Unmethylated CpG motifs are prevalent in bacterial but not vertebrate genomic DNAs. Oligodeoxynucleotides (ODN) containing CpG motifs activate host defense mechanisms leading to innate and acquired immune responses. The recognition of CpG motifs requires Toll-like receptor (TLR) 9, which triggers alterations in cellular redox balance and the induction of cell signaling pathways including the mitogen activated protein kinases (MAPKs) and NF kappa B. Cells that express TLR-9, which include plasmacytoid dendritic cells (PDCs) and B cells, produce Th1-like proinflammatory cytokines, interferons, and chemokines. Certain CpG motifs (CpG-A) are especially potent at activating NK cells and inducing IFN-alpha production by PDCs, while other motifs (CpG-B) are especially potent B cell activators. CpG-induced activation of innate immunity protects against lethal challenge with a wide variety of pathogens, and has therapeutic activity in murine models of cancer and allergy. CpG ODN also enhance the development of acquired immune responses for prophylactic and therapeutic vaccination.

CpG Motifs in Bacterial DNA and Their Immune Effects

Human and mouse plasmacytoid dendritic cells have been shown to correspond to a specialized cell population that produces large amounts of type I interferons in response to viruses, the so-called natural interferon-producing cells. As a result, intensive investigation is now focused on the potential functions of plasmacytoid dendritic cells in both innate and adaptive immunity. Here we review recent progress on the characterization of plasmacytoid dendritic cell origin, development, migration and function in immunity and tolerance, as well as their effect on human diseases.

Plasmacytoid dendritic cells in immunity.

▪ Abstract Type 1 interferon-(α, β, ω)-producing cells (IPCs), also known as plasmacytoid dendritic cell precursors (pDCs), represent 0.2%–0.8% of peripheral blood mononuclear cells in both humans and mice. IPCs display plasma cell morphology, selectively express Toll-like receptor (TLR)-7 and TLR9, and are specialized in rapidly secreting massive amounts of type 1 interferon following viral stimulation. IPCs can promote the function of natural killer cells, B cells, T cells, and myeloid DCs through type 1 interferons during an antiviral immune response. At a later stage of viral infection, IPCs differentiate into a unique type of mature dendritic cell, which directly regulates the function of T cells and thus links innate and adaptive immune responses. After more than two decades of effort by researchers, IPCs finally claim their place in the hematopoietic chart as the most important cell type in antiviral innate immunity. Understanding IPC biology holds future promise for developing cures for infectious...

IPC: professional type 1 interferon-producing cells and plasmacytoid dendritic cell precursors.

Increased concentrations of DNA-containing immune complexes in the serum are associated with systemic autoimmune diseases such as lupus. Stimulation of Toll-like receptor 9 (TLR9) by DNA is important in the activation of plasmacytoid dendritic cells and B cells. Here we show that HMGB1, a nuclear DNA-binding protein released from necrotic cells, was an essential component of DNA-containing immune complexes that stimulated cytokine production through a TLR9-MyD88 pathway involving the multivalent receptor RAGE. Moreover, binding of HMGB1 to class A CpG oligodeoxynucleotides considerably augmented cytokine production by means of TLR9 and RAGE. Our data demonstrate a mechanism by which HMGB1 and RAGE activate plasmacytoid dendritic cells and B cells in response to DNA and contribute to autoimmune pathogenesis.

Toll-like receptor 9-dependent activation by DNA-containing immune complexes is mediated by HMGB1 and RAGE.

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by a breakdown of tolerance to nuclear antigens and the development of immune complexes. Genomic approaches have shown that human SLE leukocytes homogeneously express type I interferon (IFN)–induced and neutrophil-related transcripts. Increased production and/or bioavailability of IFN-α and associated alterations in dendritic cell (DC) homeostasis have been linked to lupus pathogenesis. Although neutrophils have long been shown to be associated with lupus, their potential role in disease pathogenesis remains elusive. Here, we show that mature SLE neutrophils are primed in vivo by type I IFN and die upon exposure to SLE-derived anti-ribonucleoprotein antibodies, releasing neutrophil extracellular traps (NETs). SLE NETs contain DNA as well as large amounts of LL37 and HMGB1, neutrophil proteins that facilitate the uptake and recognition of mammalian DNA by plasmacytoid DCs (pDCs). Indeed, SLE NETs activate pDCs to produce high levels of IFN-α in a DNA- and TLR9 (Toll-like receptor 9)–dependent manner. Our results reveal an unsuspected role for neutrophils in SLE pathogenesis and identify a novel link between nucleic acid–recognizing antibodies and type I IFN production in this disease.

