the
whole culture period (Figure 5D). Of interest, no evidence of HIV
replication was obtained in PBMC cultures of PR-12 (Figure 5D). These
findings suggested a potential linkage between HTLV-II load, chemokine secretion, and HIV replication in IL-2-stimulated PBMC cultures of
these individuals. This hypothesis was further reinforced with the cell
cultures of PR-11, which showed considerable levels of chemokine
secretion and delayed kinetics of HIV replication compared with the
PBMC culture of PR-7 (Figure 6A).
Furthermore, chemokine secretion and HTLV-II expression occurred
simultaneously and preceded HIV replication in PR-11's cultures
(Figure 6A). Viral replication as well as chemokine secretion in
PR-11's cell cultures were also investigated after 3 months of
zidovudine monotherapy; HTLV-II proviral load decreased from 16 239 to
500 copies per 105 cells, whereas no substantial changes in
HIV-1 DNA load (1060 vs 1844 copies per 105 cells) occurred
before and after therapy, respectively. Quite strikingly, increased HIV
RNA plasma levels (from 4810 to 36 530 copies per milliliter, before
and after therapy, respectively) likely indicated the presence of
zidovudine-resistant strains in this individual. A more rapid kinetic
of HIV replication ex vivo in the absence of HTLV-II production and in
reduced levels of MIP-1, MIP-1, and RANTES was noted in cultures
established 90 days after initiation of zidovudine monotherapy (Figure
6B).
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| Fig 5.
Chemokine secretion in culture supernatants of
IL-2-stimulated PBMCs.
Cultures were established from a mono-HTLV-II-infected patient
(PR-1), whereas the bars indicate the range of chemokine concentrations
of PR-2 and PR-3 (A). Similar cultures were performed with PBMCs of the
HIV-1/HTLV-II-coinfected patient PR-7, with the bars indicating the
range of PR-8, (B); of HIV-1/HTLV-II-coinfected patients PR-10, with
bars indicating the chemokine levels observed with cells of PR-9 (C);
and of HIV-1/HTLV-II-coinfected patients PR-12 (D). Cells were seeded
at 1 × 105 cells per well and culture supernatants
were harvested at regular intervals and then tested for chemokine
concentrations and HIV-1 or HTLV-II p24 Gag Ags. Results are
representative of 3 independent experiments.
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| Fig 6.
Chemokine secretion in culture supernatants of
IL-2-stimulated PBMCs from mono-HTLV-II-infected patient PR-11
before (A) and after (B) 3 months of zidovudine monotherapy.
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These findings support a strong correlation between HTLV-II load and
expression, CC-chemokine secretion, and HIV-1 replication ex vivo,
which was further investigated.
When the levels of the 3 HIV-suppressive chemokines secreted in
IL-2-stimulated PBMC cultures were expressed as median values and then
plotted against HTLV-II DNA load, MIP-1, but not MIP-1 or
RANTES, was found significantly associated with the HTLV-II copy number
(Figure 7). This observation suggested that
MIP-1 could play a crucial role in controlling HIV replication in
autologous PBMC cultures of coinfected individuals.
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| Fig 7.
Correlation between the HTLV-II proviral load in PBMCs
and the MIP-1 secretion in ex vivo cultures.
HTLV-II copy number was evaluated by competitive PCR on
1 × 105 cells (horizontal axis). Median levels of
MIP-1 (A), MIP-1 (B), and RANTES (C) secreted by IL-2-stimulated
PBMCs from mono-HTLV-II-infected and HIV-1/HTLV-II-coinfected
patients are shown. Median values represents the chemokine
concentrations (vertical axis) observed by multiple determinations.
