Modulation of Dendritic Cell Apoptosis and CD8+ Cytotoxicity by Histamine: Role of Protein Kinase C.

Dendritic cells (DC) are able to present extracellular antigens associated with the molecules of the major histocompatibility complex class I. In a previous work, we demonstrated that the histamine (HIS), acting through H1/H4 receptors, increases the cross-presentation of soluble ovalbumin by murine DC and can enhance the recruitment of specific CD8+ T lymphocytes during the development of chronic inflammatory responses. Here, we studied in more depth the mechanisms underlying this enhancement. We showed that the cytotoxicity of specific CD8+ lymphocytes is increased in HIS-treated DC and it is lost by inhibition of vacuolar-ATPase that prevents endosome acidification. It is known that HIS acts through G protein-coupled receptors. The H1/H4 receptors are associated with a Gq subunit, which involves PKC signaling, a pathway related to the apoptotic process. Interestingly, we demonstrated for the first time that HIS prevents DC apoptosis induced by heat shock through the inhibition of caspase-3, a mechanism dependent on PKC activation, since it is reversed by its inhibition. By contrast, cytolytic activity of T lymphocytes induced by HIS-stimulated DC was independent of PKC pathway.

Dual activation of Toll-like receptors 7 and 9 impairs the efficacy of antitumor vaccines in murine models of metastatic breast cancer.

PURPOSE: Since combination of Toll-like receptor (TLR) ligands could boost antitumor immunity, we evaluated the efficacy of dendritic cell (DC) vaccines upon dual activation of TLR9 and TLR7 in breast cancer models. METHODS: DCs were generated from mouse bone marrow or peripheral blood from healthy human donors and stimulated with CpG1826 (mouse TLR9 agonist), CpG2006 or IMT504 (human TLR9 agonists) and R848 (TLR7 agonist). Efficacy of antitumor vaccines was evaluated in BALB/c mice bearing metastatic mammary adenocarcinomas. RESULTS: CpG-DCs improved the survival of tumor-bearing mice, reduced the development of lung metastases and generated immunological memory. However, dual activation of TLRs impaired the efficacy of DC vaccines. In vitro, we found that R848 inhibited CpG-mediated maturation of murine DCs. A positive feedback loop in TLR9 mRNA expression was observed upon CpG stimulation that was inhibited in the presence of R848. Impaired activation of NF-κB was detected when TLR9 and TLR7 were simultaneously activated. Blockade of nitric oxide synthase (NOS) and indoleamine-pyrrole-2,3-dioxygenase (IDO) improved the activation of CpG-DCs. When we evaluated the effect of combined activation of TLR9 and TLR7 in human DCs, we found that R848 induced robust DC activation that was inhibited by TLR9 agonists. CONCLUSIONS: These observations provide insight in the biology of TLR9 and TLR7 crosstalk and suggest caution in the selection of agonists for multiple TLR stimulation. Blockade of NOS and IDO could improve the maturation of antitumor DC vaccines. R848 could prove a useful adjuvant for DC vaccines in human patients.

Differential expression of the fractalkine chemokine receptor (CX3CR1) in human monocytes during differentiation.

Circulating monocytes (Mos) may continuously repopulate macrophage (MAC) or dendritic cell (DC) populations to maintain homeostasis. MACs and DCs are specialized cells that play different and complementary immunological functions. Accordingly, they present distinct migratory properties. Specifically, whereas MACs largely remain in tissues, DCs are capable of migrating from peripheral tissues to lymphoid organs. The aim of this work was to analyze the expression of the fractalkine receptor (CX3CR1) during the monocytic differentiation process. Freshly isolated Mos express high levels of both CX3CR1 mRNA and protein. During the Mo differentiation process, CX3CR1 is downregulated in both DCs and MACs. However, MACs showed significantly higher CX3CR1 expression levels than did DC. We also observed an antagonistic CX3CR1 regulation by interferon (IFN)-γ and interleukin (IL)-4 during MAC activation through the classical and alternative MAC pathways, respectively. IFN-γ inhibited the loss of CX3CR1, but IL-4 induced it. Additionally, we demonstrated an association between CX3CR1 expression and apoptosis prevention by soluble fractalkine (sCX3CL1) in Mos, DCs and MACs. This is the first report demonstrating sequential and differential CX3CR1 modulation during Mo differentiation. Most importantly, we demonstrated a functional link between CX3CR1 expression and cell survival in the presence of sCX3CL1.