We investigated the interactions between human monocyte-derived dendritic cells (DCs) and Ag-activated circulating TCR-γδ-expressing lymphocytes (Vδ2). Coculture of immature DCs (iDCs) with peripheral blood Vδ2 T cells activated with either pyrophosphomonoesters (isopentenyl pyrophosphate; IPP) or aminobiphosphonates (pamidronate; PAM) led to a significant up-modulation of CD86 and MHC class I molecules and to the acquisition of functional features typical of activated DCs. DC activation induced by both IPP- and PAM-stimulated γδ T cells was mostly mediated by TNF-α and IFN-γ secreted by activated lymphocytes. However, the effect of PAM-activated γδ T cells, but not that of IPP-activated cells, required cell-to-cell contact. Reciprocally, activation of Vδ2 T cells by PAM, but not by IPP, was dependent on cell contact with iDCs. In fact, when PAM-stimulated DC-γδ T cell cocultures were separated by a semipermeable membrane or treated with blocking anti-CD86 Abs, induction of CD25 and CD69 as well as IFN-γ and TNF-α secretion by Vδ2 cells were strongly reduced. These results demonstrate for the first time a bidirectional activating interaction between iDCs and PAM-stimulated γδ T lymphocytes, thus suggesting a potential adjuvant role of this early cross-talk in the therapeutic activity of aminobiphosphonate drugs.

Infection of host cells by viruses leads to the activation of multiple signaling pathways, resulting in the expression of host genes involved in the establishment of the antiviral state. Among the transcription factors mediating the immediate response to virus is interferon regulatory factor-3 (IRF-3) which is post-translationally modified as a result of virus infection. Phosphorylation of latent cytoplasmic IRF-3 on serine and threonine residues in the C-terminal region leads to dimerization, cytoplasmic to nuclear translocation, association with the p300/CBP coactivator, and stimulation of DNA binding and transcriptional activities. We now demonstrate that IRF-3 is a phosphoprotein that is uniquely activated via virus-dependent C-terminal phosphorylation. Paramyxoviridae including measles virus and rhabdoviridae, vesicular stomatitis virus, are potent inducers of a unique virus-activated kinase activity. In contrast, stress inducers, growth factors, DNA-damaging agents, and cytokines do not induce C-terminal IRF-3 phosphorylation, translocation or transactivation, but rather activate a MAPKKK-related signaling pathway that results in N-terminal IRF-3 phosphorylation. The failure of numerous well characterized pharmacological inhibitors to abrogate virus-induced IRF-3 phosphorylation suggests the involvement of a novel kinase activity in IRF-3 regulation by viruses.

Human peripheral blood monocytes differentiate into macrophages when cultured in vitro for a few days. In the present study, we investigated the expression of C-C chemokine and CXCR4 receptors in monocytes at different stages of differentiation. Culturing of monocytes for 7 days resulted in a progressive decrease of the mRNA that encodes for CCR2 and CCR3, whereas the expression of mRNA for other chemokine receptors (CCR1, CCR4, CCR5, and CXCR4) was not substantially affected. The loss of CCR2 mRNA expression in 7-day–cultured macrophages was associated with a strong reduction in the receptor expression at the plasma membrane, as well as in the monocyte chemotactic protein (MCP-1) binding, as compared with freshly isolated monocytes. Furthermore, the biologic response to MCP-1, as measured by intracellular calcium ions increase and chemotactic response, was lost in 7-day–cultured macrophages. Differentiation of monocytes into macrophages also resulted in an increased secretion of MCP-1 that, at least in part, was responsible for the downmodulation of its receptor (CCR2). The loss of CCR2 expression and the parallel increase of MCP-1 secretion triggered by differentiation may represent a feedback mechanism in the regulation of the chemotactic response of monocytes/macrophages.

Dendritic cells (DCs) generated by a single-step exposure of human monocytes to type I IFN and GM-CSF (IFN-DCs) are endowed with potent immunostimulatory activities and a distinctive migratory response to specific chemokines. In this study, we evaluated the effects of 1α,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), the biologically active metabolite of vitamin D₃, on the DC differentiation/activation induced by type I IFN. We found that 1,25(OH)₂D₃ prevented the generation of IFN-DCs when added to freshly isolated monocytes, and was capable of redirecting already differentiated IFN-DCs toward a more immature stage, as revealed by their immunophenotype, reduced allostimulatory activity, and impaired LPS-induced production of Th1-polarizing cytokines. Control and 1,25(OH)₂D₃-treated IFN-DCs exhibited a similar expression of vitamin D receptor, as well as comparable cell death rates. Furthermore, the chemotactic response of IFN-DCs to CCL4 and CCL19 was markedly reduced or completely abrogated by 1,25(OH)₂D₃. Despite these changes in the IFN-DC migratory behavior, the expression of CCR5 and CCR7 and the calcium fluxes triggered by CCL4 and CCL19 were not affected. These findings indicate that, in this innovative single-step DC generation model from monocytes, the suppressive effect of 1,25(OH)₂D₃ is associated with a potent impairment of DC migration in response to inflammatory and lymph node-homing chemokines, thus unraveling a novel mechanism involved in 1,25(OH)₂D₃-mediated immunomodulation.

Objective: In patients with hepatitis C virus (HCV)/HIV co-infection, a faster progression of liver fibrosis to cirrhosis has been reported. In this study, an investigation was carried out to determine whether gp120, an HIV envelope protein, modulates the biology of human hepatic stellate cells (HSCs), key cell types in the pathogenesis of fibrosis. Methods: Myofibroblastic HSCs were isolated from normal human liver tissue. Gene expression was measured by real-time PCR. Cell migration was assessed in Boyden chambers. Intracellular signalling pathways were evaluated using phosphorylation-specific antibodies or by transfection of a reporter plasmid. Results: Transcripts for the chemokine receptors CCR5 and CXCR4, which bind gp120, were detectable in human HSCs. Upon exposure to M-tropic recombinant gp120, which binds CCR5, a significant increase in HSC chemotaxis was observed (1.6±0.3-fold, p=0.03). The effects of gp120 were prevented by protein inactivation. gp120 also resulted in a significant increase in secretion (1.5±0.3-fold, p=0.03) and gene expression (1.47±0.13-fold, p=0.02) of the proinflammatory chemokine monocyte chemoattractant protein-1, and in increased gene expression of tissue inhibitor of metalloprotease-1 and interleukin-6 (2.03±0.57-fold, p=0.02). gp120-induced migration required Akt activation. gp120 also induced activation of nuclear factor-κB (NF-κB) and p38 ^MAPK . Preincubation of HSCs with TAK779, a CCR5 receptor antagonist, prevented gp120-mediated chemotaxis and monocyte chemoattractant protein-1 secretion. Expression of CCR5 was detectable in areas of inflammation and fibrogenesis in liver biopsies of patients with HCV/HIV co-infection. Conclusions: This study shows that HIV gp120 modulates different aspects of HSC biology, including directional cell movement and expression of proinflammatory cytokines. These results identify a direct pathway possibly linking HIV infection with liver fibrogenesis via envelope proteins.