Journal:

Article Title: Isoforms of Vitamin E Have Opposing Immunoregulatory Functions during Inflammation by Regulating Leukocyte Recruitment 1

doi: 10.4049/jimmunol.0803659

Figure Lengend Snippet: The αT and γT treatments did not alter BAL cytokines, tissue eotaxins, lung endothelial cell VCAM-1 expression, or lung tissue PGE2. Mice were treated with tocopherols, as in Fig. 1B. A, B, F, and G, On day 21, the BAL supernatants were examined for cytokines using the CBA Th1/Th2 kit (BD Biosciences). C and D, Lung tissue was placed in RNAlater and then examined by quantitative PCR for eotaxin 1 (CCL11) and eotaxin 2 (CCL24). E, Frozen lung tissue sections were labeled with rat anti-mouse VCAM-1 and a FITC-conjugated secondary Ab. Presented is the sum of fluorescence intensity of the endothelial cells per area of endothelium. Isotype control Ab did not label the tissue (data not shown). H, PGE2 from lung extracts. n = 8–10 animals per group. *, p < 0.05 compared with the corresponding saline control.

Article Snippet: Cells were stimulated by cross-linking VCAM-1 with 24 mg/ml rat anti-mouse VCAM-1 (clone MVCAM.A; BD Pharmingen) plus 12 mg/ml goat anti-rat IgG Ab (cat. no. 3050-01; Southern Biotechnology Associates), as we previously described ( 2 ).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Labeling, Fluorescence

Journal:

Article Title: Isoforms of Vitamin E Have Opposing Immunoregulatory Functions during Inflammation by Regulating Leukocyte Recruitment 1

doi: 10.4049/jimmunol.0803659

Figure Lengend Snippet: Tocopherols directly regulate endothelial cells, but not leukocytes, during leukocyte migration without affecting endothelial cell expression of VCAM-1 or MCP-1. A–D, Transwell assay for leukocyte migration with tocopherol left in the culture. Endothelial cells on Transwells were incubated overnight with tocopherols, and then spleen cells were added to the upper chamber of the Transwells to examine cell migration. A, Dose curve of αT. B, Dose curve of γT treatment. C, Dose curve of γT effects on αT (80 μM)-treated cells. D, Endothelial cells were treated overnight with tocopherol, and then in the presence or absence of blocking anti-VCAM-1 Abs, spleen cell migration was determined. Anti-VCAM-1 Abs were added every 4 h, as previously described (19). E and F, Expression of VCAM-1 and MCP-1 by endothelial cells. E, Mean fluorescent intensity of anti-VCAM-1 or isotype Ab control-labeled cells. F, MCP-1 in mHEVa culture supernatant. G–K, Tocopherol pretreatment of cells, followed by washing before the start of the leukocyte migration assay with physiological laminar flow. G, Mice were treated with 2 mg of tocopherol/day or vehicle (ethoxylated castor oil) for 4 days. Spleens were collected, RBC were lysed, and leukocyte tocopherol was determined. H, Spleen leukocytes from untreated mice or spleen leukocytes from mice treated with tocopherols as in G were added to the endothelial cells and examined for transendothelial migration at 15 min under physiological laminar flow. I, Spleen leukocytes from untreated mice or spleen leukocytes from mice treated with tocopherols as in G were added to the endothelial cells and examined for association with the endothelial cells at 2 min under physiological laminar flow. J and K, 85% confluent monolayers of mHEVa cells on slides were treated overnight with the tocopherols at the concentrations indicated to achieve physiological concentrations of tocopherols (Fig. 5), and then the endothelial cells were washed before addition of spleen cells. Spleen cells were isolated from untreated mice, the spleen RBC were lysed, and these spleen leukocytes were added to the endothelial cells to examine leukocyte transendothelial migration under physiological laminar flow. J, Leukocyte transendothelial migration at 15 min under laminar flow. K, Leukocyte association with endothelial cells at 2 min under laminar flow. n = 3–5. *, p < 0.05 compared with DMSO vehicle-treated controls. DMS0 (0.02%) did not affect migration (data not shown).

Article Snippet: Cells were stimulated by cross-linking VCAM-1 with 24 mg/ml rat anti-mouse VCAM-1 (clone MVCAM.A; BD Pharmingen) plus 12 mg/ml goat anti-rat IgG Ab (cat. no. 3050-01; Southern Biotechnology Associates), as we previously described ( 2 ).

Techniques: Migration, Expressing, Transwell Assay, Incubation, Blocking Assay, Labeling, Isolation

Journal:

Article Title: Isoforms of Vitamin E Have Opposing Immunoregulatory Functions during Inflammation by Regulating Leukocyte Recruitment 1

doi: 10.4049/jimmunol.0803659

Figure Lengend Snippet: Tocopherols regulate VCAM-1 activation of endothelial cell PKCα. Endothelial cells were pretreated overnight with physiological concentrations of tocopherols (80 μM α-tocopherol and/or 2 μM γ-tocopherol), as in Fig. 5. Endothelial cell VCAM-1 was stimulated with anti-VCAM-1 and a secondary Ab for 10 min. Phosphorylation of PKCα-Thr638 was examined by Western blot, developed using an ECL kit, and analyzed by densitometry, as in Materials and Methods. These concentrations of α-tocopherol (80 μM) and γ-tocopherol (2 μM) were optimal for their inhibition or enhancement, respectively, of VCAM-1 activation of PKCα (data not shown). Shown are representative blots and data from five experiments. *, p < 0.05 compared with nonstimulated groups. **, p < 0.05 compared with nonstimulated groups, and p < 0.07 compared with DMSO-treated/anti-VCAM-stimulated group.

Article Snippet: Cells were stimulated by cross-linking VCAM-1 with 24 mg/ml rat anti-mouse VCAM-1 (clone MVCAM.A; BD Pharmingen) plus 12 mg/ml goat anti-rat IgG Ab (cat. no. 3050-01; Southern Biotechnology Associates), as we previously described ( 2 ).

Techniques: Activation Assay, Western Blot, Inhibition