Immunological properties of a DNA plasmid encoding a chimeric protein of herpes simplex virus type 2 glycoprotein B and glycoprotein D.

A DNA plasmid containing a chimeric sequence encoding both herpes simplex virus type 2 (HSV-2) glycoprotein B (gB) and glycoprotein D (gD) external domains (pcgDB) was used to immunize BALB/c mice against genital HSV-2 infection. To determine the efficacy of this vaccine, groups of mice immunized with the pcgDB plasmid were compared with animals immunized with plasmids corresponding to the individual proteins (pcgBt or pcgDt), administered separately or in combination (pcgBt + pcgDt). We studied the response of the different mouse groups to viral challenge by analyzing clinical disease (vaginitis), serum antibody levels, as well as lymphoproliferative responses and cytokine production by spleen cells. Increased IFN-gamma levels correlated with prolonged survival in mice immunized with the plasmid pcgDB, relative to mice immunized with plasmids coding for the individual proteins alone or in combination. Our results show that immunization with the plasmid encoding the chimeric protein is advantageous over separate proteins. These findings may have important implications for the development of multivalent DNA vaccines against HSV and other complex pathogens.

Phosphodiesterase 4 inhibitors prevent cytokine secretion by T lymphocytes by inhibiting nuclear factor-kappaB and nuclear factor of activated T cells activation.

Blockade of phosphodiesterase 4 with rolipram reduced the production of tumor necrosis factor (TNF)-alpha, interleukin (IL)-5, IL-10, and IL-2 but poorly inhibited cell proliferation and interferon-gamma (IFN-gamma) production by activated human T cells. Addition of dibutyryl cAMP mimicked rolipram inhibitions on proliferation, IL-2, TNF-alpha, and IFN-gamma but not on IL-10 or IL-5 production. Moreover, the inhibitory effects of rolipram on proliferation, IFN-gamma, and TNF-alpha but not of IL-10 production can be prevented by a specific protein kinase A inhibitor. Rolipram and pentoxifylline, a nonspecific phosphodiesterase inhibitor, decreased transcription of IL-2 and TNF-alpha promoters in transiently transfected normal T cells. Moreover, they inhibited the activation of nuclear factor-kappaB (NF-kappaB) and nuclear factor of activated T cells (NFAT) and stimulated activator protein-1 (AP-1) and cAMP response element-binding proteins (CREBs). In contrast, dibutyryl cAMP inhibited NF-kappaB but not NFAT activation. Thus, our data indicate that blockade of phosphodiesterase 4 regulates transcription of a particular cytokine through inhibition of NF-kappaB and NFAT, and stimulation of AP-1 and CREB.

HIV-1 Tat inhibits IL-2 gene transcription through qualitative and quantitative alterations of the cooperative Rel/AP1 complex bound to the CD28RE/AP1 composite element of the IL-2 promoter.

Dysregulation of cytokine secretion plays an important role in AIDS pathogenesis. Here, we demonstrate that expression of HIV-1 Tat protein in Jurkat cells induces a severe impairment of IL-2 but not TNF gene transcription. Interestingly, this inhibition correlates with the effect of the viral protein on the transactivation of the CD28RE/AP1 composite element (-164/-154), but not with that observed on the NFAT/AP1 site of the IL-2 gene promoter, neither with the effect on NF-kappa B- nor AP1-independent binding sites. Endogenous expression of Tat induced a decrease in the amount of the specific protein complex bound to the CD28RE/AP1 probe after PMA plus calcium ionophore stimulation. This effect was accompanied by qualitative alterations of the AP1 complex. Thus, in wild-type Jurkat cells, c-jun was absent from the complex, whereas in Tat-expressing cells, c-jun was increasingly recruited overtime. By contrast, similar amounts of c-rel and a small amount of NFAT1 were detected both in wild type and in Jurkat Tat(+) cells. Furthermore, Tat not only induced the participation of c-jun in the cooperative complex but also a decrease in its transactivation activity alone or in combination with c-rel. Thus, the interaction of Tat with the components of this rel/AP1 cooperative complex seems to induce quantitative and qualitative alterations of this complex as activation progresses, resulting in a decrease of IL-2 gene transcription. Altogether our results suggest the existence of tuned mechanisms that allow the viral protein to specifically affect cooperative interactions between transcription factors.

An essential role of the nuclear factor of activated T cells in the regulation of the expression of the cyclooxygenase-2 gene in human T lymphocytes.

