Plasmacytoid dendritic cells (PDC) produce high levels of type I IFN upon stimulation with viruses, while monocytes and monocyte-derived dendritic cells (MDDC) produce significantly lower levels. To find what determines the high production of type I IFN in PDC, we examined the relative levels of IRF transcription factors, some of which play critical roles in the induction of IFN. Furthermore, to determine whether the differences could result from expression of distinct IFNA subtypes, the profile of IFNA genes expressed was examined. PDC responded equally well to stimulation with HSV-1 and Sendai virus (SV) by producing high levels of type I IFN, whereas the MDDC and monocyte response to SV were lower, and neither responded well to HSV-1. All three populations constitutively expressed most of the IRF genes. However, real-time RT-PCR demonstrated increased levels of IRF-7 transcripts in PDC compared with monocytes. As determined by intracellular flow cytometry, the PDC constitutively expressed significantly higher levels of IRF-7 protein than the other populations while IRF-3 levels were similar among populations. Analysis of the profile of IFNA genes expressed in virus-stimulated PDC, monocytes and MDDC demonstrated that each population expressed IFNA1 as the major subtype but that the range of the subtypes expressed in PDC was broader, with some donor and stimulus-dependent variability. We conclude that PDC but not MDDC are uniquely preprogrammed to respond rapidly and effectively to a range of viral pathogens with high levels of IFN-alpha production due to the high levels of constitutively expressed IRF-7.

Plasmacytoid dendritic cells (PDC) are the natural type I IFN-producing cells that produce large amounts of IFN-α in response to viral stimulation. During attempts to isolate PDC from human PBMC, we observed that cross-linking a variety of cell surface receptors, including blood DC Ag (BDCA)-2, BDCA-4, CD4, or CD123 with Abs and immunobeads on PDC leads to inhibition of IFN-α production in response to HSV. To understand the mechanisms involved, a number of parameters were investigated. Cross-linking did not inhibit endocytosis of soluble Ag by PDC. Flow cytometry for annexin V and activated caspase-3 indicated that PDC are not undergoing apoptosis after receptor cross-linking. Cross-linking of CD123, but not the other receptors, caused the up-regulation of costimulatory molecules CD80 and CD86, as well as the down-regulation of CD62L, indicating PDC maturation. Thus, anti-CD123 Ab may be acting similar to the natural ligand, IL-3. Anti-phosphotyrosine Ab, as well as Ab to the IFN regulatory factor, IRF-7, was used in intracellular flow cytometry to elucidate the signaling pathways involved. Tyrosine phosphorylation occurred after cross-linking BDCA-2 and BDCA-4, but not CD4. Cross-linking did not affect IRF-7 levels in PDC, however, cross-linking BDCA-2, BDCA-4, and CD4, but not CD123, inhibited the ability of IRF-7 to translocate to the nucleus. Taken together, these results suggest that cross-linking BDCA-2, BDCA-4, and CD4 on PDC regulates IFN-α production at the level of IRF-7, while the decrease in IFN-α production after CD123 cross-linking is due to stimulation of the IL-3R and induction of PDC maturation.

Plasmacytoid dendritic cells (pDC) are well-known for their ability to produce large quantities of interferon-alpha (IFN-alpha) in response to viruses. In addition, pDC produce IFN-alpha in response to HSV-infected cells. We demonstrate that both tonsil and PBMC contain pDC that respond to stimulation with HSV either in suspension or in tonsil tissue-fragment culture. We hypothesized that other DC subsets acquire virus in the periphery and deliver the interferongenic signals to the pDC in the draining lymphoid tissue. As a model for pDC/myeloid DC interaction, we studied the interaction of pDC derived from blood with HSV-infected and uninfected monocyte derived dendritic cells (MDDC). Infected, but not uninfected, MDDC induced IFN-alpha in pDC. To further study pDC/infected MDDC interactions, we labeled MDDC with fluorescent cell trackers PKH67 or CFSE prior to infection with HSV and co-cultured with pDC. Cells were then analyzed using conventional and imaging flow cytometry. In addition, we infected MDDC with a GFP-expressing HSV prior to co-culture with pDC. Using traditional flow cytometry, we observed that pDC became fluorescent after co-incubation with uninfected or infected, fluorescently labeled MDDC, indicating that MDDC transferred fluorescent protein and membrane to pDC. By imaging flow cytometry, we observed formation of conjugates between pDC and MDDC as well as transfer and internalization of cellular components from the labeled MDDC by pDC, with preferential uptake from, and association with, infected vs. uninfected MDDC. These studies demonstrate that MDDC infected with HSV are able to stimulate IFN-alpha and chemokine production by pDC through the transfer of cellular materials from the HSV-infected MDDC to the pDC. Together, these observations indicate that heterogeneous populations of DC interact to generate an effective IFN-alpha response.