Search

The addition of low concentrations (0.1-1 nM) of extracellular HIV-1 Tat protein to PC12 neuronal cells stimulated a rapid (peak at 5 min) elevation of the cAMP intracellular levels, which in turn induced the phosphorylation of CREB transcription factor (peak at 15 min) on serine-133 (Ser-133). On the contrary, at later time points (60-120 min) Tat induced a significant decline of intracellular cAMP with respect to the basal levels observed in control cells treated with bovine serum albumin. In blocking experiments performed with pharmacological inhibitors, Tat decreased the intracellular levels of cAMP and CREB Ser-133 phosphorylation through a signal transduction pathway involving the sequential activation of phosphatidylinositol 3-kinase, AKT, and cyclic nucleoside phosphodiesterases. Moreover, in transient transfection experiments, Tat inhibited transcription of CREB promoter in a manner strictly dependent on the presence of the cAMP-responsive elements (CRE) in the CREB promoter. Consistently, the expression of endogenous CREB protein was significantly reduced in PC12 cells by prolonged (24-48 h) treatment with Tat. This decline in the expression of CREB, which plays an essential role in the survival and function of neuronal cells, anticipated a progressive increase of apoptosis in Tat-treated cells. Although obtained in a neuronal cell line, our findings might help to explain some aspects of the pathogenesis of HIV-1-associated dementia.

We show here that HIV type 1 (HIV-1) Tat protein, in combination with anti-CD3/CD28 mAbs, promotes IL-2 production and proliferation of primary CD4(+) T lymphocytes, obtained from HIV-1-seronegative donors. This effect was observed when Tat was immobilized on a solid support, but it was not observed with soluble Tat. Such hyperactivation was accomplished by recruiting the rolipram-sensitive cyclic nucleoside phosphodiesterase 4 and resulted in increased susceptibility to HIV-1 infection. Accordingly, rolipram potently inhibited HIV-1 replication in cultures stimulated by anti-CD3/CD28 +/- Tat. These results add to the concept that decreasing Tat activity is an important addition to anti-HIV-1 therapy, and they suggest a target for anti-HIV-1 chemotherapy, phosphodiesterase 4.

Treatment of dopaminergic rat PC12 cells with human immunodeficiency virus, type 1 (HIV-1) Tat protein or tat cDNA inhibited the expression of tyrosine hydroxylase (TH), the rate-limiting enzyme for the dopamine biosynthetic pathway, as well as the production and release of dopamine into the culture medium. Moreover, the Tat addition to PC12 cells up-regulated the expression of the inducible cAMP early repressor (ICER), a specific member of the cAMP-responsive element modulator transcription factor family, in a cAMP-dependent manner. In turn, ICER overexpression abrogated the transcription activity of the TH promoter in PC12 cells, strongly suggesting ICER involvement in Tat-mediated inhibition of TH gene expression. In vivo injection of synthetic HIV-1 Tat protein into the striatum of healthy rats induced a subclinical Parkinson's-like disease that became manifested only when the animals were treated with amphetamine. As early as one week postinjection, the histochemical examination of the rat substantia nigra showed a reduced staining of neurons expressing TH followed by a loss of TH(+) neurons at later time points. As Tat protein can be locally released into the central nervous system by HIV-1-infected microglial cells, our findings may contribute to the explanation of the pathogenesis of the motorial abnormalities often reported in HIV-1 seropositive individuals.

Objective: To investigate the intracellular signals elicited by extracellular HIV-1 Tat protein in lymphoid CD4 T cells., Methods: CD4 Jurkat T cells were treated with a series of glutathione S-transferase (GST)-Tat fusion proteins: full-length two-exon GST-Tat (GST-Tat2E); one-exon Tat, in which the second exon of Tat was deleted (GST-Tat1E); two-exon Tat, in which the seven arginine residues have been changed to alanine residues (GST-TatArg(mut)), GST-TatdeltaN, which shows a deletion of the N-terminal 21 amino acids. The cells were either treated with soluble GST-Tat proteins or seeded on plates coated with GST-Tat proteins immobilized on plastic. At various time points, Jurkat cells were lysed and examined for c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) activity., Results: Soluble and immobilized GST-Tat2E, but not GST-Tat1E, GST-TatArg(mut) and GST-TatdeltaN, activated JNK in a dose-dependent manner, induced a rapid phosphorylation of c-Jun on Ser63 and promoted the de novo synthesis of c-Jun protein. Moreover, both GST-Tat2E and GST-Tat1E also stimulated ERK/MAPK. However, the activation of JNK was maximal at concentrations of 100 nM of GST-Tat2E and was blocked by the S6-kinase inhibitor rapamycin, whereas the activation of ERK/MAPK was already maximal at 1 nM of GST-Tat2E and was enhanced by rapamycin., Conclusions: Tat-mediated activation of JNK requires the second exon of Tat, which is dispensable for the activation of ERK/MAPK. The ability to stimulate JNK and ERK/MAPK does not require Tat internalization.