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|
Enhanced inhibitory effect of CD8+ T cells from
HTLV-II-monoinfected patients on HIV-1 replication in
CD4+ T cells from HIV-1-monoinfected individuals:
dominant role of MIP-1
A critical role for a noncytolytic anti-HIV activity of
CD8+ T cells in controlling HIV replication has been
reported by several authors36; the 3 CC-chemokines
investigated here have previously been implicated as major players for
this antiviral activity.37 Of interest, HTLV-II is known to
express a preferential tropism for CD8+ T lymphocytes in
vivo.38 Therefore, CD8+ T cells from HTLV-II
monoinfected PR-1 and PR-3 individuals (characterized by a high
proviral load) were cocultivated with CD4+ cells of PR-5
and PR-6 HIV-1 monoinfected individuals (characterized by high HIV
proviral DNA and viremia), separated by a semipermeable membrane. The
ability of different numbers of allogeneic CD8+ T cells
inhibiting HIV replication in this system was compared with that of
autologous CD8+ T cells. Results of a typical experiment
(representative of 3 independently performed experiments) are shown in
Figure 8. As expected, HIV-1 p24 Gag
production occurred in CD8-depleted CD4+ cells of PR-5 and
PR-6, and peaked at day 24 of cultures (Figure 8). In contrast,
CD4+ T cells from PR-5 or PR-6, incubated with equal
numbers (1:1 ratio) of either purified autologous (PR-5/PR-5 and
PR-6/PR-6) or allogeneic (PR-1/PR-5 and PR-3/PR-6) CD8+ T
cells, showed a drastic reduction of viral expression. Interestingly, autologous CD8+ T cells from both HIV-1-monoinfected
individuals lost their inhibitory capacity at a 1:5 ratio with
CD4+ T cells, whereas CD8+ T cells of
HTLV-II-monoinfected individuals maintained a potent suppressive
effect up to a 1:20 ratio with CD4+ cells (Figure 8). A
clear, although only partial, HIV inhibitory activity in mixed CD8/CD4
cocultures was present even at 1:50 (Figure 8). Of note is the fact
that a cocktail of antihuman MIP-1, MIP-1, and RANTES polyclonal
antibodies completely reverted the suppressive effects exhibited by
CD8+ T cells of HTLV-II-monoinfected individuals on HIV-1
replication (Figure 8).
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| Fig 8.
CD8+/CD4+ cell-dependent
suppressive effect of CD8+ T cells from HIV-1 (PR-5 and
PR-6, respectively) or HTLV-II (PR-1 and PR-3, respectively)
monoinfected patients on HIV-1 p24 Ag production in cultures of
CD4+ cells purified from a mono-HIV-1-infected
individual.
From PR-1 and PR-5 (A), and from PR-6 and PR-3 (B), respectively.
CD8+ cells were separated from HIV-infected
CD4+ cells by a semipermeable membrane in a Transwell
system. Cultures were performed in the absence or presence of
anti-RANTES, anti-MIP-1, and anti-MIP-1 polyclonal Abs (2 µg/mL
each).
|
|
Similar results were obtained when unfractionated PBMCs, instead of
purified CD4+ and CD8+ cells, were used in the
same cocultivation system. PBMCs from PR-4 or PR-5 HIV-1 monoinfected
and from PR-1 or PR-2 HTLV-II monoinfected were cocultivated in the
same wells but separated by a semipermeable membrane up to 30 days in
the presence of IL-2. The results of a typical experiment are shown in
Figure 9. HIV-1 p24 Ag production was
promptly observed in PBMC cultures of PR-5, which was fully suppressed
in the presence of PR-2 PBMCs on the other side of the chamber (Figure
9), as previously observed with purified CD4+ and
CD8+ cells (Figure 8). Antichemokine neutralizing Abs
(Nabs) were added either individually or as cocktails in the attempt to
identify whether some chemokines were playing a dominant role in terms of HIV suppression in this system. Reversion of HTLV-II PBMC
suppressive effect was indeed observed when either a cocktail of all 3 Nabs or anti-MIP-1 Nab alone was added to the cocultures (Figure
9). In contrast, and quite surprisingly, no reversion of PBMC-mediated anti-HIV effect was observed when anti-RANTES or anti-MIP-1 Nabs were added alone or in combination to the cultures (Figure 9). As
previously observed in terms of correlation between chemokine secretion
in culture and HTLV-II proviral load in vivo, MIP-1 seems to play a
crucial role in mediating HIV suppression by HTLV-II-infected cells.
