A mixture of 48 TaqMan Gene Expression assays (Applied Biosystems) was prepared for use with the TaqMan PreAmp Master Mix Kit (Applied Biosystems). 1 interferon production by all human cells. strong class=”kwd-title” Keywords: Type 1 interferons, Systemic lupus erythematosus, Myositis, Plasmacytoid dendritic cells Introduction The type 1 interferons, which include interferon-alpha (IFN) and interferon-beta (IFN), may play a significant role in several autoimmune diseases, particularly systemic lupus erythematosus (SLE; reviewed in [1; 2; 3]) and dermatomyositis (DM; reviewed in [4; 5]). Anti-IFN neutralizing antibody therapy is currently being studied in clinical trials for both NOD-IN-1 SLE NOD-IN-1 [6; 7] NOD-IN-1 and myositis [8], and various other approaches to modulating the type 1 interferon pathway have been considered. Plasmacytoid dendritic cells (pDCs) are immune system cells capable of producing large amounts of type 1 interferons. pDCs have been observed to concentrate in diseased tissue sites, in DM muscle [9; 10] and skin [11; 12], and in SLE glomeruli [13] and skin [14; 15]. The effects of such concentration is unknown but it is notable that DM muscle shows marked enrichment of NOD-IN-1 type 1 interferon-inducible transcripts in comparison to blood, even though both compartments are highly dominated by such transcripts (accounting for 85% of the most abundant 25 transcripts in both blood and muscle, of 18,000 measured). For example, ISG15 (interferon-stimulated gene 15) transcript was 570-fold increased in muscle but 9-fold increased in blood; Mx1 (myxovirus resistance protein 1) transcript was 281-fold increased in muscle but 6-fold increased in blood [16]. To further understand the consequences of human pDC accumulation in autoimmune disease tissue sites, we studied the Rabbit polyclonal to NFKB3 effects of increasing pDC cell number on type 1 interferon production. We found that IFN production by human pDCs proceeds exponentially, not linearly, within a physiological range of increasing cell numbers, an effect in part mediated by the type 1 interferon receptor (IFNAR). We demonstrate directly that type 1 interferons substantially augment their own production by pDCs, effects that have previously been demonstrated in non-human cells [17; 18; 19; 20; 21; 22], but only hypothesized for human pDCs [23; 24] based on indirect experiments and on mouse experiments [20; 21; 22; 25; 26; 27; 28]. Methods pDC isolation, purification, stimulation, and viability Normal donor peripheral blood mononuclear cells (PBMCs) were isolated over Ficoll-Paque (Amersham Biosciences). Plasmacytoid dendritic cells (pDCs) were purified using a NOD-IN-1 BDCA-4 cell isolation kit (Miltenyi Biotec) with 2 steps of pDC enrichment on magnetic columns. The purity of isolated BDCA-2+CD123+ pDC was 92.02 % 1.02% by flow cytometry (n = 5). For measurement of IFN protein, varying number of pDCs as described below were stimulated in 200 l assays in 96-well or 50 l or 75 l assays in 384-well plates with CpG oligodeoxynucleotide (ODN) type A, human toll-like receptor 9 (TLR9) ligand ODN2216 (InvivoGen) 5 g/ml for 24 hours, and the supernatants removed and immediately assayed by ELISA. TLR9 agonists were used because TLR9 activating immunostimulatory DNA complexes are believed to be directly relevant to the mechanism of type 1 interferon production in SLE and DM [1; 4]. For measurement of priming and IFNAR blocking effects, these experiments were done in the absence and presence of IFN-2a protein (PBL Biomedical, Product #11100-1) at varying doses as indicated and absence or presence of anti-IFNAR2 antibody (PBL Biomedical, Product #21385-1), with isotype control matched IgG2a antibody, both at 5 g/ml (BD Pharmingen Product #554126). For harvesting of RNA for transcript experiments, 10,000 pDCs in wells of 384-well microtiter plates were stimulated with ODN2216 at 5 g/ml in the absence and presence of 25 pg/ml IFN-2a for 1, 3, 6, 16, and 24 hours and then frozen at ?80C for subsequent RNA extraction. In separate experiments, PBMCs were compared with both the BDCA-4 positively selected fraction (pDCs) and the remaining BDCA4-depleted PBMC fraction. Triplicate wells of 10,000 PBMCs, 10,000 BDCA4-depleted PBMCs, and 10,000 BDCA4-positively selected cells (pDCs) from the same purification were stimulated with ODN2216 at 5 g/ml for 6 hours. All cells were then frozen at ?80C for subsequent RNA extraction. For harvesting of proteins for western blots, pDCs (BDCA4+; mean of 4 experiments 1.19 106 cells), PBMCs (mean of 2 experiments 8.40 105 cells), or BDCA4-depleted PBMCs.
