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*
Institute of Human Virology, University of Maryland Biotechnology Institute, Baltimore, MD 21201;
Human Anatomy Section, Department of Morphology and Embryology, University of Ferrara, Ferrara, Italy; and
Institute of Normal Morphology, Chieti, Italy
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The aim of this study was to investigate the mechanisms by which extracellular Tat protein may favor viral infection. As CXC-chemokine receptor 4 (CXCR4)4 and CC-chemokine receptor 5 (CCR5) serve as primary coreceptors mediating the entry of the HXB2 T cell line-tropic (X4-tropic) and BaL macrophage-tropic (M-tropic) strains of HIV-1, respectively 22 , we have analyzed the expression of both CXCR4 and CCR5 in primary resting CD4+ T cells following exposure to extracellular Tat.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PBMC were isolated by Ficoll-Hypaque density-gradient centrifugation (Pharmacia, Uppsala, Sweden) of heparinized leukocyte units obtained from healthy adult donors, who gave their informed consent to this research according to the Helsinki declaration of 1975. Resting CD4+ T cells were isolated by stringent immunomagnetic negative selection with Dynabeads M450 (Dynal, Oslo, Norway). For this purpose, we used a mixture of mAbs against CD19 and CD20 present on B cells; CD16, CD56, and CD57 on NK cells; CD14 on monocytes; and CD8 (all mAb were from Coulter-Immunotech, Miami, FL). The final cultures of CD4+ T cells thus obtained were always >85% pure, as determined by two-color flow cytometry analysis using a phycoerythrin (PE)-conjugated anti-CD4 mAb (Becton Dickinson, San Jose, CA) in combination with a PC5-conjugated anti-CD3 mAb (Coulter-Immunotech). After purification, cells were resuspended in AIM-V serum-free medium (Life Technologies, Grand Island, NY) at 1.8 x 106 cells/ml and seeded in 48-well flat-bottom plates (0.6 ml/well).
Adherence of viral proteins to microtiter plates
Full-length synthetic Tat was from Technogen (Caserta, Italy), and recombinant p24 protein and anti-Tat rabbit polyclonal IgG were from Intracell (Cambridge, MA). Viral proteins were resuspended in PBS containing 0.1% BSA (Sigma, St. Louis, MO) and stored in aliquots at -70°C before use. Flat-bottom (48-well) polystyrene plates (Costar, Cambridge, MA) were coated overnight at 4°C with either viral proteins or BSA at the concentrations indicated in the text. Plates were then rinsed with AIM-V serum-free medium to remove nonadherent proteins, and medium was immediately added to the plates after the final wash. The amount of Tat coated to each well was estimated by ELISA by using anti-Tat polyclonal IgG (Intracell), followed by horseradish peroxidase-conjugated goat anti-rabbit IgG (Dako, Copenhagen, Denmark), as previously described 19 . Tat-coated plates were examined before and after 24 h of culture with CD4+ T cells.
In some cases, anti-Tat IgG were added to the wells (5 µg/well) after the final wash and left in the culture medium for all the time of the experiment. In other cases, 200 µg/ml of cycloheximide (Sigma) were added to the culture medium.
Flow cytometric analysis of cell surface molecules
Surface expression of CXCR4, CCR5, CD29, CD49, and CD69 was evaluated by direct staining with the PE-conjugated anti-CXCR4, anti-CCR5, anti-CD29, anti-CD49 (all from PharMingen, San Diego, CA), and PC5-conjugated anti-CD69 (Coulter-Immunotech) mAbs. Briefly, aliquots of 3 x 105 cells were stained with 5 µl of each mAb in 200 µl of PBS containing 2% FCS at 4°C for 30 min. Nonspecific fluorescence was assessed by using isotype-matched controls. After staining procedures, samples were analyzed using a FACSCalibur flow cytometer (Becton Dickinson). Data collected from 10,000 cells are presented as either histograms or mean fluorescence intensity (MFI) values.
In three experiments, cell-associated immunofluorescence was calculated by using the Quantum 27 fluorescence quantitation kit (Flow Cytometry Standards, San Juan, PR) following manufacturer’s instructions. This system allows for the direct quantitation of the fluorescence intensity of a test sample in terms of number of molecules of equivalent soluble fluorochrome (MESF) by comparison with a set of calibrated fluorescent standards consisting of four populations of microbeads, having different levels of fluorescence intensity (expressed as units of MESF) and matching the emission and excitation spectra of specimens labeled with the same fluorochrome, and one reference blank population.