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| Fig 9.
Neutralizing activity of antichemokine Abs in cocultures
of unfractionated PBMCs from HIV-1 (PR-5) and HTLV-II (PR-2)
monoinfected patients.
PBMCs from the 2 individuals were cocultivated in the same chamber but
separated by a semipermeable membrane up to 30 days in the presence or
absence of antichemokine Abs (2 µg/mL each) added either individually
or in combination.
|
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Recombinant (r) MIP-1 inhibits HIV replication in CD8-depleted
PBMC cultures of HIV-1-monoinfected individuals
To further demonstrate the capacity of MIP-1 to suppress HIV-1
replication, PBMC cultures from HIV-1-monoinfected individuals were
depleted of CD8+ T cells, stimulated with
phytohemagglutinin (PHA-P, Sigma Chemical) and IL-2 (20 U/mL;
Boehringer-Mannheim) in the presence or absence of rMIP-1 (100 ng/mL; R & D Systems). The excess of PHA was removed by
cell centrifugation after 72 hours and cells were resuspended in medium
enriched with IL-2 as described above. As shown in Figure 10, rMIP-1 exerted profound inhibitory
effects in these PBMC cultures established from different
HIV-1-monoinfected individuals, thus confirming and extending our
findings with cell cultures of HTLV-II/HIV-1-infected persons.
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| Fig 10.
Recombinant MIP-1 inhibits HIV-1 replication in
CD8-depleted PBMC cultures of monoinfected individuals.
PBMCs were isolated from 3 individuals with progressive HIV-1 disease,
and CD8+ T cells were removed by a single round of
immunomagnetic beads (Dynal, Oslo, Norway). rMIP-1 (100 ng/mL) that
was added twice (time 0 and 72 hours from the establishment of the
culture) inhibited virus replication in these autologous cell cultures,
although with variable potency.
|
|
| |
Discussion |
In this work we describe that simultaneous replication of both HIV-1
and HTLV-II can be investigated in cultures of PBMCs obtained from
coinfected individuals and maintained in IL-2-enriched medium. HTLV-II
expression occurred earlier than that of HIV-1, and its kinetics were
directly correlated to the number of HTLV-II copies present in patient
PBMCs (viral load). Furthermore, evidence of HTLV-II interference on
HIV-1 replication was obtained both in terms of kinetics of virus
production and, more importantly, of cytokine and chemokine secretion,
which were consistent with their respective roles of positive and
negative regulators of HIV replication. CD8+ T cells from
HTLV-II-monoinfected individuals released soluble factors that
suppressed HIV-1 replication more potently than autologous CD8+ T lymphocytes in the cultures of both unfractionated
PBMCs and purified CD4+ cells of mono HIV-1-infected
individuals. Antichemokine Nabs reverted the suppressive effects of
CD8+ cells of HTLV-II-monoinfected individuals. Among
others, MIP-1 appeared to play a major role in these culture
systems, also supported by the ability of rMIP-1 to inhibit HIV
replication in the cultures of HIV-1-monoinfected individuals. Of
note, MIP-1 secretion, but not that of either RANTES or MIP-1,
was significantly correlated to the HTLV-II load in vivo.
A major determinant of HIV-1 infection is the efficiency of virus
entry. In contrast to what was observed for HTLV-I,39 in
our ex vivo system of PBMCs of HIV-1/HTLV-II-coinfected individuals, we have not observed any down-regulation of the CD4 surface molecules after HTLV-II infection. In fact, expression of CD3 and CD8
Ag was unchanged, whereas CD4 was down-regulated only in concomitance with HIV-1 replication in the cocultures of both
HTLV-II-monoinfected and HTLV-II/HIV-1-coinfected individuals.