Here we report that synthetic HIV-1 Tat protein, immobilized on a solid substrate, up-regulates the surface expression of the CXC-chemokine receptor 4 (CXCR4), but not of the CC-chemokine receptor 5 in purified populations of primary resting CD4+ T cells. The Tat-mediated increase of CXCR4 occurred in a well-defined range of concentrations (1-10 nM of immobilized Tat) and time period (4-8 h postincubation). Moreover, the increase of CXCR4 was accompanied by an increased entry of the HXB2 T cell line-tropic (X4-tropic), but not of the BaL macrophage-tropic strain of HIV-1. The ability of Tat to up-regulate CXCR4 expression was abrogated by the protein synthesis inhibitor cycloheximide, clearly indicating the requirement of de novo synthesis. As Tat protein is actively released by HIV-1 infected cells, our data indicate a potentially important role for extracellular Tat in rendering bystander CD4+ T cells more susceptible to infection with X4-tropic HIV-1 isolates.

Many viruses have evolved genes encoding proteins that regulate cell death by apoptosis. The human immunodeficiency virus type 1 (HIV-1) Nef protein alters T-cell development and signaling and is required for optimal viral replication and pathogenicity in vivo. To analyze the interference of Nef with cell survival, we used both regulated and constitutively expressed nef alleles in stably transfected T-cell lines. Nef-expressing cells were sensitized to cell death by apoptosis, which was specifically exacerbated by an anti-CD95 IgM monoclonal antibody (MoAb). Flow cytometric analysis showed that the surface expression of both CD95 and CD95 ligand (CD95L) was upregulated by endogenous Nef expression. Nef-mediated apoptosis was almost completely suppressed by the addition in culture of an anti-CD95 Fab' IgG MoAb, which specifically blocks CD95/CD95L interactions. Lastly, mutation of a proline motif in the core region of the nef gene, which disrupts its ability to interact with cellular kinases and reduces HIV-1 replication in vitro, completely abrogated the Nef-mediated induction of apoptosis as well as its ability to upregulate surface CD95 and CD95L. These findings may provide molecular insight into the role of endogenous Nef in the T-cell depletion observed in vivo, particularly HIV-specific cytotoxic CD8(+) T cells.

Objective: To evaluate the signal transduction potential of HIV-1 Tat in a neuronal cell model., Methods: The tyrosine phosphorylation levels of the focal adhesion kinase p125FAK and its association with phosphoinositide 3-kinase (PI 3-K) were evaluated in serum-starved rat pheochromocytoma PC12 cells, either treated with low concentrations (0.1-1 nM) of extracellular HIV-1 Tat protein or stably transfected with Tat cDNA., Results: Extracellular Tat induced a rapid increase of p125FAK tyrosine phosphorylation and p125FAK-associated PI 3-K activity. By using recombinant mutated Tat proteins, it was found that deletion of amino acids 73-86 encoded by the second exon of the tat gene resulted in a significant decrease of the ability of Tat to induce p125FAK tyrosine phosphorylation. Paradoxically, mutations in the basic region encoded by the first exon of tat, which is essential for nuclear localization and HIV-1 LTR transactivation, increased the ability of Tat to stimulate p125FAK tyrosine phosphorylation. Moreover, in comparison with cells transfected with a control vector, PC12 cells stably transfected with tat cDNA showed greater amounts of p125FAK protein, an increase in p125FAK tyrosine phosphorylation and higher levels of p125FAK-associated PI 3-K activity. The addition of anti-Tat neutralizing antibody to tat-transfected PC12 cells in culture blocked both the p125FAK tyrosine phosphorylation and its association with PI 3-K but did not affect the total amount of p125FAK., Conclusion: HIV-1 Tat protein enhanced both the expression and the functionality of p1 25FAK in PC12 neuronal cells. Whereas the first event required intracellular Tat, the increased p125FAK phosphorylation was strictly dependent upon extracellular Tat.

