1. The heat shock protein Gp96 binds to human neutrophils and monocytes and stimulates effector functions
by Markus P. Radsak, Norbert Hilf, Harpreet Singh-Jasuja, Sibylla Braedel, Peter Brossart, Hans-Georg Rammensee, and Hansjoerg Schild
Blood
Volume 101(7):
April 1, 2003
©2003 by American Society of Hematology

2. Binding of FITC-labeled Gp96 to PMNs and monocytes
Binding of FITC-labeled Gp96 to PMNs and monocytes.PBLs (2 × 105) were incubated with Gp96-FITC (50 μg/mL) and analyzed by FACS gating on PMNs (CD66b+) or monocytes (CD14+).
Binding of FITC-labeled Gp96 to PMNs and monocytes.PBLs (2 × 105) were incubated with Gp96-FITC (50 μg/mL) and analyzed by FACS gating on PMNs (CD66b+) or monocytes (CD14+). Gp96-FITC binding was competed in the presence of 5 times excess of unlabeled Gp96 on PMNs (A) or monocytes (C), but not by IgG2b as irrelevant protein (PMNs [B] or monocytes [D]). (E) The summarized results of normalized LPS-FITC or Gp96-FITC binding to PMNs from 4 independent experiments are presented with standard deviation. LPS-FITC or Gp96-FITC binding was competed using fucoidan (1 mg/mL), unlabeled LPS (5-fold), or Gp96 (5-fold) (*P < .004; **P < .05, by Student t test).
Markus P. Radsak et al. Blood 2003;101:
©2003 by American Society of Hematology

3. Enhanced binding of Gp96-FITC or LPS-FITC by activated PMNs
Enhanced binding of Gp96-FITC or LPS-FITC by activated PMNs.Unstimulated PMNs (○) or PMNs prestimulated with PMA (10 ng/mL) for 10 minutes at 37°C (●) were incubated with the indicated concentrations of gp96-FITC (A) or LPS-FITC (B) and analyzed by FACS. Th...
Enhanced binding of Gp96-FITC or LPS-FITC by activated PMNs.Unstimulated PMNs (○) or PMNs prestimulated with PMA (10 ng/mL) for 10 minutes at 37°C (●) were incubated with the indicated concentrations of gp96-FITC (A) or LPS-FITC (B) and analyzed by FACS. The increase in mean fluorescence was statistically significant for both Gp96-FITC (50 μg/mL) or LPS-FITC (30 μg/mL) binding in 5 independent experiments (n = 5; P < .05 orP < .001, respectively, by Student ttest).
Markus P. Radsak et al. Blood 2003;101:
©2003 by American Society of Hematology

4. Phagocytosis of fluorochrome-labeled polystyrene beads by PMNs
Phagocytosis of fluorochrome-labeled polystyrene beads by PMNs.Purified PMNs (2 × 105) were incubated with the indicated stimuli in the presence of 5 × 106fluorochrome-labeled microspheres for 45 minutes at 37°C.
Phagocytosis of fluorochrome-labeled polystyrene beads by PMNs.Purified PMNs (2 × 105) were incubated with the indicated stimuli in the presence of 5 × 106fluorochrome-labeled microspheres for 45 minutes at 37°C. Cells were washed and fixed in 1% paraformaldehyde. Fluorescence intensity was evaluated by FACS. The results shown are representative of 4 independent experiments with different donors. Statistical analysis of all experiments showed a significant increase in mean fluorescence with PMA, LPS, or Gp96 compared with the medium control (n = 4;P < .05 by Student t test).
Markus P. Radsak et al. Blood 2003;101:
©2003 by American Society of Hematology

5. Analysis of IL-8 in supernatant of PMNs or MNCs by ELISA
Analysis of IL-8 in supernatant of PMNs or MNCs by ELISA.Cells (4 × 105) were incubated in the presence of LPS, Gp96, or flanking fractions of the Gp96 purification scheme.
Analysis of IL-8 in supernatant of PMNs or MNCs by ELISA.Cells (4 × 105) were incubated in the presence of LPS, Gp96, or flanking fractions of the Gp96 purification scheme. Culture supernatants were harvested after 6 hours at 37°C with 7.5% CO2 and assayed for IL-8 by ELISA. To evaluate the contribution of cells other than PMNs to the release of IL-8, supernatants from PMNs (▪), PMNs with 5% MNCs added (■) (A) or MNCs only (B) were analyzed. All samples were taken in duplicates. The results shown are representative of 3 independent experiments. Statistical analysis of all 3 experiments indicated a significant increase in the release of IL-8 in the presence of Gp96 (at 100 μg/mL or 20 μg/mL for PMNs, P < .001; for PMNs+MNC or MNCs alone, P < .05) or LPS (at 10 ng/mL for PMNs,P = .0017; for PMNs+MNCs or MNCs, P < .05; flanking fraction+LPS, 10 ng/mL for PMNs, P < .001; for MNCs, P < .05), but not for flanking fraction alone (by Student t test).
Markus P. Radsak et al. Blood 2003;101:
©2003 by American Society of Hematology

6. Intracellular IL-8 staining in PMNs and monocytes
Intracellular IL-8 staining in PMNs and monocytes.Purified PMNs or MNCs (2 × 105) were incubated for 4 hours at 37°C with 7.5% CO2 with the indicated stimuli in the presence of brefeldin A (1 μg/mL) or monensin (GolgiStop, 2 μM).
Intracellular IL-8 staining in PMNs and monocytes.Purified PMNs or MNCs (2 × 105) were incubated for 4 hours at 37°C with 7.5% CO2 with the indicated stimuli in the presence of brefeldin A (1 μg/mL) or monensin (GolgiStop, 2 μM). Cells were washed and stained as described in “Materials and methods.” FITC-labeled mAbs against CD66b or CD14 were used to identify PMNs or monocytes, respectively, and a PE-labeled mAb against IL-8 was used to evaluate the amount of IL-8 produced by the cells. The shaded histograms represent the fluorescence of stimulated cells; the white histograms, the respective medium control. The results shown are representative of 3 independent experiments. The increase in mean fluorescence of Gp96 or LPS-stimulated monocytes was statistically significant compared with the medium control (P < .05 by Student ttest).
Markus P. Radsak et al. Blood 2003;101:
©2003 by American Society of Hematology