We have previously reported that transcriptional induction of cyclooxygenase-2 (COX-2) isoenzyme occurs early after T cell receptor triggering, suggesting functional implications of cyclooxygenase activity in this process. Here, we identify the cis-acting elements responsible for the transcriptional activation of this gene in human T lymphocytes. COX-2 promoter activity was induced upon T cell activation both in primary resting T lymphocytes and in Jurkat cells. This induction was abrogated by inhibition of calcineurin phosphatase with the immunosuppressive drug cyclosporin A, whereas expression of an active calcineurin catalytic subunit enhanced COX-2 transcriptional activation. Moreover, cotransfection of nuclear factor of activated T cells (NFAT) wild type protein transactivated COX-2 promoter activity. Conversely, dominant negative mutants of NFATc or c-Jun proteins inhibited COX-2 induction. Electrophoretic mobility shift assays and site-directed mutagenesis allowed the identification of two regions of DNA located in the positions -117 and -58 relative to the transcriptional start site that serves as NFAT recognition sequences. These results emphasize the central role that the Ca(2+)/calcineurin pathway plays in COX-2 transcriptional regulation in T lymphocytes pointing to NFAT/activator protein-1 transcription factors as essential for COX-2 promoter regulation in these cells.

HIV-1 infection induces differentiation of immature neural cells through autocrine tumor necrosis factor and nitric oxide production.

Immature neural cell lines could be productively infected by HIV-1. Interestingly, this infection was associated with a differentiation to a mature neuronal phenotype, characterized by the expression of mature neurofilaments and cell adhesion molecules, intercellular cell adhesion molecule-1, and vascular cell adhesion molecule-1. Infection also induced TNF-alpha and IL-1beta mRNA expression, as well as the synthesis of inducible nitric oxide synthase by neuroblastoma cells. Exogenous addition of TNF-alpha, but not of IL-1beta or many other cytokines, including nerve growth factor, mimicked those effects induced by infection. Moreover, blocking endogenous TNF-alpha or NO production in cultures of infected cells with a neutralizing anti-TNF-alpha antibody or inducible nitric oxide synthase inhibitors prevented the expression of the mature cell phenotype as well as expression of intercellular cell adhesion molecule-1 and vascular cell adhesion molecule-1. Addition of NO generators and TNF-alpha activated NF-kappaB- and intercellular cell adhesion molecule-1-dependent promoter transcription, whereas inducible nitric oxide synthase inhibitors prevented the transcriptional activation of intercellular cell adhesion molecule-1 promoter that was induced by TNF-alpha. Those results suggest that HIV can infect immature neural cells and this infection induces their neural development via a TNF-alpha- and NO-mediated mechanism.

Induction of cyclooxygenase-2 on activated T lymphocytes: regulation of T cell activation by cyclooxygenase-2 inhibitors.

Cyclooxygenase (COX), known to exist in two isoforms, COX-1 and COX-2, is a key enzyme in prostaglandin synthesis and the target for most nonsteroidal anti-inflammatory drugs. In this study, we show that human T lymphocytes express the COX-2 isoenzyme. COX-2 mRNA and protein were induced in both Jurkat and purified T cells stimulated by TCR/CD3 or PMA activation. COX-2 mRNA was induced very early after activation and superinduced by protein synthesis inhibitors, whereas it was inhibited by the immunosuppressive drug cyclosporin A, identifying it as an early T cell activation gene. Interestingly, treatment with COX-2-specific inhibitors such as NS398 or Celecoxib severely diminished early and late events of T cell activation, including CD25 and CD71 cell surface expression, IL-2, TNF-alpha, and IFN-gamma production and cell proliferation, but not the expression of CD69, an immediate early gene. COX-2 inhibitors also abolished induced transcription of reporter genes driven by IL-2 and TNF-alpha promoters. Moreover, induced transcription from NF-kappaB- and NF-AT-dependent enhancers was also inhibited. These results may have important implications in anti-inflammatory therapy and open a new field on COX-2-selective nonsteroidal anti-inflammatory drugs as modulators of the immune activation.

Inhibition of phosphodiesterase type IV suppresses human immunodeficiency virus type 1 replication and cytokine production in primary T cells: involvement of NF-kappaB and NFAT.