Extracellular HIV-1 Tat protein (0.1-100 ng/ml) induced a rapid (peak at 30 min) increase in the Ser133 phosphorylation levels of the transcription factor CREB in serum-starved Jurkat cells, as revealed by Western blot and indirect immunofluorescence analyses. Nuclear cAMP-responsive element (CRE) binding activity in electrophoretic mobility shift assays was constitutive in unstimulated Jurkat cells, showing only a small increase upon Tat treatment. However, transient transfection experiments performed with various chloramphenicol acetyl-transferase (CAT) constructs showed that Tat produced a fourfold induction of CAT activity only in the presence of a CRE-dependent CAT construct. Moreover, the use of plasmids encoding for GAL4-CREB fusion proteins demonstrated that Tat induction of pG4-CAT reporter gene required the CREB moiety of the GAL4-CREB fusion protein and that Ser133 CREB was essential for Tat activity. Extracellular Tat also stimulated Ser133 CREB phosphorylation in freshly isolated PBMC; this effect was completely blocked by either staurosporin, a broad-spectrum inhibitor of various protein kinases, or PD 98059, a specific inhibitor of mitogen-activated protein kinases (MAPK). Furthermore, extracellular Tat induced a rapid (peak at 5-15 min) stimulation of the MAPK catalytic activity in primary PBMC. Altogether, these findings suggest that HIV-1 Tat protein activates CREB in lymphoid cells through a signal cascade involving the MAPK pathway.

The addition in culture of extracellular HIV-1 Tat protein (0.1-1 nM) to PC12 cells induced a rapid increase of the bulk protein kinase C (PKC) catalytic activity. Among various PKC isoforms (alpha, beta I, beta II, delta, epsilon, eta, theta, and zeta) expressed in PC12 cells, Tat selectively stimulated alpha, epsilon, and zeta, as judged by activities in immunoprecipitates. Activation of these isoforms was suppressed by the tyrosine kinase inhibitor genistein. Moreover, PKC-zeta showed the fastest kinetics of activation in response to Tat, but PKC-alpha and PKC-epsilon showed the highest levels of activation. PKC-alpha activation was accompanied by a rise of intracellular IP3, while the PI 3-kinase inhibitors wortmannin and LY294002 suppressed PKC-epsilon activation. Taken together, these findings demonstrate that extracellular Tat shows a cytokine-like activity in PC12 cells, being able to trigger an intracellular signalling cascade which involves PKC-alpha, -epsilon, and -zeta.

The regulatory Tat protein of the human immunodeficiency virus type-1 (HIV-1) is essential for viral replication and also shows pleiotropic activities on various cell functions. To get further insights into the molecular mechanisms underlying the biological activity of Tat, we investigated the effect of endogenous and exogenous Tat protein on c-fos gene expression in T lymphoblastoid (Jurkat) and monocytic (U937) cell lines, as well as in primary peripheral blood mononuclear cells (PBMC). Transient cotransfection of tat cDNA in sense orientation (tat/S), together with a plasmid containing the c-fos promoter (FC3, from -711 to +42) in front of the bacterial chloramphenicol acetyltransferase (CAT) gene significantly enhanced CAT activity in Jurkat cells activated by the addition of 15% fetal calf serum (FCS) or 5 micrograms/mL phytohemagglutinin plus 10(-7) mol/L phorbol myristate acetate (PMA) and U937 cells activated by 15% FCS or 10(-7) mol/L PMA. This effect was specifically due to Tat, since Jurkat and U937 cells cotransfected either with tat cDNA in antisense orientation (tat/AS), tat carrying a mutation in the aminoacid cys22-gly22 (tat 22/S) or with the backbone vector alone (pRPneo-SL3) did not show any significant difference in c-fos promoter activity as compared to cells transfected with FC3 plasmid alone. By using deletion mutants of the c-fos promoter, we found that the minimal DNA sequence required for Tat activity was located between nucleotides -404/-220 and that the serum responsive element (SRE, -317/-288), present within this region, was still responsive to Tat. A single point mutation in the SRE completely abrogated the responsiveness to tat/S. Exogenous recombinant Tat protein was also able to upregulate c-fos promoter activity in serum-activated Jurkat and U937 cells, as well as endogenous c-fos mRNA expression and c-Fos protein synthesis in both serum-activated cell lines and primary PBMC. c-Fos protein was shown essential for an optimal transactivation of the HIV-1 long terminal repeat (LTR) by Tat: incubation of Jurkat cells with antisense, but not sense, c-fos oligonucleotides significantly reduced either the Tat-enhanced expression of an LTR-CAT reporter construct or the levels of gag p24 in the culture supernatants of Jurkat cells and PBMC acutely infected with HIV-1. Our data suggest that the c-fos upregulation mediated by Tat might play a significant role in the control of viral gene transactivation.

