We studied the secretion of gelatinase B by dendritic cells (DC) generated by culturing human peripheral blood monocytes in granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). First, we found the intracellular expression of gelatinase B on sections of fixed DC pellets. Zymography analysis of the supernatants of DC cultured for 72 h demonstrated the presence of gelatinase B. To determine if DC produce net enzymatic activity, bioactive gelatinase, a novel sensitive fluorescent-activated substrate conversion (FASC) assay was used to complement the zymography data. Culture media of unstimulated DC demonstrated reproducible net gelatinolytic activity. Tumor necrosis factor-α (TNF-α) IL-1β but not lipopolysaccharide (LPS) stimulation caused a significant increase in gelatinase B production in zymography analysis. Both types of stimulation failed to increase net gelatinase activity in FASC assay. Interestingly, interferon-β (IFN-β) significantly diminished both the total zymolytic production and the net bioactive gelatinase produced by DC in a dose-dependent manner. We conclude that human monocyte-derived DC secrete bioactive gelatinase B and that IFN-β inhibits this production.
The fetus and infant are highly susceptible to viral infections. Several viruses, including human cytomegalovirus (CMV), cause more severe disease in early life compared with later life. It is generally accepted that this is a result of the immaturity of the immune system. gammadelta T cells are unconventional T cells that can react rapidly upon activation and show major histocompatibility complex-unrestricted activity. We show that upon CMV infection in utero, fetal gammadelta T cells expand and become differentiated. The expansion was restricted to Vgamma9-negative gammadelta T cells, irrespective of their Vdelta chain expression. Differentiated gammadelta T cells expressed high levels of IFN-gamma, transcription factors T-bet and eomes, natural killer receptors, and cytotoxic mediators. CMV infection induced a striking enrichment of a public Vgamma8Vdelta1-TCR, containing the germline-encoded complementary-determining-region-3 (CDR3) delta1-CALGELGDDKLIF/CDR3gamma8-CATWDTTGWFKIF. Public Vgamma8Vdelta1-TCR-expressing cell clones produced IFN-gamma upon coincubation with CMV-infected target cells in a TCR/CD3-dependent manner and showed antiviral activity. Differentiated gammadelta T cells and public Vgamma8Vdelta1-TCR were detected as early as after 21 wk of gestation. Our results indicate that functional fetal gammadelta T cell responses can be generated during development in utero and suggest that this T cell subset could participate in antiviral defense in early life.
Abstract Conventional PKC (cPKC)‐α regulates TRIF‐dependent IFN response factor 3 (IRF3)‐mediated gene transcription, but its role in MyD88‐dependent TLR signaling remains unknown. Herein, we demonstrate that PKC‐α is induced by several MyD88‐dependent TLR/IL‐1R ligands and regulates cytokine expression in human and murine DC. First, inhibition of cPKC activity in human DC by cPKC‐specific inhibitors, Gö6976 or HBDDe, downregulated the production of classical inflammatory/immunomodulatory cytokines induced by TLR2, TLR5 or IL‐1R but not by TLR3 stimulation. Similarly, dominant negative PKC‐α repressed Pam 3 CSK 4 induced NF‐κB‐ and AP‐1‐driven promoter activities in TLR2‐expressing human embryonic kidney 293 T cells. Dominant negative PKC‐α inhibited NF‐κB reporter activity mediated by overexpression of MyD88 but not TRIF. Unexpectedly, BM‐derived DC from PKC‐α −/− mice exhibited decreased TNF‐α and IL‐12p40 production induced by both MyD88‐ and TRIF‐dependent ligands. Furthermore, PKC‐α is coupled to TLR2 signaling proximal to MyD88 since MAPK and IκB kinase‐α/β phosphorylations and IκBα degradation were inhibited in PKC‐α −/− BM‐derived DC. Finally, co‐immunoprecipitation assays revealed that PKC‐α physically interacts with Pam 3 CSK 4 activated TLR2 in WT but not in MyD88 −/− DC. Collectively this study identifies a species‐specific role of PKC‐α as a key component that controls MyD88‐dependent cytokine gene expression in human and mouse but differentially regulates production of TRIF‐dependent cytokines.
