Fig 1.
The effect of anesthetics on cross-talk of Fc receptor and Mac-1 for phagocytosis.
Targets opsonized with IgG and Mac-1 ligand will be captured by macrophages via their Fc receptors (FcRs) and Mac-1. Mac-1 consists of α- and β-subunits as shown in the scheme. Rap 1 is activated by FcRs after FcRs engage with IgG. Activated Rap1 can activate protein called RapL, which interacts with α subunit of Mac-1. Activated Mac-1 can activate Rap1 via protein Talin. As shown in the scheme, Rap1 will receive signal from both FcRs and Mac-1 to form phagocytic cup.
Fig 2.
The effect of anesthetics on macrophage phagocytosis and receptor expression level.
(A) Macrophage cell line RAW264.7 cells were used to test phagocytosis of sheep erythrocytes opsonized by IgG under different anesthetics (Isoflurane 1 and 2%, Sevoflurane 1.5 and 3%, Propofol 20 and 100 μM). Propofol was dissolved in DMSO. DMSO at the final concentration was 0.1%. Neutrophil inhibitory factor (NIF) is a Mac-1 inhibitor and used at 10 μM. Data are shown as mean +/- S.D. of quadruplicates. Representative figures from two independent experiments are shown. Statistical analysis was performed using one-way ANOVA with Bonferroni post hoc analysis. *, ** and *** represent p<0.05, p<0.01, and p<0.001, respectively. n.s. = not significant. (Β) Macrophage cell line RAW264.7 cells were used to test phagocytosis of sheep erythrocytes opsonized by IgG under different anesthetics. %Phagocytosis was calculated as in the Methods. Data are shown as mean +/- S.D. of quadruplicates. Representative figures from two independent experiments are shown. Statistical analysis was performed using one-way ANOVA with Bonferroni post hoc analysis. *, ** and *** represent p<0.05, p<0.01, and p<0.001, respectively. n.s. = not significant. (C) The expression levels of complement receptors and Fc receptors on RAW264.7 cells were detected by flow cytometry when co-incubated with IgG opsonized sheep erythrocytes as described in the method. Expression levels were analyzed with FlowJo software. Data are shown as mean +/- S.D. of quadruplicates. Representative figures from two independent experiments are shown. Statistical analysis was performed using one-way ANOVA with Bonferroni post hoc analysis. n.s. = not significant. CR4; complement receptor 4. MFI; mean fluorecense intensity.
Fig 3.
The effect of isoflurane and sevoflurane on E. coli phagocytosis.
RAW264.7 cells (A), thioglycollate-induced mouse peritoneal macrophages (B) and PMA-stimulated THP-1 cells (C) were used to test macrophage phagocytosis of fluorescent E. coli opsonized with serum under Isoflurane (1%) or Sevoflurane (1.5%). Phagocytosis was assessed using flow cytometry as described in Methods. Data are shown as mean +/- S.D. of quadruplicates. Representative figures from two independent experiments are shown. Statistical analysis was performed using one-way ANOVA with Bonferroni post hoc analysis. *** represents p<0.001, respectively.
Fig 4.
The effect of anesthetics on Rap1 activation of RAW264.7 cells.
Rap1 activation was probed using Rap1 activation assay kit as described in the method section. RAW cells were co-incubated either with non-opsonized sheep erythrocytes or IgG opsonized sheep erythrocytes. Rap1 activation was tested with or without (A) isoflurane (1%) and (B) sevoflurane (1.5%). Neutrophil inhibitory factor (NIF) was used at 10 μM. Representative blots are shown from two independent experiments with the same pattern. Immunoreactive proteins were visualized by enhanced chemiluminescence and intensities were quantified with Image J software. Data are shown as mean+/- S.D. of Rap1 GTP/ total Rap1 from three independent measurements. Statistical analysis was performed using one-way ANOVA with Bonferroni post hoc analysis. ** and *** represents p< 0.01 and p<0.001, respectively.
Fig 5.
Photolabeling of Rap1 by using azi-sevoflurane.
(A) Photolabeling experiment was performed using azi-sevoflurane. Sequences covered by mass spectrometry is highlighted in yellow. The adducted residue is shown in red. Red box denotes high conserved GTP-binding domain among Ras-related proteins. The blue line denotes effector binding site. (B) Rigid docking of sevoflurane was performed on Rap1. Arrow shows docked sevoflurane. GTP binding domains were indicated in red. The effect binding domain was shown in blue (overlapping G2 domain is shown in red). Residues within 4A from the docked sevoflurane were shown in orange. (C) Sevoflurane binding pocket is shown. The surface is shown in light gray.
Fig 6.
Rap1 activation of alanine mutants at the sevoflurane binding site.
Alanine scanning mutagenesis was performed at the sevoflurane binding site using Rap1 pcDNA3.1 plasmid. Mutated plasmid was transiently transfected into HEK293 cells and Rap1 activation was tested. Representative blot is shown from two independent experiments from the same pattern. Immunoreactive proteins were visualized by enhanced chemiluminescence and intensities were quantitated with Image J software. Data are shown as mean+/- S.D. of Rap1 GTP/ total Rap1 from three independent measurements. Statistical analysis was performed using one-way ANOVA with Bonferroni post hoc analysis. *, ** and *** represent P<0.05, p< 0.01 and p<0.001, respectively.
Fig 7.
Phagocytosis assay of Rap1 mutant.
RAW 264.7 cells were stably transfected with WT and deactivating mutant L161A. Phagocytosis was performed using fluorescence labeled E. coli. Phagocytosis was assessed using flow cytometry as described in Methods. Data are shown as mean +/- S.D. of quadruplicates. Representative figures from two independent experiments are shown. Statistical analysis was performed using one-way ANOVA with Bonferroni post hoc analysis. *** represents p<0.001, respectively.