We have here investigated the effect of the regulatory Tat protein of the human immunodeficiency virus type 1 (HIV-1) on the PI 3-kinase catalytic activity in PC12 rat pheochromocytoma cells. After as early as 1 min from the beginning of the treatment with recombinant HIV-1 Tat protein, a significant increase in the tyrosine phosphorylation levels of the p85 regulatory subunit of PI 3-kinase was noticed in 48 h serum-starved PC12 cells. Moreover, the addition of Tat to PC12 cells induced a great increase in PI 3-kinase immunoprecipitated with an anti-phosphotyrosine antibody with a peak of activity (19-fold increase with respect to the basal levels) after a 15-min treatment. This increase in PI 3-kinase activity was significantly higher in PC12 cell cultures supplemented with Tat protein than in cultures stimulated by 100 ng/ml nerve growth factor (NGF; 8-fold increase with respect to the basal levels). Further experiments showed that Tat protein was able to specifically activate PI 3-kinase at picomolar concentrations. In fact: (i) maximal activation of PI 3-kinase was observed at concentrations as low as 1 ng/ml and was specifically blocked by anti-Tat neutralizing antibody; (ii) a Tat-dependent activation was also observed in experiments in which PI 3-kinase activity was evaluated in either anti-Tyr(P) or anti-p85 immunoprecipitates; (iii) 100 nM wortmannin completely blocked the Tat-mediated increase in PI 3-kinase activity both in vitro and in vivo. Our data strongly support the concept that extracellular Tat acts as a cell stimulator, inducing intracellular signal transduction in uninfected cells.

CD3 mAb and HIV-1 Tat protein co-immobilized on plastic were able to induce a strong proliferation of resting human CD4 T cells, cultured in a serum-free chemically defined medium. Blocking studies performed with heparin or peptides containing the RGD sequence demonstrated that the heparin-binding basic domain of Tat plays a predominant role in CD4+ T cell activation. Moreover, the enhanced proliferative response of CD4+ T cells to immobilized Tat appeared to be mediated by alpha 5, beta 1, and alpha v subunits of surface integrin receptors. In contrast, soluble Tat showed a dose-dependent inhibitory activity on the proliferative response of resting CD4+ T cells stimulated by CD3 mAb co-immobilized with Tat or fibronectin, but not with CD28 mAb. In transient transfection assays performed with an HIV-1 long terminal repeat (LTR)-chloramphenicol acetyltransferase (CAT) plasmid CD3 mAb co-immobilized with Tat or fibronectin or CD28 mAb significantly stimulated CAT activity over the background. On the other hand, while immobilized Tat alone had no effects on LTR transactivation, soluble Tat was able to transactivate LTR-CAT in a dose-dependent manner. When CD4+ T cells activated by CD3 mAb co-immobilized with Tat were recovered, cultured for 7 days with 25 U/ml recombinant IL-2, and given an additional activation signal by recross-linking CD3 mAb, a marked increase of apoptosis was observed with respect to cells not subjected to CD3 mAb recross-linking. While co-immobilized Tat plus CD3 mAb did not show any significant effect on activation-induced cell death, high concentrations of soluble Tat synergized with immobilized CD3 mAb in the induction of apoptosis.

Peripheral blood cytopenias and bone marrow abnormalities are frequently observed in HIV-1-seropositive subjects. Two major mechanisms have been proposed to explain the hematopoietic failure often observed in patients with advanced HIV-1 disease: (i) infection of cells composing the bone marrow microenvironment with a deranged production of hematopoietic growth factors; (ii) direct suppression of hematopoietic progenitor cells mediated by HIV-1 virions and/or viral proteins. In vivo and in vitro experimental evidence supports a combination of both mechanisms. In fact, it has been shown that: (i) infection with HIV-1 and/or exposure of bone marrow accessory cells to envelope glycoprotein 120 (env gp 120) increases the production of inhibitory cytokines such as tumor necrosis factor alpha; (ii) a subset of CD34+ hematopoietic progenitor cells co-expresses the CD4 antigen and may be infected in vivo with HIV-1; (iii) HIV-1 virions or immune complexes containing env gp 120 are able to induce apoptosis of uninfected CD34+ hematopoietic progenitors. This last inhibitory effect appears to be mediated by the upregulation of transforming growth factor beta 1, which is endogenously produced by hematopoietic progenitors. Both the load and the biological characteristics of the virus play an important role in causing these suppressive effects, since different HIV-1 isolates display varying abilities to suppress hematopoiesis, and some isolates are not cytopathic at all.

