1. Osteopontin functionally activates dendritic cells and induces their differentiation toward a Th1-polarizing phenotype
by Andreas C. Renkl, Julia Wussler, Thomas Ahrens, Käthe Thoma, Shigeyuki Kon, Toshimitsu Uede, Stefan F. Martin, Jan C. Simon, and Johannes M. Weiss
Blood
Volume 106(3):
August 1, 2005
©2005 by American Society of Hematology

2. OPN induces the emigration of human Langerhans cells from the epidermis and their activation with up-regulated expression of MHC class II molecules.
OPN induces the emigration of human Langerhans cells from the epidermis and their activation with up-regulated expression of MHC class II molecules. Split thickness sheets of human skin (2 cm2) were floated on c-RPMI supplemented with (closed symbols) or without (open symbols) OPN (0.5 μg/mL). Sheets were removed after 24 hours. Emigrated cells were counted microscopically following lyses of contaminating erythrocytes. Cells were double stained by FITC-labeled antibodies against CD1a- and PE-labeled antibodies against HLA-DR. (A) The relative number of LCs was determined by flow cytometry by positive CD1a staining. The absolute number of emigrated LCs per cm2 of skin was determined by the following formula: percentage of CD1a+ cells × microscopically counted cells/cm2 of skin. Data are expressed as absolute number of LCs that had emigrated from 1 cm2 skin. The mean of 6 independent experiments ± SEM is shown. Each pair of closed and corresponding open symbols represents data from the same donor. In 5 of 6 donors, OPN stimulated LC migration. When calculating the percentage of OPN-induced migration for each donor and performing a Wilcoxon signed rank test, a P value of .094 was obtained for this set of experiments. (B) In FACS analysis CD1a and HLA-DR, double-stained emigrated cells were gated for CD1a expression and HLA-DR expression of CD1a+ cells was analyzed. The dotted curves give the isotype control staining. Shown is 1 representative experiment of 5, each with tissues from different donors.
Andreas C. Renkl et al. Blood 2005;106:
©2005 by American Society of Hematology

3. OPN-stimulated DCs show morphologic signs of activation.
OPN-stimulated DCs show morphologic signs of activation. DCs derived from peripheral blood monocytes were left untreated (A-C), or stimulated with 0.5 μg/mL OPN (D-F) or 10 μg/mL LPS (G-I) on day 5 of culture. Photographs of cultures were taken 24 hours (A,D,G) and 48 hours (B,E,H) after addition of the stimulus. Bar in panel A for magnification in panels A-B, D-E, G-H: 150 μM. For morphologic evaluation of DCs, cells were cultured as described in “Materials and methods,” and after 48 hours of treatment, unstimulated DCs (C), OPN-stimulated DCs (F), or LPS-stimulated DCs (I) were fixed on adhesion slides and stained immunohistochemically with antibodies against HLA-DR. Bar in panel C for magnification in panels C,F,I: 15 μM. Panels A-B, D-E, G-H: Cell cultures were analyzed with an Axiovert microscope (Zeiss, Göttingen, Germany) using a Zeiss 10.0 objective lens providing a 10-fold magnification. Panels C, F, I: Adhesion slides were analyzed with an Axioskop microscope (Zeiss) with oil immersion using a 100× objective lens (Plan Neofluar objective, Zeiss). Photos were taken with a Canon EOS 300V (Canon, Krefeld, Germany) used as an onboard camera without additional optical lenses. All slides were scanned with CanoScan (Canon), and images were assembled with CorelDraw 11.
Andreas C. Renkl et al. Blood 2005;106:
©2005 by American Society of Hematology

4. OPN induces terminal DC differentiation with up-regulated expression of MHC class II, costimulatory, and adhesion molecules.
OPN induces terminal DC differentiation with up-regulated expression of MHC class II, costimulatory, and adhesion molecules. DCs (1 × 106) were cultured in the presence or absence of either OPN or LPS from days 5 to 7 of culture. Cells were stained with antibodies against HLA-DR, CD40, CD80, CD86, CD44, and CD54 (A) and with antibodies against CD1a and CD14 to determine their DC phenotype (B) and were analyzed by FACS after 24 and 48 hours of stimulation. FACS analysis of 1 representative of 5 experiments is shown. The dotted line on the left in panels A and B represents isotype control staining of unstimulated DCs. (C) To demonstrate that the OPN added for DC activation induces HLA-DR expression independent of the endogenous OPN, DCs from day 5 (▪) were cultured for 24 hours in 100% (▩) or 50% (▨) DC-conditioned medium or in fresh medium without GM-CSF or IL-4 (0%; □), with (+) or without (–) exogenous OPN. Corrected mean fluorescence (cMFI) of HLA-DR expression from 1 representative of 3 independent experiments is given.
Andreas C. Renkl et al. Blood 2005;106:
©2005 by American Society of Hematology

