Measurement of Foxp3 gene expression by qRT-PCR RNA of digested lung cells was isolated using the Absolutely RNA Mini Prep kit (Stratagene, La Jolla, CA), according to the manufacturers protocol


Measurement of Foxp3 gene expression by qRT-PCR RNA of digested lung cells was isolated using the Absolutely RNA Mini Prep kit (Stratagene, La Jolla, CA), according to the manufacturers protocol. pulmonary CD4+ T cells, expressing Foxp3-protein or FR4 remained stable. Therefore, the inhibition by V1+ T cells might not be targeting Foxp3+ Treg but rather CD4+ T cells destined to produce IL-10. treatment with anti V1 mAbs resulted in an increase in the lung of inducible CD4+CD25+ T cells capable of generating IL-10, associated with an increase in anti-inflammatory and SIRT3 a decrease in proinflammatory cytokines, but not with an increase in Foxp3-protein+ Treg. 2. Materials and Methods 2.1. Animals, sensitization and airway challenge Female C57BL/6 mice were obtained from OrientBio (Sungnam, Korea) or from Jackson Laboratories (Bar Harbor, Maine), were managed on ovalbumin (OVA)-free diets, and were investigated at an age of 8C12 wks, under protocols approved either by the Institutional Animal Care and Use Committees of the Chungbuk National University or college, or of the National Jewish Medical and Research Center, Denver Colorado. Mice were sensitized by i.p. injection of 20 g OVA (Grade V; Sigma Chemical Co., St. Louis, MO) emulsified in 2.25 mg aluminum hydroxide (AlumImuject; Pierce Chemical, Rockford, IL) in a total volume of 100 ml on days 0 and 14, and challenged via the airways with OVA (1% in saline) for 20 min on days 28, 29 and 30 by ultrasonic nebulization (particle size 1C5 mm; De Vilbiss, Somerset, PA). Mice were sacrificed for further assay at 48 h after the last allergen challenge. 2.2. Administration of anti-TCR mAbs Hamster anti-TCR V1 mAb 2.11 [23] and anti V4 mAb UC3 [24] were purified from hybridoma culture supernatants using a Protein G-Sepharose affinity column (Pharmacia, Uppsala, Sweden). T cell functional depletion/inactivation was achieved after Bimatoprost (Lumigan) injection of 200 g hamster anti-TCR V4 or V1 mAbs into the tail veins of mice, 3d before each of the two OVA sensitizations, and the effect of the antibody treatment on TCR expression was monitored as previously explained [12]. Using this method, we effectively remove TCR-expression ( 80% reduction based on antibody staining) and specifically remove the function of the targeted T cells (based on functional tests and comparison of the antibody treatment and cell transfer experiments). Whether this treatment also eliminates the targeted cells is not obvious. Sham antibody treatments were performed with nonspecific hamster IgG (Jackson Laboratories, Bar Harbor, ME). Notice: Throughout this paper, we use the nomenclature and numbering system for murine V genes launched by Heilig et al. [25]. 2.3. Bronchoalveolar lavage and Measurement of cytokines in BAL fluid Lungs were lavaged with Hanks balanced salt answer (HBSS, 1 ml) via an intratracheal tube, and total leukocyte figures were measured using a Coulter Counter (Coulter Corporation, Hialeah, FL). Differential cell counts were performed by counting at least 200 cells on cytocentrifuged preparations (Cytospin 2; Cytospin, Shandon Ltd., Runcorn, Cheshire, UK), stained with Leukostat (Fisher Diagnostics, Fair Lawn, NJ), and differentiated by standard hematological criteria [3]. IL-10, IL-13, and TGF- in BAL fluid samples were detected by ELISA using commercial packages (R&D Systems, Minneapolis, MN), as previously described [12]. Cytokine levels were determined by comparison with known requirements. The limits of detection were 7.8 pg/ml for IL-13 and 15.6 pg/ml for Bimatoprost (Lumigan) the other cytokines. 2.4. Circulation cytometric analysis Lung tissue cells, prepared as previously explained in detail [10], were resuspended in RPMI medium supplemented with Bimatoprost (Lumigan) 2 mM L-glutamine, 20 M 2-mercaptoethanol, 100 models of penicillin/ml, 50 g of streptomycin per ml, and 10% FBS, and stimulated overnight at 37 C in the presence of 50 ng/ml phorbol 12-myristate 13-acetate (PMA), 500 ng/ml of calcium ionomycin, and 10 g/ml brefeldin A (BFA). Cells were harvested, washed, and resuspended at 2105/well in staining buffer (BSS made up of 1% sodium azide and 2% FBS). Cells were first stained with FITC-conjugated hamster anti-mouse CD4 and CD25 mAbs (Pharmingen, San Diego, CA) for 30 min at 4C, and then fixed in 2% paraformaldehyde for 20 min. For intra-cytoplasmic staining, cells were washed and incubated in staining buffer made up of 0.1% saponin for 10 minutes. Continuously exposed to saponin, cells were then stained with PE-conjugated mouse IgG1 anti-murine IL-10 mAb (Pharmingen) for 30 min at 4C. After washing with staining buffer made up of saponin, cells were washed again with staining buffer without saponin to allow membrane closure. In the experiment described in Table 1, freshly isolated cells from lung and spleen were exceeded over nylon.