Human CD34+ hematopoietic progenitor cells, stringently purified from the peripheral blood of 20 normal donors, showed an impaired survival and clonogenic capacity after exposure to either heat-inactivated human immunodeficiency virus (HIV) 1 (strain IIIB) or cross-linked envelope gp120. Cell cycle analysis, performed at different times in serum-free liquid culture, showed an accumulation in G0/G1 in HIV-1- or gp120-treated cells and a progressive increase of cells with subdiploid DNA content, characteristic of apoptosis. In blocking experiments with anti-transforming growth factor (TGF) beta 1 neutralizing serum or TGF-beta 1 oligonucleotides, we demonstrated that the HIV-1- or gp120-mediated suppression of CD34+ cell growth was almost entirely due to an upregulation of endogenous TGF-beta 1 produced by purified hematopoietic progenitors. Moreover, by using a sensitive assay on the CCL64 cell line, increased levels of bioactive TGF-beta 1 were recovered in the culture supernatant of HIV-1/gp120-treated CD34+ cells. Anti-TGF-beta 1 neutralizing serum or TGF-beta 1 oligonucleotides were also effective in inducing a significant increase of the plating efficiency of CD34+ cells, purified from the peripheral blood of three HIV-1-seropositive individuals, suggesting that a similar mechanism may be also operative in vivo. The relevance of these findings to a better understanding of the pathogenesis of HIV-1-related cytopenias is discussed.

In HIV-1-infected cell cultures, a relatively low concentration (5 micrograms/ml) of monoclonal antibody (mAb) against HIV-1-transactivating Tat protein was an efficient inhibitor of HIV-1 replication both in HIV-1(IIIB)-infected Jurkat cell and peripheral blood mononuclear cell (PBMC) cultures and significantly reduced the expression of a Tat-responsive CAT-reporter construct in HIV-1(IIIB)-infected Jurkat cells. Anti-Tat mAb also caused a significant reduction and a consistent delay in HIV-1 replication when added to PBMCs from HIV-1-infected patients cocultivated with phytohemagglutinin (PHA)-stimulated normal PBMCs. These data indicate that an autocrine-paracrine loop sustained by extracellular Tat protein, which is actively released by HIV-1-infected cells, may affect HIV-1 replication in cell cultures in vitro. An inverse relationship between natural anti-Tat antibody levels and p24 antigenemia was demonstrated by retrospective analysis of serial serum samples obtained from 10 HIV-1-seropositive hemophiliac patients followed over a 7-9-year period. This datum points to a possible influence of anti-Tat antibody on the progression of HIV-1 disease in vivo. These findings have strong implications for Tat protein as a possible target for specific immunotherapy in HIV-1-infected patients.

The regulatory Tat protein of human immunodeficiency virus type-1 (HIV-1) exerts a pleyotropic activity on the survival and proliferation of different cell types in culture. In this report, we investigated the effect of either endogenous or exogenous Tat on Bcl-2 proto-oncogene expression and cell survival in Jurkat T-cell lines and primary peripheral blood mononuclear cells. Stable and transient transfections of Jurkat cells with the cDNA of tat and a plasmid containing Bcl-2 promoter in front of CAT (Bcl-2 Pr/CAT) stimulated CAT activity and showed an increase of Bcl-2 mRNA and protein expression. This effect was specifically related to tat, because Jurkat cells transfected with the cDNA of tat in antisense orientation, tat carrying a mutation in the amino acid cys22-gly22, or the control vector alone (pRPneo-SL3) did not show any significant difference in Bcl-2 promoter activity with respect to parental Jurkat cells. We also observed a specific correlation between tat-induced Bcl-2 gene expression and inhibition of apoptosis induced by serum withdrawal. Our results suggest that the structural integrity of the activation domain of Tat was required for the promotion of the Bcl-2 promoter and Jurkat cell survival, because a single mutation in the aminoacid cys22 was sufficient to completely block the upregulation of Bcl-2 and inhibition of apoptosis. Moreover, picomolar concentrations of native or recombinant Tat were able to upregulate Bcl-2 expression both in Jurkat and primary peripheral blood mononuclear cells, suggesting that extracellular Tat, actively released by infected cells, may also play a significant role in suppressing apoptosis. An aberrant cell survival of lymphoid cells consequent to the upregulation of Bcl-2 may represent an additional pathogenetic mechanism that could help explain both the dysregulated immune response and the frequent occurrence of hyperplastic/neoplastic disorders in HIV-1-seropositive individuals.