5. OPN induces the secretion of TNF-α and IL-12 by DCs
OPN induces the secretion of TNF-α and IL-12 by DCs. DCs were cultured in the presence or absence of OPN from days 5 to 7 of culture.
OPN induces the secretion of TNF-α and IL-12 by DCs. DCs were cultured in the presence or absence of OPN from days 5 to 7 of culture. Supernatants were analyzed by ELISA after 24 hours and 48 hours of OPN stimulation for the secretion of TNF-α (A), IL-12p40 (B), bioactive IL-12p70 (C, ▪), and IL-10 (C, □). Cytokine secretion of 1 × 106 DCs in pg/mL ± SEM of 8 (A,C) or 6 (B) independent experiments is shown.
Andreas C. Renkl et al. Blood 2005;106:
©2005 by American Society of Hematology

6. OPN-stimulated DCs exhibit enhanced T-cell allostimulatory capacity.
OPN-stimulated DCs exhibit enhanced T-cell allostimulatory capacity. Mixed lymphocyte reaction (MLR) was performed with allogeneic T cells (10 × 104/200μL) and immature DCs (10 × 103/200 μL) on day 5 of DC culture in the presence or absence of OPN, added throughout the MLR (A). Alternatively (B), DCs were stimulated with OPN from day 5 to day 7 of DC culture (48 h DC OPN) or left untreated for control (48 h DC wo OPN), prior to their addition to MLR. T-cell proliferation was measured by [H3]-thymidine incorporation in the last 18 hours of the experiment. (A-B) One representative experiment with T cells and DCs from the same donor pair is shown. This is representative of 7 independent experiments. Data are expressed as the mean counts per minute ± SD of quadruplicate cultures. (C) MLR was performed with DCs cultured for 5 days and OPN was added throughout the experiment (○). Alternatively, DCs were prestimulated with OPN for 48 hours prior to addition to MLR (•). The allostimulatory capacity in both settings was compared with that of immature DCs from day 5 (open gray diamonds). For statistical analysis of all experiments, the percent induction of T-cell proliferation by OPN was calculated by the following formula: T-cell proliferation induced by prestimulated DCs or DCs in the presence of OPN (CPM)/T-cell proliferation induced by immature DCs of day 5 (CPM) × 100%. Statistical analysis of 7 independent experiments ± SEM (*P < .05, one-way analysis of variance [ANOVA], Student-Neuman-Keuls test). (D) The stimulatory capacity of 48-hour OPN-stimulated DCs (•) is compared with the T-cell stimulatory capacity of unstimulated DCs from day 7 of DC culture (closed gray diamonds). Statistical analysis of 10 independent experiments ± SEM (#P < .05, Kruskal-Wallis one-way ANOVA on Ranks, Student-Neuman-Keuls test) is shown.
Andreas C. Renkl et al. Blood 2005;106:
©2005 by American Society of Hematology

7. In the presence of allogeneic T cells, OPN augments the production of IL-12p70 by DCs and IFN-γ by T cells.
In the presence of allogeneic T cells, OPN augments the production of IL-12p70 by DCs and IFN-γ by T cells. MLR was performed with immature DCs (10 × 103/200 μL), cultured for 5 days prior to MLR and allogeneic T cells (10 × 104/200 μL) in the presence or absence of OPN, added throughout the MLR (A,C). (B,D) DCs were prestimulated with OPN from day 5 to day 7 of DC culture or left untreated as controls prior to addition to MLR. Supernatants were obtained after 48 hours of coculture and analyzed for their content of IL-12p70 and IL-10 (A-B), or IFN-γ and IL-13 (C-D) by ELISA. Data are given as mean pg/mL ± SD of duplicate measurements. Shown is 1 representative of 7 independent experiments.
Andreas C. Renkl et al. Blood 2005;106:
©2005 by American Society of Hematology

8. OPN-activated DCs polarize T cells toward a Th1- and IFN-γ–secreting phenotype.
OPN-activated DCs polarize T cells toward a Th1- and IFN-γ–secreting phenotype. Naive CD45RA+ Th cells (10 × 103/200 μL) isolated by immunomagnetic negative depletion were polarized by coculture with 12-hour ± OPN-stimulated DCs (5 × 103/200 μL). On day 5 of coculture, IL-2 (50 U/mL) was added. Cytokine secretion was induced on day 9 by the addition of PMA and ionomycin. Supernatants were obtained after 24 hours and analyzed by cytokine-specific ELISA. Experiments were performed with allogeneic (A) or autologous (B) DCs and Th cells. As positive control for DC-induced Th1 differentiation, DCs were polarized in the allogeneic system by IFN-γ (A); and in the autologous model, with a combination of IL-1β, TNF-α (maturation factor, MF), plus IFN-γ.20,28 Shown is 1 representative experiment of 2 (A) and 4 (B) independent experiments. Data are expressed as mean pg/mL ± SD of duplicate measurements. Absolute cytokine amounts in the autologous and the allogeneic system cannot be compared because no crossover experiments with matched donor pairs were performed.
Andreas C. Renkl et al. Blood 2005;106:
©2005 by American Society of Hematology