HIV infection assay
At 4 h postseeding, cells were infected with HIV-1 (HXB2 and BaL strains; Advanced Biotechnologies, Columbia, MD; multiplicity of infection of 0.01) for 3 h and then washed three times with PBS. Of note, viral stocks were treated before use with RNase-free DNase I (Boehringer Mannheim, Indianapolis, IN) to remove contaminating DNA. Fourteen hours postinfection, CD4+ T cells were collected and lysed in proteinase K-lysis buffer and allowed to incubate at 56°C for 60 min, then at 98°C for 20 min. Serial dilutions of cell lysates were subjected to HIV-1 DNA PCR by using the following primers designed based on previously published sequences 23 : 5'-primer, 5'-TCTCTCTGGTTAGACCAGATCTG; 3'-primer, 5'-ACTGCTAGAGATTTTCCACACTG. These primers, which amplify a 180-bp fragment in the long terminal repeat (LTR) R/U5 region, were designed to detect early steps in reverse transcription 23 . Samples were subjected to 40 cycles of amplification (95°C for 1 min, 50°C for 1 min, and 72°C for 1 min). Negative controls were represented by samples containing buffer only or uninfected cells. The PCR products were separated on a 2% agarose gel, transferred to a nylon membrane, and hybridized with a 32P-labeled oligonucleotide probe (5'-CTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGC) against an internal sequence of the HIV PCR product and analyzed after exposure to x-ray film. To normalize for the quantity of DNA in each sample, ß-globin PCR was conducted using ß-globin primers (Stratagene, La Jolla, CA). ß-Globin PCR products were visualized under UV light after staining of agarose gels with ethidium bromide. Each sample was amplified in duplicate or triplicate.
Statistical analysis
The results were expressed as means ± SD of three or more experiments performed in duplicate. Statistical analysis was performed using the two-tailed Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To investigate the role of Tat protein on the expression of surface CXCR4 and CCR5, we have chosen a previously established model system that allows us to assess the response of resting CD4+ T cells to truly extracellular Tat protein by using protein immobilized on plastic 5 . The doses of Tat reported in all experiments are those added overnight to the plates to coat the wells and do not represent the amount of protein bound to each well. The HIV-1 protein p24 and/or BSA were used as controls.
The purity of freshly isolated CD4+ T cell populations
ranged from 85 to 98% in different experiments, reaching 95% in the
representative experiment illustrated in Fig. 1
, A–D. CD4+ T
cells constantly expressed detectable, albeit variable, levels of
surface CXCR4 that were rapidly enhanced following incubation at 37°C
in serum-free medium (Fig. 1B). Culture of CD4+
T cells in Tat-coated plates resulted in a clearly detectable increase
in CXCR4 expression with respect to the spontaneous CXCR4 induction
within 2–8 h, with maximal (p < 0.01) effect
being observed after 4 h (Figs. 1, B–C). On the other
hand, when T cells were seeded in p24-coated or BSA-coated plates, no
effect on the spontaneous induction of CXCR4 expression was observed.
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The effect of Tat on CXCR4 was selective in that CCR5, which was
normally expressed at very low levels in freshly purified resting
CD4+ T cells, was not affected by immobilized Tat nor by
immobilized p24 (Fig. 1
B). Of note, the early activation
marker CD69, whose expression was transiently up-regulated when freshly
isolated CD4+ T cells were seeded in culture, also did not
show significant variations in Tat-treated cells with respect to those
treated with BSA (Fig. 1B).
When the phenotypic analysis was conducted for longer time periods (up
to 72 h), no significant differences in the expression of CXCR4,
CCR5, and CD69 could be observed between Tat- and BSA-treated cells
(Fig. 1D). Taken together, these data indicate that the
Tat-mediated up-regulation of CXCR4 in resting CD4+ T cells
is transient but specific. In this respect, other surface Ags (CD4, and
the integrin markers CD29 and CD49) were also unaffected by Tat
treatment at any time point considered (data not shown).
In selected experiments, an accurate analysis of the relative number of
anti-CXCR4 mAb binding sites per cell was performed using
quantitative fluorescein microbeads standards (Fig. 2). With this approach, a >2-fold
(p < 0.01) increase in MESF corresponding to
CXCR4 was observed in cells seeded for 4 h on Tat-coated plates
(14,100 ± 2,230) with respect to cells seeded on BSA-coated
plates (5700 ± 950).
