Figure 1.
IFNγ-primed macrophages show Rituximab-dependent lysis of B cell lymphoma Raji cells.
A. Raw 264.7 cells or B. peritoneal macrophages were primed with 25 ng/ml IFNγ over-night. 51Cr- labeled Raji cells coated with 10 µg/ml Rituximab were added to IFNγ primed macrophages at E∶T ratios of 0∶1, 12.5∶1, 25∶1 and 50∶1. The amount of 51Cr released was measured at the end of 8 hours and is shown as % cytotoxicity. C. 51Cr-labeld Raji cells were coated with increasing concentrations of Rituximab as indicated in the figure or treated with Herceptin (10 µg/ml). They were then co-cultured with IFNγ-primed Raw 264.7 cells for 8 hours. The supernatants were harvested and the amount of 51Cr released was measured. The graph shows % cytotoxicity values obtained at 50∶1 E∶T ratio. D. To test the effect of IFNγ priming, Raw 264.7 cells were primed with increasing concentration of IFNγ and co-cultured with 51Cr- labeled Rituximab-coated Raji targets for 8 hours. % cytotoxicity values obtained at the E∶T ratio of 50∶1 are shown in the graph.
Figure 2.
Rituximab-coated targets induce significant activation of Syk kinase and the PtdIns 3-kinase/Akt pathway in macrophages.
IFNγ-primed peritoneal macrophages were stimulated with paraformaldehyde-fixed Rituximab-coated or control antibody-treated Raji cells at E∶T ratio of 1∶1 for indicated time points. Protein-matched whole cell lysates were analyzed by Western blotting with A. phospho-Syk antibody (upper) and B. phospho-Akt antibody (upper). The same membranes were reprobed with actin antibody (lower). The graphs in the lower panels show mean and SD of the band intensity of phospho-Syk and phospho-Akt from three independent experiments. Data were analyzed by student's t-test (* = p value≤0.05).
Figure 3.
Activation of the PtdIns 3-kinase/Akt pathway is critical for macrophage ADCC.
A. IFNγ-primed Raw 264.7 cells were treated with DMSO, 10 µM Ly294002 and 20 nM Rapamycin for 30 minutes. They were then incubated with 51Cr-labeled Rituximab-coated Raji cells for 8 hours. The graph shows % cytotoxicity at 50∶1 E∶T ratio. B. To test the specificity of the inhibitors used, Raw 264.7 cells were incubated with DMSO, 10 µM Ly294002 or 20 nM Rapamycin for 30 minutes. The cells were then stimulated with immune-complexes for 7 minutes. Protein-matched cell lysates were analyzed by Western blotting with phospho-Akt antibody and phospho-p70S6K antibody (upper). R indicates resting samples and A indicates cells activated with immune-complexes. The same membranes were reprobed with actin antibody (lower).
Figure 4.
Over-expression of active Akt enhances macrophage ADCC.
A. Wild-type and Myr-Akt expressing peritoneal macrophages were activated with IFNγ over-night. 51Cr- labeled Rituximab-coated Raji targets were added and supernatants harvested at the end of 8 hours. The graph shows % cytotoxicity values obtained at E∶T ratio of 50∶1. Data was analyzed using student's t-test (* = p value≤0.05). B. To confirm the expression of the Myr-Akt transgene, cell lysates from wild-type and Myr-Akt peritoneal macrophages were analyzed by Western blotting with phospho-Akt antibody (upper). The same membrane was reprobed with actin antibody (lower).
Figure 5.
Influence of the PtdIns 3-kinase/Akt pathway on mediators of cytotoxicity: TNFα and nitric oxide.
A. Raw 264.7 cells were left untreated or treated with IFNγ for 16 hours. They were then incubated with DMSO or 10 µM Ly294002 for 30 minutes followed by stimulation with Rituximab-coated Raji cells (indicated as RR in the figure) at E∶T ratio of 1∶1 for 8 hours. Controls conditions consisted of Raw 264.7 cells stimulated with media alone (UT) or IFNγ (25 ng/ml) alone or Rituximab-coated Raji cells alone. Supernatants were harvested and levels of TNFα were measured using ELISA. B. Raw 264.7 cells were cultured in presence of IFNγ and increasing concentrations of TNFα neutralizing antibody (0–1.6 µg/ml) or control antibody (1.6 µg/ml). After 16 hours of IFNγ priming, 51Cr- labeled Rituximab-coated Raji targets were added to Raw 264.7 cells and incubated for 8 hours. The graph shows % cytotoxicity at E∶T ratio of 50∶1. C. Raw 264.7 cells were primed with IFNγ in presence of varying concentrations of L-NMMA (0–10 mM). Next day 51Cr- labeled Rituximab-coated Raji targets were added and amount of 51Cr released was measured at the end of 8 hours. The graph shows % cytotoxicity at E∶T ratio of 50∶1. D. Raw 264.7 cells were pre-treated with IFNγ (25 ng/ml) for 16 hours. They were then stimulated with immune-complex (indicated as IC in the figure) at concentration of 350 µg/ml for 8 hours in presence of DMSO or 10 µM Ly294002. Controls conditions consisted of Raw 264.7 cells cultured with media alone (UT) or IFNγ (25 ng/ml) alone. Supernatant were harvested at the end of 24 hours and levels of NO produced were measured.
Figure 6.
The PtdIns 3-kinase/Akt pathway promotes conjugate formation between macrophages and tumor cells.
A. Raw 264.7 cells were primed with IFNγ overnight followed by treatment with DMSO or 10 µM Ly294002 or 20 nM Rapamycin for 30 minutes. They were then mixed with Rituximab-coated Raji cells at the E∶T ratio of 5∶1 and adhered to poly-L-lysine coated cover-slips for 1 hour. Raji cells were labeled with CD37 antibody followed by anti-mouse Alexa Fluor 594 (red fluorescence). F-actin was labeled with FITC-phalloidin. Stained cells were observed under the microscope for action polarization at the synapse. B. Samples were processed as described in A. 100 red cells were analyzed per cover-slip and a total of 300 red cells per condition were scored for conjugate formation. The results were expressed as % conjugates formed. The data indicate % conjugates formed in one experiment. Similar results were obtained in three independent experiments. C. Conjugate formation by WT and Myr-Akt expressing peritoneal macrophages was determined by processing the samples as described in A and B.
Figure 7.
The PtdIns 3-Kinase/Akt pathway enhances the spreading of macrophages on antibody-coated surfaces.
A. Spreading of thioglycollate-elicited peritoneal macrophages on Rituximab-coated cover-slips in the presence of DMSO or 10 µM Ly294002. B. Peritoneal macrophages, pre-incubated with DMSO or 10 µM Ly294002 were allowed to spread on Rituximab-coated cover-slips at 37°C for indicated times before fixation. Data are presented as mean surface area occupied by cells. Shown are data from one experiment. Similar results were obtained from three independent experiments. C. Mean surface area occupied by Wt and Myr-Akt peritoneal macrophages.