Activation with TLR ligands did not alter IGIP transcription when combined with VIP in culture. mRNA expression could be up-regulated as much as thirty five-fold above non-stimulated samples within 12C48 hours. Na?ve B cells cultured with exogenous rhIGIP produced IgA in greater quantities than non-stimulated controls. Finally, we demonstrate that IGIP activation drives the production of – switch circles from IgM+/IgD+ na?ve human B cells, indicating its role as an IgA switch factor. explained a novel protein, IgA-Inducing Protein (IGIP), isolated from a bovine Peyers patch and mesenteric lymph node combined cDNA library with cells activated under various conditions (35). Bovine IGIP, like BAFF and APRIL, was found to be produced primarily by DCs, in response to CD40L activation. Further, we found that recombinant bovine IGIP enhances IgA expression in IgM+ peripheral blood B cells in an culture system. Here, we demonstrate the effects of this peptide on human B lymphocytes and characterize the conditions under which IGIP is usually expressed and the conditions required for IGIP function. Materials and Methods Reagents The following reagents were utilized for mDC or B cell activation experiments, and are outlined with their working concentrations and suppliers. Pam3CSK4 (TLR2 ligand, 1g/ml, Alexxa Biochemicals), Poly I:C (TLR3 ligand, 10g/ml, Sigma), LPS (TLR4 ligand, 100ng/ml, Sigma), Flagellin (TLR5 ligand, 1g/ml, Alexxa Biochemicals), Oxethazaine CpG ODN 2006 (TLR9 ligand, 10g/ml, IDT DNA) or CD40L(30ng/ml, Alexxa Biochemicals), VIP (1M, Calbiochem), rhTGF- (10ng/ml, R&D Systems), TACI-Fc(soluble BAFF and APRIL neutralizing receptor, 20ng/ml, R&D Systems), rhIL-10 (25ng/ml, R&D Systems), anti-TGF- mAb (30g/ml, R&D Systems) Phorbol-myristate acetate (PMA)(10ng/ml,Sigma) and Ionomycin(1g/ml, Sigma). Treatment with PMA and Ionomycin is usually abbreviated PMA/I. Oxethazaine Isolation of Na?ve Human B cells from peripheral blood Total PBMCs were isolated from 100 ml whole blood from normal healthy donors via Accuprep (Accurate Chemical and Scientific Corp., Westbury, NY) gradient centrifugation, as previously explained (36). Peripheral blood mono-nuclear cells (PBMCs) were treated with biotinylated mouse monoclonal antibody (MAb) to human IgD (Southern Biotechnology Associates, Birmingham, AL), followed by anti-biotin Microbeads (Miltenyi Biotec, Auburn, CA). All reactions were carried out in sorting buffer (PBS pH 7.4, 0.5% BSA, 2mM EDTA) at 4C. Oxethazaine sIgD+ B cells were then purified with an automated magnetic cell sorter (AutoMACS, Miltenyi). The purity of sorted sIgD+ cells, tested by circulation cytometry and the absence of RT-PCR Rabbit Polyclonal to TF3C3 Oxethazaine for IgG and IgA transcripts as previously explained, exceeded 95% in all experiments (data not shown). Isolation of peripheral blood mononuclear cell subsets PBMCs were isolated by density gradient centrifugation. Populations of T cells, B cells, monocytes and NK cells were isolated with anti-human CD3, CD19, CD14 and CD56 Microbeads, respectively, by double-column sorting with an AutoMACS magnetic cell separator (Miltenyi). Isolated populations were shown to be 98% real by FACS analysis (data not shown). Preparation of monocyte-derived dendritic cells PBMCs were labeled with anti-CD14 Microbeads (Miltenyi) and monocytes were separated by AutoMACS (Miltenyi). CD14+ monocytes were cultured with 10 ng/ml rhIL-4 (R&D Systems) and 1400 U/ml rhGM-CSF (Leukine, Immunex, Seattle, WA) for 6 days as previously explained (37, 38). Non-adherent cells were removed and cultured for an additional 14 hours in total RPMI 1640 (cRPMI), with or without 300 ng/ml of soluble rhuCD40L. Purity of isolated mDC populations was verified by circulation cytometry with antibodies to DC-SIGN (R&D Systems) and HLA-DR (Southern Biotech). Amplification of human IGIP Pure mDC cultures were stimulated with 500 ng/ml rhCD40L (Axxora LLC, San Diego, CA ), 20 ng/ml LPS or 10 ng/ml PMA and 1g/ml Ionomycin for 14 hours. Total RNA was extracted with an RNeasy RNA extraction kit (Qiagen) and DNase-treated with a DNA Free DNase kit (Ambion, Austin, TX) as per manufacturer instructions. Human IGIP transcripts were amplified with a Titan One Tube RT-PCR kit (Roche, Indianapolis, IN) according to manufacturers instructions with forward primer 5-AATATCATTAATTTGCACTGT-3 and reverse primer 5-TTTTGCCTACTTTATTTCA-3. Heat cycling conditions were as follows: 50C for 50min.; 95C for 5 min.; 35 cycles of 95C for 30 sec., 50C for 1 min., 72C for 2 min.; 72C for 7 min.; 4C hold. Fragments were visualized in a 1% w/v agarose gel made up of 0.5g/ml ethidium bromide. RT-PCR assays RT-PCR for hIGIP, BAFF, APRIL and IL-10 was performed at the UTMB Real-Time PCR Core Facility with primer and probe units from BD Biosciences as follows: hIGIP: Fwd-5CCCATCTCAGTGCTGGGAAA3 Rev-5CTGATGCACAACACGTTTGCT3, Probe-5CACCATGTGGAAACC3, BAFF: Fwd-5ACCGCGGGACTGAAAATCT3 Rev-5GTTCTGACTGGAGTTGCCTTCTC3, Probe-5TGAACCACCAGCTCC3, APRIL: Fwd-5CACTCTGTCCTGCACCTGGTT3 Rev-5TCTGTCACATCGGAGTCATCCT3, Probe-5CATTAACGCCACCTCC3, IL-10: Fwd-5CCCCAAGCTGAGAACCAAGAC3 Rev-5TCCCCCAGGGAGTTCACA3 Probe-5CAGACATCAAGGCGC3 Densitometry Densitometry measurements were made with AlphaEaseFC Software (AlphaInnotech Corp., San Leandro, CA). Integrated Density Values (IDVs) were determined for each band in the gel, with the software.