Recently, it has been shown that both CCR5 and CXCR4 are up-regulated
in response to IL-2.40 In this regard, we have previously reported that PBMC stimulation with IL-2 without prior mitogen (PHA)
activation represented an in vitro system more physiologic in
comparison with standard mitogen-activated T-cell blasts, in that both
NSI and SI viruses, including primary HIV isolates, could efficiently
replicate.31 Furthermore, under an IL-2-dependent condition, both monocytes and endogenous proinflammatory cytokines were
shown to play an important role in virus spreading,31
likely resembling in vivo events. Thus, it can be inferred that the
expression levels of HIV-1 coreceptors in our culture systems were not
the limiting factor for HIV-1 replication. This hypothesis was
confirmed by the observation that the HIV primary isolates obtained
from the IL-2-stimulated PBMCs of coinfected PR-7, PR-8, and PR-9
individuals were all defined as R5, with the sole exception of a second
passage virus from PR-11, which was dualtropic (R5/X4).19
Unlike HTLV-I and HIV-1 coinfection,39,41 the analysis of
viral expression and cellular phenotypes in the primary cultures of
HIV-1- and HTLV-II-coinfected patients suggests that the 2 viruses
were not coinfecting the same cell type because no evidence of
reciprocal activation was observed.
The role of IL-2 in the expression of HIV-1 and HTLV-II is correlated
to lymphocyte proliferation and activation, resulting in cytokine
production.31,42 Resting T cells can be infected by HIV-1,
but production of high levels of virus in T cells requires further
cellular activation/proliferation.43-46 Autocrine and
paracrine regulation of viral expression by endogenous cytokines has
been demonstrated in cell lines either acutely or chronically infected by HIV-1,47,48 and in primary cells, both infected in
vitro, as well as during viral isolation from patient
PBMCs.49-51 T-cell lines infected by HTLVs are known to
constitutively express various cytokines, including IL-2, IL-6, and
TNF-,33,34 which together induce activation of resting
CD4+ T cells.52 Proinflammatory cytokines such
as TNF-, IL-1, IFN- , and IL-6 were shown to profoundly affect
the capacity of HIV to replicate because they act as potent
up-regulators of viral expression in both T-lymphocytic and monocytic
cells.47,51,53-55 Of interest, in our unstimulated primary
cultures from coinfected patients, substantial levels of
proinflammatory cytokines were observed. However, the lack of both IL-2
production and of CD25 expression indicated that TNF- was not
sufficient to activate either the IL-2R-chain or HIV-1 gene expression.
IL-2-stimulation of PBMCs induces autocrine/paracrine release of
proinflammatory cytokines as well as HIV replication.31,56 HIV-1 expression occurred in the IL-2-stimulated PBMCs of coinfected individuals after HTLV-II replication and when cytokine production was
at its peak, confirming and extending previous
observation,31 but also suggesting that proinflammatory
cytokines may contribute to, but are not the only determinants of, HIV
replication, at least in the presence of HTLV-II-infected cells.
HTLV-I-infected T cells expressed a wide spectrum of chemokines and
that the virally encoded transcriptional activator Tax is capable of
inducing a number of chemokine genes.23 The results reported here extend these findings to HTLV-II-infected cells. The
levels of the CC-chemokines, RANTES, MIP-1, and MIP-1, earlier identified as components of the CD8+ T-cell-derived
nonlytic suppressor factor for HIV-1 replication,19,22 in
supernatants of the IL-2-stimulated PBMC cultures, were indeed correlated to the patterns of HIV-1 replication. HIV-1 expression was
not detected until chemokine secretion was maximal, whereas it emerged
when a decrease of their secretion occurred. This negative correlation
was confirmed in the peculiar case of cultures established from cells
obtained from coinfected PR-11 before and after zidovudine monotherapy,
which was associated with a remarkable decrease of HTLV-II proviral
load, but also with an increase of HIV-1 viral load. Ex vivo, a
substantial loss of chemokine production in the primary culture
established after therapy was reflected by earlier HIV
replication kinetics and no HTLV-II expression.