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To evaluate the dose-dependence of Tat-mediated CXCR4
up-regulation, plates were next coated with synthetic Tat at a
concentration range comprised between 0.01 and 100 nM (Fig. 3). After 4 h of incubation,
immobilized Tat up-regulated the surface expression of CXCR4 in a
bell-shaped fashion. Plates coated with 1–10 nM Tat showed a
statistically significant (p < 0.01) increase
of CXCR4 levels with respect to plates coated with 0.01, 0.1, and 100
nM Tat or with 10 nM p24 or 0.1% BSA. The specificity of the effect
was demonstrated by the ability of anti-Tat polyclonal IgG to
abrogate (p < 0.01) the Tat-mediated
up-regulation of surface CXCR4. Of note, when soluble Tat was used, a
greater variability in CXCR4 modulation was noticed with respect to the
experiments performed with immobilized Tat. In general, the
concentrations of soluble Tat required to observe up-regulation of the
surface CXCR4 after 4 h of culture were one log higher (100 nM)
than those required to obtain similar effects with immobilized Tat. To
further ascertain whether the Tat-mediated up-regulation of CXCR4 was
truly due to extracellular Tat, the amount of protein immobilized on
plastic was evaluated before and after seeding CD4+ T cells
on Tat-coated plates for 24 h by ELISA. In three separate
experiments, the enzyme immune adsorbance OD values were similar before
(0.93 ± 0.23) and after (0.85 ± 0.15) performing the cell
cultures. Taken together, these data indicate that internalization of
extracellular Tat by CD4+ T cells is not required to
up-regulate CXCR4.
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To determine whether the Tat-induced up-regulation of CXCR4
surface expression was due to de novo protein synthesis or merely
relocation of preexisting receptor to the cell surface, we performed
the experiments in presence of cycloheximide, a pharmacological
inhibitor of protein synthesis. When primary CD4+ T cells
were seeded in culture for 4 h in the presence of 200 µg/ml of
cycloheximide, a significant (p < 0.01)
inhibition of surface CXCR4 was observed (Fig. 4), indicating that neosynthesis of CXCR4
takes place as soon as freshly isolated CD4 cells are seeded in
culture. Of note, the Tat-mediated up-regulation of CXCR4 was also
almost completely abolished by the addition of cycloheximide (Fig. 4).
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To define the role of CXCR4 up-regulation in CD4+ T
cell infection by HIV-1, we used an X4-tropic strain of HIV-1. Primary
CD4+ T cells cultured for 4 h in BSA- or Tat-coated
plates were infected with HXB2 (multiplicity of injection (MOI) = 0.01)
for 3 h. After an additional 14 h of culture in fresh medium,
samples were analyzed by PCR for the presence and amount of viral DNA
as a measurement of viral entry. Semiquantitative PCR of strong-stop
DNA (with LTR R/U5 primers), an early product of reverse transcription,
revealed a significant higher level of proviral DNA in cells seeded on
Tat-coated plates with respect to those seeded on control (BSA-coated)
plates (Fig. 5). In contrast, infection
with 0.01 MOI of the M-tropic strain BaL was undetectable in both the
BSA- and Tat-treated cultures (data not shown). These results indicate
that the Tat-induced up-regulation of CXCR4 affects the susceptibility
of CD4+ T cells to infection by X4 HIV-1 strains.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The specificity of the Tat-mediated CXCR4 up-regulation was underlined by the fact that Tat was unable to up-regulate other surface markers, such as CD69, CD4, CD29, and CD49. In particular, the inability of Tat to up-regulate CD69, which represents an early activation Ag, clearly indicates that the Tat-mediated CXCR4 up-regulation cannot be considered the consequence of a generic CD4+ T cell activation. In this respect, we have previously demonstrated that immobilized Tat was unable to induce the proliferation of resting CD4+ T cells by itself and required the presence of a second stimulus 5 .
In agreement with Jourdan et al. 25 , we observed that freshly isolated CD4+ T cells expressed low levels of cell surface CXCR4 that were rapidly enhanced following incubation of the cells in medium alone, even in absence of serum. Extracellular Tat induced a further increase in CXCR4 expression, with maximal effect observed after 4 h of incubation. The efficient inhibitory effect of cycloheximide on the surface expression of CXCR4 induced by extracellular Tat suggests that the Tat-mediated CXCR4 up-regulation is not due to merely relocation of preexisting receptor to the cell surface, but requires de novo protein synthesis of CXCR4.
