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. 2000 Dec 19;97(26):14620-5.
doi: 10.1073/pnas.011512398.

Pivotal Role of Cyclic Nucleoside Phosphodiesterase 4 in Tat-mediated CD4+ T Cell Hyperactivation and HIV Type 1 Replication

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Free PMC article

Pivotal Role of Cyclic Nucleoside Phosphodiesterase 4 in Tat-mediated CD4+ T Cell Hyperactivation and HIV Type 1 Replication

P Secchiero et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

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.

Figures

Figure 1
Figure 1
Decrease in the intracellular cAMP levels in CD4+ T cells by Tat. Primary CD4+ T lymphocytes were pretreated with either IBMX or equivalent volumes of DMSO dilution buffer for 45 min and then seeded in wells coated with dilution buffer (BSA), Tat alone, or anti-CD3/CD28 ± Tat. Cell extracts were analyzed for the amount of intracellular cAMP after 4 h of culture. Data are expressed as means ± SD of four independent experiments performed in triplicate.
Figure 2
Figure 2
Costimulatory effect of Tat on IL-2 production in primary CD4+ T lymphocytes. The amount of IL-2 was evaluated after 16 h in both cell lysates (intracellular IL-2) and in the supernatant (extracellular IL-2) of cultures seeded on wells coated as indicated. Data are expressed as means ± SD of 4–9 separate experiments performed in triplicate.
Figure 3
Figure 3
Costimulatory effect of immobilized Tat on CD4+ T cell proliferation (A) and Tat peptides on IL-2 production and cell proliferation (B and C). Primary CD4+ T cells were seeded in wells coated with dilution buffer (BSA), anti-CD3, anti-CD3/CD28, anti-CD3/CD28 + Tat, or anti-CD3/CD28 + Tat peptides (Pept. 1–5). In A and C, [3H]thymidine incorporation was measured by liquid scintillation, and results are expressed as arithmetic mean cpm of triplicate cultures. The results represent the means ± SD of 4–6 separate experiments, performed in duplicate. In B, the amount of IL-2 was evaluated after 16 h in the culture supernatant of cultures seeded on wells coated as indicated. Data are expressed as means ± SD of five separate experiments performed in triplicate.
Figure 4
Figure 4
Expression of PDEs in CD4+ T cells. The expression of PDEs was examined in freshly purified primary CD4+ T cells by specific RT-PCR. For RT-PCR, equivalent amounts of RNA samples, before (−) and after (+) RT, were used as a template for the amplification reaction with the specific primers. The expected amplification products (PDE3 = 938 bp, PDE4A = 272 bp, PDE4B = 363 bp, and PDE7 = 285 bp) are visualized by ethidium bromide staining. Lane M, 100-bp ladder of molecular weight marker. The data are representative of three experiments performed on separate cell preparations.
Figure 5
Figure 5
Effect of PDE inhibitors on the Tat-mediated increase of IL-2 production in CD4+ T cells. The amount of released IL-2 was evaluated after 4 and 16 h in the culture supernatant of CD4+ T cells seeded on wells coated with anti-CD3, anti-CD3/CD28 ± Tat in the presence of DMSO, IBMX, rolipram, cilostimide, and Rp-8-Br-cyclic AMPS. Data are expressed as means ± SD of five separate experiments performed in triplicate.
Figure 6
Figure 6
Effect of PDE inhibitors on the Tat-mediated increase in [3H]thymidine incorporation in CD4+ T cells. [3H]Thymidine incorporation was measured in CD4+ T cells treated with DMSO, IBMX, rolipram, and cilostimide. Data are expressed as means ± SD of four separate experiments performed in triplicate.
Figure 7
Figure 7
Effect of PDE inhibitors on HIV-1 replication. p24 levels were evaluated in cultures of primary CD4+ T lymphocytes, seeded on wells coated with anti-CD3, anti-CD3/CD28 ± Tat, or Pept. 4 and then infected or not with HIV-1. The index of HIV-1 replication was calculated as the ratio of p24 at day 8/day 1 after infection (fold of p24 increase). Data are expressed as means ± SD of three separate experiments performed in duplicate.

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