In view of the fact that HIV-1 replication in our cellular system could
be critically influenced by CC-chemokines, particularly in
consideration that all viruses with the possible exception of 1 were
R5, the effect of chemokine neutralization on HIV-1 expression was
further investigated. Our results show unambiguously that soluble
factors from HTLV-II-infected CD8+ T cells from
HTLV-II-monoinfected patients were able to modulate HIV-1 expression
in naturally infected CD4+ T cells in a negative fashion.
Strikingly, either unfractionated PBMCs or CD8+ T cells
from HTLV-II-monoinfected individuals were more potent than autologous
CD8+ T cells in suppressing HIV replication. The major
component of these factors was represented by CC-chemokines, because
direct addition of antichemokine NAbs in cultures strongly decreased the soluble anti-HIV activity of HTLV-II-infected cells. These findings confirm and extend the previous observation that CC-chemokines released from HTLV-I-infected MT-2 cells are able to suppress HIV-1
replication in CD4+ T cells.57
Quite surprisingly, MIP-1 alone seemed to account for most the
anti-HIV activity, and its levels of secretion in culture, unlike those
of RANTES and MIP-1, were correlated significantly with HTLV-II load
in vivo. Of interest, CD8+ T lymphocytes have recently been
shown to represent a biologically relevant source of MIP-1 in
vivo,58 whereas spontaneous CC-chemokine production
by a CD8+ lymphocyte subpopulation from HTLV-II-infected
individuals, but not from individuals infected with HIV-1 alone or
unstimulated normal donors, has been demonstrated.59 In
addition, evidence that CD8+ cytotoxic T lymphocytes
produce chemokines after engagement of viral Ags and that MIP-1 is
required for an inflammatory response to virus challenge suggests that
these molecules are key elements in the generation of effective
antiviral immunity.60 Human MIP-1 is encoded by 2 highly
related nonallelic genes producing 2 different isoforms designed
LD78 and LD78.61 The MIP-1 isoform differs only in
3 amino acids (a.a.): the penultimate NH2-terminal residue and a.a. 39 and 47. Important is the NH2-terminal regions
for their biologic activity, because the difference in 1 residue may be
important for receptor binding.62 LD78 signals
predominantly via CCR1; in contrast, LD78 activates CCR5 more
efficiently than LD78 or RANTES.63,64 On PBMCs, LD78
isoforms show 10- to 50-fold higher antiviral activity against M-tropic
HIV-1 strains compared with RANTES and LD78.63,64 It is
conceivable that different isoforms of MIP-1 could account for its
biologic relevance in our culture system.
Taken together, these results provide evidence that HTLV-II may exert a
potent control over HIV replication via secretion of MIP-1. The high
HTLV-II proviral load might favor HIV-1 latency in coinfected patients
through a chemokine-dependent mechanism.
Several questions remain unanswered in terms of the in vivo interaction
between human pathogenic retroviruses. The optimization of an
appropriate in vitro system of primary cells directly established from
coinfected patients represents an important step for the understanding
of these interactions and pathogenesis in vivo.
| |
Footnotes |
Submitted September 13, 1999; accepted December 17, 1999.
Supported in part by grants of the National AIDS Research Program
Against AIDS of the Istituto Superiore di Sanità, and by the AIRC
program 1998. The Universities of Parma and of Verona participated in
the HERN concerted action, supported by the BIOMED Program of the
European Commission. A.C. is currently at the Columbia University of
New York, NY.
Reprints: Claudio Casoli, Istituto di Patologia Medica,
Facoltà di Medicina, Università di Parma, v. Gramsci, 14, I-43100 Parma, Italy; e-mail: claucaso{at}ipruniv.cce.unipr.it.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Abstract
Introduction