A number of studies have shown that CXCR4 expression can be rapidly up-regulated or down-regulated depending upon the conditions used to stimulate resting T cells 25, 26, 27, 28 . While down-regulation of surface CXCR4 appears to be the consequence of rapid endocytosis of cell surface receptor molecules 29 , the mechanisms responsible for CXCR4 up-regulation are less clear. Nevertheless, the Tat-mediated up-regulation of CXCR4 surface expression was not accompanied by modifications of surface CCR5. Consistent with our data, other authors have previously shown that CXCR4 and CCR5 are differentially regulated on T lymphocytes 27, 28 . CXCR4 and CCR5 are the predominant chemokine receptors used as coreceptors in HIV-1 entry, and as such, their expression is important for determining viral tropism. In the course of HIV-1 infection, M-tropic viral strains predominate during the early phase of infection, while dual-tropic and T cell line-tropic viral strains appear late during disease progression to AIDS.
Since CXCR4 was already expressed by resting CD4+ T cells, a crucial issue was to evaluate whether the Tat-mediated increase of surface CXCR4 could impact HIV-1 infectivity. We were able to demonstrate that immobilized Tat was associated to an increased entry of X4-tropic but not of an M-tropic strain of HIV-1 into primary resting CD4+ T cells. Thus, extracellular Tat may contribute to determine the shift from macrophage- to dual- and T cell line-tropic virus strains, rendering a larger population of lymphocytes more susceptible to HIV-1 late in the course of infection.
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Footnotes |
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2 These authors contributed equally to this study.
3 Address correspondence and reprint requests to Dr. Paola Secchiero, Institute of Human Virology, University of Maryland Biotechnology Institute, 725 West Lombard Street, Baltimore, MD 21201-1192. E-mail address:
4 Abbreviations used in this paper: CXCR4, CXC chemokine receptor 4; CCR5, CC-chemokine receptor 5; X4-tropic, HXB2 T cell line-tropic; M-tropic, BaL macrophage-tropic; PE, phycoerythrin; MFI, mean fluorescence intensity; MESF, molecules of equivalent soluble fluorochrome; LTR, long terminal repeat.
Received for publication October 23, 1998. Accepted for publication November 6, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and -
isoforms in PC12 cells. Biochem. Biophys. Res. Commun. 242:332.[Medline]
and Tat involvement in the immunosuppression of uninfected T cells and C-C chemokine decline in AIDS. Proc. Natl. Acad. Sci. USA 95:3851.This article has been cited by other articles:
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S. Opi, J.-M. Peloponese Jr., D. Esquieu, G. Campbell, J. de Mareuil, A. Walburger, M. Solomiac, C. Gregoire, E. Bouveret, D. L. Yirrell, et al. Tat HIV-1 Primary and Tertiary Structures Critical to Immune Response Against Non-homologous Variants J. Biol. Chem., September 20, 2002; 277(39): 35915 - 35919. [Abstract] [Full Text] [PDF]
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J. D. Estes, B. F. Keele, K. Tenner-Racz, P. Racz, M. A. Redd, T. C. Thacker, Y. Jiang, M. J. Lloyd, S. Gartner, and G. F. Burton Follicular Dendritic Cell-Mediated Up-Regulation of CXCR4 Expression on CD4 T Cells and HIV Pathogenesis J. Immunol., September 1, 2002; 169(5): 2313 - 2322. [Abstract] [Full Text] [PDF]
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 S. M. Agwale, M. T. Shata, M. S. Reitz Jr., V. S. Kalyanaraman, R. C. Gallo, M. Popovic, and D. M. Hone A Tat subunit vaccine confers protective immunity against the immune-modulating activity of the human immunodeficiency virus type-1 Tat protein in mice PNAS, July 23, 2002; 99(15): 10037 - 10041. [Abstract] [Full Text] [PDF]
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V. Bres, R. Kiernan, S. Emiliani, and M. Benkirane Tat Acetyl-acceptor Lysines Are Important for Human Immunodeficiency Virus Type-1 Replication J. Biol. Chem., June 14, 2002; 277(25): 22215 - 22221. [Abstract] [Full Text] [PDF]