Human immunodeficiency virus type 1 (HIV-1) transactivating Tat protein is pivotal to virus replication. Tat's potential effects on HIV-1 pathogenesis, however, go well beyond its role in the virus's life cycle. Current data indicate that biologically active Tat is released from HIV-1-infected cells and readily endocytosed and targeted to the nucleus of nearby, or perhaps distant, cells, where it may exert a series of pleiotropic effects. This paracrine action has been extensively investigated, and depending on the amounts of exogenously added Tat, its effects may extend from the suppression of immunocompetent cells to transactivation of heterologous genes to the promotion of growth of Kaposi's sarcoma spindle cells. We have already observed that various cell lines, either permanently transfected with an expressive HIV-1 tat gene construct or cultured in the presence of exogenously added Tat protein, are protected from programmed cell death after serum withdrawal or other apoptotic stimuli. The present article shows that various types (lymphoblastoid, epithelial, neuronal) of permanently tat-transfected cell lines actively release fully bioactive Tat protein. The addition of anti-Tat antibody to the culture medium completely abolishes their increased survival/proliferation capacity in serum-free culture. In these conditions, therefore, the enhanced survival/proliferation potential of permanently tat-transfected cells seems entirely dependent on a Tat-protein autocrine loop. The finding that anti-Tat antibody, added to culture medium, exerts a negative influence on the expression of a Tat-responsive HIV-1 long terminal repeat chloramphenicol-acetyltransferase construct, transiently transfected into permanently tat-transfected cells, suggests that the Tat autocrine loop may also be required for optimal HIV-1 long terminal repeat transactivation.

We investigated whether cells belonging to the megakaryocytic lineage could be infected in vitro with human immunodeficiency virus type-1 (HIV-1). Primary GPIIb/IIIa+ bone marrow (BM) cells and HEL continuous cell line were first phenotypically characterized for the presence of megakaryocytic markers and CD4 antigen, then challenged in vitro with the laboratory strain IIIB of HIV-1. Both GPIIb/IIIa+ BM and HEL cells expressed significant levels of CD4 receptor (> 50%) and were efficiently infected with HIV-1, as judged by the presence of proviral DNA after polymerase chain reaction analysis and by quantitative evaluation of gag p24 antigen in the culture supernatants. Of note, infection with HIV-1 in both primary BM megakaryocytes and HEL cells was specifically blocked by soluble recombinant CD4. To ascertain whether the CD4 receptor was essential for infection of megakaryocytic cells, HEL were subcloned into CD4+ and CD4- cells. Although unfractionated and CD4+ HEL cells were productively infected with HIV-1, CD4- HEL cells could not be infected. Infection of HEL cells did not induce gross cytotoxic effects or a significant increase of apoptosis. On the other hand, treatment of unfractionated or CD4+ HEL cells with cross-linked recombinant env gp120 or Leu3a anti-CD4 monoclonal antibody markedly (P < 0.01) increased the degree of apoptosis with respect to HEL cells infected with HIV-1 or treated with cross-linked gag p24 or anti-GPIIb/IIIa antibody. Taken together, these data indicate that the CD4 receptor represents the main route of infection in cells belonging to the megakaryocytic lineage. Moreover, an inappropriate engagement of CD4 by either free env gp120 or anti-CD4 monoclonal antibody could be more relevant than a direct infection with HIV-1 in the induction of the frequent BM megakaryocyte abnormalities found in HIV-1 seropositive thrombocytopenic patients.

Human CD4+ T lymphoblastoid Jurkat cells were stably transfected with two different plasmid vectors containing the cDNA of human immunodeficiency virus-type 1 (HIV-1) tat gene under the control of either the promoter of simian virus 40 (pRPneo/tat) or the long terminal repeat region of SL3 murine leukaemia virus (pRPneo/SL3/tat). Both pRPneo/tat and pRPneo/SL3/tat Jurkat cell lines showed a constant and high production of bioactive Tat in transient co-transfection assays with an HIV-1 long terminal repeat (LTR)-chloramphenicol acetyltransferase (CAT) reporter plasmid. Tat-positive and mock-transfected Jurkat cells were cultured with various cytotoxic agents, which have been associated to the progressive loss of CD4 T-lymphocytes characteristic of HIV-1 disease. In the presence of recombinant tumour necrosis factor-alpha (TNF-alpha), anti-fas antibody, Leu3a anti-CD4 antibody, the percentage of apoptosis, evaluated in a 24-72 h short-term assay, was lower (P < 0.05) in tat-positive Jurkat cells than in mock-transfected controls. The low susceptibility to the cytotoxic activity of TNF-alpha and anti-fas antibody of tat-transfected cells was confirmed by counting viable cells up to 15 d of culture. Also, recombinant Tat protein was able to prevent the increase of apoptosis induced in mock-transfected Jurkat by TNF-alpha. Of note, tat-expressing cells showed a better survival with respect to mock-transfected control cells even when acutely infected with high doses (500,000 cpm of reverse transcriptase) of HIV-1 (strain IIIB) or treated with heat-inactivated HIV-1. These data demonstrate that the expression of the regulatory HIV-1 Tat protein is able to rescue Jurkat lymphoblastoid cells from apoptosis induced by a variety of cytotoxic agents. Since Tat protein expression is restricted to the initial phases of an active HIV-1 replication, the anti-apoptotic effect of Tat could have the physiological significance of selectively protecting HIV-1 producing cells from death, at least for the time necessary to allow virus production and spreading.