Rolipram, a phosphosdiesterase type IV-specific inhibitor, prevented p24 antigen release from anti-CD3-activated human immunodeficiency virus (HIV)-infected T cells and CD4(+)-cell depletion associated with viral replication in a dose-responsive manner but minimally inhibited T-cell proliferation. Moreover, rolipram reduced the production of tumor necrosis factor alpha (TNF-alpha) and interleukin-10 (IL-10) by HIV-infected T cells. The transcriptional ability of a luciferase reporter gene under control of the HIV long terminal repeat, induced by phorbol myristic acetate plus ionomycin or by TNF-alpha, in primary T and Jurkat cells was also inhibited by rolipram. Rolipram inhibited NF-kappaB and NFAT activation induced by T-cell activation in Jurkat and primary T cells, as measured by transient transfection of reporter genes and electrophoretic mobility shift assays. Exogenous addition of TNF-alpha in the presence of rolipram restored NF-kappaB but not NFAT activation or p24 release. Addition of dibutyryl-cyclic AMP (dBcAMP) mimicked the effects of rolipram on p24 antigen release, NF-kappaB activation, and TNF-alpha secretion, but it did not affect NFAT activation or IL-10 production. The protein kinase A inhibitor KT5720 prevented the inhibition of TNF-alpha secretion but not that of HIV type 1 (HIV-1) replication caused by rolipram. Our data indicate that blockade of phosphodiesterase type IV could be of benefit against HIV-1 disease by modulating cytokine secretion and transcriptional regulation of HIV replication, and they suggest an important role of NFAT in HIV replication in primary T cells. Some of those activities cannot be ascribed solely to its ability to increase cAMP.

Alteration of macrophage function by a Trypanosoma cruzi membrane mucin.

Cytokines secreted by macrophages play important roles in the immune response to Trypanosoma cruzi. Here, we report that a purified glycosylphosphatidylinositol (GPI)-anchored mucin from the T. cruzi membrane, Ag C10, is able to bind to the macrophage cell surface and blocks the subsequent binding of mAb against CD62L/L-selectin, whereas binding of mAbs directed against other monocyte surface molecules is unaffected. In addition, Ag C10 binding to macrophages triggered a CD54/ICAM-1-mediated cell adhesion as well as an increase in intracellular Ca2+, which was further augmented by cross-linking the Ag C10-bound surface receptors by mAb against Ag C10. Interestingly, Ag C10-treated monocytes secreted IL-1beta, but not TNF-alpha or IL-12. Moreover, these cells could secrete IL-1beta, but not TNF-alpha or IL-12, after activation with LPS. T. cruzi-infected macrophages displayed similar alterations in cytokine secretion, with an impaired ability to secrete IL-12 and TNF-alpha, but not IL-1beta, upon LPS activation. These effects were substantially inhibited by neutralizing mAb against Ag C10. These effects appeared to take place at the transcriptional level, since mRNA for TNF-alpha, but not that for IL-1beta, was drastically reduced in LPS-stimulated infected cells treated with Ag C10. Conceivably, inhibition of TNF-alpha and IL-12 by T. cruzi could be involved in the evasion of the immune response by this parasite.

Replication of human immunodeficiency virus-1 in primary human T cells is dependent on the autocrine secretion of tumor necrosis factor through the control of nuclear factor-kappa B activation.

Tumor necrosis factor (TNF)-alpha controls T-cell activation and is a major inducer of human immunodeficiency virus (HIV)-1 replication in chronically infected cells. Therefore, we have investigated its role in primary cultures of HIV-infected human T lymphocytes by using neutralizing anti-TNF-alpha antibodies or TNF-alpha. Primary resting T lymphocytes produced TNF-alpha and supported HIV replication after T-cell receptor activation. Addition of neutralizing anti-TNF-alpha antibodies drastically reduced p24 antigen release and prevented CD4+ cell depletion associated with infection. Anti-TNF-alpha also prevented nuclear factor-kappa B (NF-kappa B) activation, and a good correlation between this inhibition and inhibition of HIV replication was observed. Moreover, supplementing the cultures with high doses of IL-2 reverted anti-TNF-alpha inhibition of cell proliferation but did not affect the inhibition of HIV p24 antigen release or NF-kappa B activation in the same cultures. Moreover, anti-TNF-alpha inhibited HIV-1 long terminal repeat (LTR)-driven transcription of a reporter gene in primary T cells in response to activation, either in the presence or the absence of HIV-1 Tat. Our results support an important role for autocrine TNF-alpha secretion in controlling HIV replication in primary T cells because of its ability to maintain NF-kappa B elevated in the nucleus of T cells.