Netting Neutrophils Are Major Inducers of Type I IFN Production in Pediatric Systemic Lupus Erythematosus

An ongoing production of IFN-alpha may be of etiopathogenic significance in systemic lupus erythematosus (SLE). It may be due to the natural IFN-producing cells (NIPC), also termed plasmacytoid dendritic cells (PDC), activated by immune complexes that contain nucleic acids derived from apoptotic cells. We here examined the role of FcgammaR in the IFN-alpha production in vitro by PBMC induced by the combination of apoptotic U937 cells and autoantibody-containing IgG from SLE patients (SLE-IgG). The Fc portion of the SLE-IgG was essential to induce IFN-alpha production, because Fab fragments or F(ab')(2) were ineffective. Normal, especially heat-aggregated, IgG inhibited the IFN-alpha production, suggesting a role for FcgammaR on PBMC. Using blocking anti-FcgammaR Abs, the FcgammaRIIa,c (CD32) but not FcgammaRI or FcgammaRIII were shown to be involved in the IFN-alpha induction by apoptotic cells combined with SLE-IgG, but not by HSV or CpG DNA. In contrast, the action of all of these inducers was inhibited by the anti-FcgammaRIIa,b,c mAb AT10 or heat-aggregated IgG. Flow cytometric analysis revealed that approximately 50% of the BDCA-2-positive PBMC, i.e., NIPC/PDC, expressed low but significant levels of FcgammaRII, as did most of the actual IFN-alpha producers activated by HSV. RT-PCR applied to NIPC/PDC purified by FACS demonstrated expression of FcgammaRIIa, but not of FcgammaRIIb or FcgammaRIIc. We conclude that FcgammaRIIa on NIPC/PDC is involved in the activation of IFN-alpha production by interferogenic immune complexes, but may also mediate inhibitory signals. The FcgammaRIIa could therefore have a key function in NIPC/PDC and be a potential therapeutic target in SLE.

Fc gamma RIIa is expressed on natural IFN-alpha-producing cells (plasmacytoid dendritic cells) and is required for the IFN-alpha production induced by apoptotic cells combined with lupus IgG.

Anti-double-stranded DNA antibodies and immunostimulatory plasmid DNA in combination mimic the endogenous IFN-alpha inducer in systemic lupus erythematosus

Anti-double-stranded DNA antibodies and immunostimulatory plasmid DNA in combination mimic the endogenous IFN-alpha inducer in systemic lupus erythematosus.

A sensitive dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA) was evaluated for ability to detect interferon-alpha (IFN-alpha) in serum of patients with acute infectious disease of less than one week's duration and a fever of > 38 degrees C. None of 36 patients with confirmed or probable bacterial disease was IFN-alpha positive. In contrast, 13/26 patients with viral infections had detectable levels of IFN-alpha in serum, all clearly positive (> or = 10 U/ml). The IFN-alpha positive serum samples were obtained early after onset of clinical disease, after a mean of 2.4 days. The IFN-alpha positive samples were obtained from 10 of the 12 patients with influenza or flu-like infection, and 3 of the 5 patients with varicella or herpes zoster. The IFN-alpha negative patients with viral disease (n = 9) included five patients with mononucleosis. The DELFIA should be useful in further studies of the value of IFN-alpha determinations in the identification of acute viral infections.

Detection of serum interferon-α by dissociation-enhanced lanthanide fluoroimmunoassay

A purification process was developed to obtain a human interferon- α (IFN-α) product that contains all major IFN-α subtypes produced by human leukocytes. The purification was accomplished by immunoaffinity chromatography using two monoclonal antibodies (mAb) and gel filtration. The process comprised two effective virus inactivation steps, solvent detergent treatment, and incubation at low pH, and the purified product was filtered with a 15-nm pore size virus removal filter. The overall yield of IFN-α in the process was about 60% when starting from the culture supernatant of Sendai virus-induced human leukocytes. The specific activity was about 1.0 × 108 IU/mg. The level of DNA and protein impurities including mouse IgG was very low. The product contained seven main subtypes: IFN-α 1, IFN-α 2, IFN-α 8, IFN-α 10, IFN-α 14, IFN-α 17, and IFN-α 21. The subtypes IFN-α 4 and IFN-α 7 were minor components. Reverse-phase HPLC indicated a constant subtype composition for the product from batch to batch. Stabilizat...

Anders Perers

Papers

Fc gamma RIIa is expressed on natural IFN-alpha-producing cells (plasmacytoid dendritic cells) and is required for the IFN-alpha production induced by apoptotic cells combined with lupus IgG.

Anti-double-stranded DNA antibodies and immunostimulatory plasmid DNA in combination mimic the endogenous IFN-alpha inducer in systemic lupus erythematosus.

Detection of serum interferon-α by dissociation-enhanced lanthanide fluoroimmunoassay

Development of a highly purified multicomponent leukocyte IFN-alpha product.