In this study we evaluated the effect of human immunodeficiency virus type 1 (HIV-1) recombinant Tat protein on mRNA expression and protein synthesis of two inflammatory cytokines-interleukin-6 (IL-6) and transforming growth factor-beta 1 (TGF-beta 1)-by peripheral blood (PB) monocytes. Whereas maximal levels of IL-6 protein were recovered in PB monocyte culture supernatants after 24-48 h from the addition of 1 micrograms/ml of recombinant Tat, TGF-beta 1 showed a slower and progressive increase, reaching maximal levels only after 72-96 h of culture. Consistently, the analysis of the steady-state levels of mRNA showed a sharp increase of IL-6 mRNA expression after 24h of culture, with a slow decline thereafter. On the other hand, TGF-beta 1 mRNA expression showed a slow increase only after 72-96 h of culture. Moreover, IL-6 appeared involved in the up-regulation of TGF-beta 1, because the addition of a neutralizing anti-IL-6 antibody to Tat-treated PB monocyte cultures significantly reduced the amounts of TGF-beta 1 recovered in the culture supernatants after 96 h. The present demonstration that HIV-1 Tat protein directly up-regulates IL-6 expression and stimulates TGF-beta 1 production both directly and indirectly, through early IL-6 production, could have important implications in the pathogenesis of HIV-1 disease.

Starting from our previous observations that the HIV-1-mediated engagement of CD4 induced apoptotic death of TF-1 hematopoietic progenitor cells, in this study we evaluated PKC activity and intracellular Ca2+ levels in TF-1 cells treated with viable and heat-inactivated HIV-1 (strain IIIB) or anti-CD4 Leu3a monoclonal antibody (mAb). Both viable and heat-inactivated HIV-1 or anti-CD4 mAb, but not anti-human cytomegalovirus (HCMV) 66kD protein or anti-CD8 mAb induced a rapid (5-10 min) increase in PKC activity under both serum-containing and serum-free conditions. The same treatment also induced both a transient and a long-lasting (48 hours) decrease (p < 0.05) in intracellular Ca2+ levels in serum-containing cultures. We propose that the observed changes in PKC activity and intracellular Ca2+ levels might be involved in the HIV-1 mediated apoptosis of hematopoietic progenitor cells.

In this study, we evaluated the effect of a short-term exposure (2 hours) to two different lymphocytotropic strains of human immunodeficiency virus type 1 (HIV-1; HIVIIIB and ICR-3) on the survival of a factor-dependent CD34+ hematopoietic progenitor cell line (TF-1). At flow cytometry analysis, a significant (P < .05) increase in the frequency of apoptotic cell death was observed in HIV-1-treated TF-1 cells, supplemented with low doses of either interleukin-3 (IL-3; 0.02 to 1 ng/mL) or granulocyte-macrophage colony-stimulating factor (GM-CSF; 0.02 to 0.2 ng/mL) with respect to mock-treated cells. On the other hand, higher doses of both cytokines or combinations of suboptimal concentrations of IL-3 plus GM-CSF (eg, 0.2, plus 0.2 ng/mL) completely reversed the HIV-1-induced increase of apoptosis. Remarkably, no signs of productive or latent virus replication were ever observed in HIV-1-treated TF-1 cells up to 16 days of liquid culture. In parallel experiments, the in vitro exposure to HIVIIIB induced a significant and progressive increase of apoptotic death in purified bone marrow CD34+ cells, seeded in liquid cultures in the presence of 1 ng/mL IL-3. The HIV-1-induced apoptosis of TF-1 cells was likely triggered by the simple interaction of HIV-1 envelope glycoprotein gp120 with CD4 receptor, which was expressed at a low level on the surface of TF-1 cells. In fact, treatment of TF-1 cells with recombinant gp120 plus a polyclonal anti-gp120 antibody or with anti-CD4 monoclonal antibody plus rabbit antimouse IgG significantly increased the percentage of apoptotic death. These data suggest that HIV-1, and perhaps also free gp120 in the presence of anti-gp120 antibody; could play a direct role in the pathogenesis of peripheral blood cytopenias in acquired immunodeficiency syndrome patients by inducing apoptotic death of hematopoietic progenitor cells without the need of a direct infection.