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E. Fanales-Belasio, S. Moretti, F. Nappi, G. Barillari, F. Micheletti, A. Cafaro, and B. Ensoli Native HIV-1 Tat Protein Targets Monocyte-Derived Dendritic Cells and Enhances Their Maturation, Function, and Antigen-Specific T Cell Responses J. Immunol., January 1, 2002; 168(1): 197 - 206. [Abstract] [Full Text] [PDF]
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R. L. Caldwell, B. S. Egan, and V. L. Shepherd HIV-1 Tat Represses Transcription from the Mannose Receptor Promoter J. Immunol., December 15, 2000; 165(12): 7035 - 7041. [Abstract] [Full Text] [PDF]
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A. de Paulis, R. De Palma, L. Di Gioia, M. Carfora, N. Prevete, G. Tosi, R. S. Accolla, and G. Marone Tat Protein Is an HIV-1-Encoded {beta}-Chemokine Homolog That Promotes Migration and Up-Regulates CCR3 Expression on Human Fc{epsilon}RI+ Cells J. Immunol., December 15, 2000; 165(12): 7171 - 7179. [Abstract] [Full Text] [PDF]
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P. Secchiero, D. Zella, S. Curreli, P. Mirandola, S. Capitani, R. C. Gallo, and G. Zauli Pivotal role of cyclic nucleoside phosphodiesterase 4 in Tat-mediated CD4+ T cell hyperactivation and HIV type 1 replication PNAS, December 8, 2000; (2000) 11512398. [Abstract] [Full Text]
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H. Xiao, C. Neuveut, H. L. Tiffany, M. Benkirane, E. A. Rich, P. M. Murphy, and K.-T. Jeang Selective CXCR4 antagonism by Tat: Implications for in vivo expansion of coreceptor use by HIV-1 PNAS, October 10, 2000; 97(21): 11466 - 11471. [Abstract] [Full Text] [PDF]
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P. Secchiero, D. Zella, S. Curreli, P. Mirandola, S. Capitani, R. C. Gallo, and G. Zauli Engagement of CD28 Modulates CXC Chemokine Receptor 4 Surface Expression in Both Resting and CD3-Stimulated CD4+ T Cells J. Immunol., April 15, 2000; 164(8): 4018 - 4024. [Abstract] [Full Text] [PDF]
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 C. M. McManus, K. Weidenheim, S. E. Woodman, J. Nunez, J. Hesselgesser, A. Nath, and J. W. Berman Chemokine and Chemokine-Receptor Expression in Human Glial Elements : Induction by the HIV Protein, Tat, and Chemokine Autoregulation Am. J. Pathol., April 1, 2000; 156(4): 1441 - 1453. [Abstract] [Full Text] [PDF]
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D. Zella, F. Romerio, S. Curreli, P. Secchiero, C. Cicala, D. Zagury, and R. C. Gallo IFN-{alpha}2b Reduces IL-2 Production and IL-2 Receptor Function in Primary CD4+ T Cells J. Immunol., March 1, 2000; 164(5): 2296 - 2302. [Abstract] [Full Text] [PDF]
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M. J. Orsini, J.-L. Parent, S. J. Mundell, and J. L. Benovic Trafficking of the HIV Coreceptor CXCR4. ROLE OF ARRESTINS AND IDENTIFICATION OF RESIDUES IN THE C-TERMINAL TAIL THAT MEDIATE RECEPTOR INTERNALIZATION J. Biol. Chem., October 22, 1999; 274(43): 31076 - 31086. [Abstract] [Full Text] [PDF]
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R. C. Gallo Tat as one key to HIV-induced immune pathogenesis and Pat toxoid as an important component of a vaccine PNAS, July 20, 1999; 96(15): 8324 - 8326. [Full Text] [PDF]
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P. Secchiero, D. Zella, S. Curreli, P. Mirandola, S. Capitani, R. C. Gallo, and G. Zauli Pivotal role of cyclic nucleoside phosphodiesterase 4 in Tat-mediated CD4+ T cell hyperactivation and HIV type 1 replication PNAS, December 19, 2000; 97(26): 14620 - 14625. [Abstract] [Full Text] [PDF]
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C. D. Pauza, P. Trivedi, M. Wallace, T. J. Ruckwardt, H. Le Buanec, W. Lu, B. Bizzini, A. Burny, D. Zagury, and R. C. Gallo Vaccination with Tat toxoid attenuates disease in simian/HIV-challenged macaques PNAS, March 28, 2000; 97(7): 3515 - 3519. [Abstract] [Full Text] [PDF]
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