Rat pheochromocytoma PC12 cells were permanently transfected with a plasmid vector, containing the tat gene of human immunodeficiency virus type 1 (HIV-1). Various clones were obtained showing the production of different levels of bioactive Tat protein (Tat) after transient cotransfection with an HIV-1 long terminal repeat-chloramphenicol acetyltransferase reporter plasmid. Under conditions of serum starvation, tat-positive PC12 clones expressing high levels of Tat showed a significantly (P < 0.05) higher proliferation rate with respect to both mock-transfected PC12 cells and tat-positive PC12 cells expressing lower levels of Tat. Moreover, all tat-positive PC12 cell clones showed a partial morphological differentiation into sympathetic-like neurons, when seeded in low density (5 x 10(3) cells/cm2) cultures. On the other hand, mock-transfected PC12 cells showed the round shaped morphology typical of untreated PC12 cells and displayed signs of neuronal differentiation only after treatment with 100 ng/ml of nerve growth factor. The addition of 5 micrograms/ml of anti-Tat monoclonal antibody to the culture medium of tat-positive PC12 cell clones almost completely blocked their increased proliferation rate (P < 0.05), but did not affect neuronal differentiation. A significant (P < 0.05) increase in cell proliferation was consistently observed in PC12 cells supplemented with low concentrations of Tat (5 to 25 ng/ml), whereas neuronal differentiation was hardly affected by exogenous Tat. Our data strongly suggest that Tat exerts a complex influence on the proliferation and differentiation of PC12 cells, and this might help in increasing understanding of the pathogenesis of the frequent neurological disorders observed in AIDS patients.

We report here that the tat gene product of human immunodeficiency virus type 1 was able to protect lymphoblastoid (Jurkat), epithelial (293) and neuronal (PC12) cell lines from apoptotic death induced by serum withdrawal. The rescue from apoptosis by Tat was reflected by an increased expression of Bcl-2 protein in tat-positive Jurkat cells with respect to mock-transfected Jurkat cells after 3-6 days of serum-free cultures. We propose that the ability of the regulatory human immunodeficiency virus type 1 Tat protein to suppress apoptosis might have important implications in understanding the pathogenesis of frequent neoplastic disorders observed in human immunodeficiency virus type 1-seropositive individuals.

Objective: To determine the mechanism underlying the poor growth in vitro of haematopoietic progenitor cells isolated from HIV-1-infected patients., Method: Apoptotic death in liquid culture of bone-marrow CD34+ cells obtained from 11 HIV-1-seropositive patients and 18 HIV-1-seronegative donors was quantitatively monitored by a flow cytometry procedure., Results: No significant differences in the percentage of apoptotic cells were noted between the two groups immediately after purification. When CD34+ cells were placed in liquid cultures supplemented with 2 ng/ml interleukin-3, the number of apoptotic cells progressively and significantly (P < 0.05) increased in all HIV-1-seropositive patients, while it remained constant in HIV-1-seronegative individuals. Although all HIV-1-seropositive patients showed signs of active viral replication in the bone-marrow micro-environment, progenitor CD34+ cells did not show the presence of active and/or latent HIV-1 infection., Conclusion: Our data demonstrate that CD34+ cells isolated from AIDS patients with active HIV-1 replication in bone-marrow accessory cells are committed to apoptotic death without being directly affected by productive infection.

In this study we evaluated the effect of human immunodeficiency virus type 1 (HIV-1) recombinant tat-protein on the production of interleukin-6 (IL-6), granulocyte/macrophage colony stimulating factor (GM-CSF) and tumor necrosis factor-alpha (TNF-alpha) by purified peripheral blood monocytes. Whereas no effects were observed on TNF-alpha and GM-CSF production, recombinant tat-protein was able to induce the production of IL-6 by peripheral blood monocytes in a dose-dependent fashion for concentrations ranging from 1 ng/ml to 1 micrograms/ml. Pre-exposure of tat-protein with a polyclonal neutralizing anti-tat antibody (dilution 1:100) completely abrogated the tat-dependent increase in IL-6 production. The ability of tat-protein to selectively stimulate the production of IL-6 by peripheral blood monocytes could help to explain the presence of elevated levels of IL-6 in the serum of HIV-1 seropositive individuals, especially in patients in advanced stages of the disease with an active viral replication.