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Neutrophils acquire antigen-presenting cell features after phagocytosis of IgG-opsonized erythrocytes

Sanne M. Meinderts, Gabriella Baker, Stan van Wijk, Boukje M. Beuger, Judy Geissler, Machiel H. Jansen, Anno Saris, Anja ten Brinke, Taco W. Kuijpers, Timo K. van den Berg and Robin van Bruggen

Data supplements

Article Figures & Data

Figures

  • Figure 1.

    Neutrophils upregulate MHC-II and costimulatory molecules following RBC phagocytosis. (A-B) RBC phagocytosis by neutrophils was measured by incubating neutrophils ± DiD-labeled unopsonized RBCs (RBC) or anti-GPA RBC-ops (RBC-ops) for 45 minutes. Subsequently, RBCs were lysed and the percentage of RBC-phagocytosing neutrophils was measured by flow cytometry. (A) Approximately 45% of neutrophils phagocytose RBC-ops (mean ± standard error of the mean [SEM]; n = 5). (B) Phagocytosis of RBC-ops by neutrophils was confirmed by cytospin. These images where blindly chosen and are representative for 5 different experiments. May-Grünwald Giemsa stain; original magnification ×500. (C-E) Surface expression of HLA-DR (MHC-II), CD40, and CD80 on freshly isolated neutrophils (T = 0) and neutrophils cultured for 20 hours (T = 20) with or without RBCs or RBC-ops. (F-H) Surface expression of HLA-DR, CD40, and CD80 on freshly isolated neutrophils (T = 0) and neutrophils stimulated with RBC-ops, IFNγ, TNFα, or GM-CSF and cultured for 20 hours (T = 20). The data represent the MFI values of HLA-DR, CD40, and CD80 (mean ± SEM; n = 5-7). Asterisks above straight-line bars represent significant differences compared with T = 0 control; asterisks above the inverted U–shaped spanner bars represent significant differences between the indicated bars (****P < .0001; ***P < .001; **P < .01; *P < .05). IFN, interferon; ns, nonsignificant.

  • Figure 2.

    MHC-II and costimulatory molecules are specifically upregulated on neutrophils that have phagocytized RBCs. (A) Schematic overview showing magnetic selection of the neutrophils that have phagocytosed RBC-ops. RBCs were biotinylated and coupled to magnetic streptavidin beads. RBCs were opsonized and incubated with neutrophils for 1 hour. Next, RBCs were lysed and phagocytosing neutrophils were obtained by magnetic selection. (B) Cytospins of the nonphagocytosing and phagocytosing neutrophils after magnetic separation. These images were blindly chosen and are representative for 5 different experiments. May-Grünwald Giemsa stain; original magnification ×500. (C-G) Surface expression of HLA-DR, CD40, and CD80 on nonphagocytosing and phagocytosing neutrophils. Data represent the MFI values of HLA-DR, CD40, and CD80 (mean ± SEM; n = 4-10). (H) Relative levels of mRNA for HLA-DR in neutrophils ± RBC-ops measured by quantitative PCR. Data are normalized for GUS expression (mean ± SEM; n = 4). Asterisks above the straight-line bars represent significant differences compared with T = 0 control; asterisks above the inverted U–shaped spanner bars represent significant differences between the indicated bars (****P < .0001; **P < .01; *P < .05).

  • Figure 3.

    Mature DCs have far greater expression levels of MHC-II and costimulatory molecules. Surface expression of HLA-DR, CD40, and CD80 on freshly isolated neutrophils (T = 0), neutrophils plus RBC-ops cultured for 20 hours (T = 20), monocytes, immature or mature DCs. Data represent the MFI values of HLA-DR (A), CD40 (B), and CD80 (C) (mean ± SEM; n = 3-5). Asterisks above the straight-line bars represent significant differences compared with T = 0 neutrophils; asterisks above the inverted U–shaped spanner bars represent significant differences between the indicated bars (****P < .0001; **P < .01; *P < .05).

  • Figure 4.

    Phagocytosis of RBCs by neutrophils induces a strongly diminished respiratory burst. (A) Release of H2O2 was measured by Amplex Red assay for neutrophils incubated ± RBC or RBC-ops. No burst was measured in the 3 conditions (mean ± SEM; n = 10-12). To control for the ability to produce a respiratory burst, neutrophils from all 3 conditions were stimulated with zymosan or PMA (mean ± SEM; n = 10-12). (B) Release of intracellular H2O2 production was measured using a DHR assay with neutrophils incubated ± RBC or RBC-ops. Neutrophils incubated with zymosan served as a positive control. Addition of PMA at 180 minutes showed neutrophils in all conditions were capable of producing ROS (mean ± SEM; n = 4-13). Asterisks above data points represent significant differences for zymosan compared with control (**P < .01; *P < .05). (C) Respiratory burst was visualized using NBT, which produces a blue precipitate in presence of intracellular ROS. Neutrophils were incubated plus RBC or RBC-ops. Neutrophils plus PMA served as a positive control and neutrophils alone served as a negative control. Images are representative for 3 individual experiments. May-Grünwald Giemsa stain; original magnification ×500. RFU, relative fluorescence unit.

  • Figure 5.

    Neutrophils can activate T cells following RBC phagocytosis. (A) Schematic overview of the autologous TT-specific T-cell assay. On day 0, (CFSE-labeled) T cells were incubated with neutrophils ± RBCs or RBC-ops TT. On day 4, T cells were restimulated with neutrophils ± RBCs or RBC-ops and ± TT. Flow cytometry was used as readout on days (1, 4, and) 8. (B) T-cell activation was measured by flow cytometry by determining the percentage of CD25+, CD38, or HLA-DR+ CD4+ T cells after 1, 4, and 8 days. Results are shown for T cells incubated with neutrophils (Neutro) ± RBCs or RBC-ops. All conditions are plus TT. T cells plus RBCs served as a negative control and T cells plus PBMCs as positive control (mean ± SEM; n = 3). (C) Histograms showing CD25, CD38, and HLA-DR staining on CD4+ T cells on days 1, 4, and 8 of a representative donor. As a positive control for CD25, CD38 and HLA-DR staining T cells stimulated with anti-CD3 and anti-CD28 were used. An isotype control was used as negative control. (D) CD4+ T-cell proliferation was measured by flow cytometry by determining the percentage of CFSE low T cells after 8 days. Percentage proliferation is shown for T cells incubated with neutrophils ± RBCs or RBC-ops and ± TT. T cells ± RBCs served as a negative control and T cells plus PBMCs as positive control (mean ± SEM; n = 8). (E) The experiment was repeated with the addition of an MHC-II blocking antibody to show that neutrophil-induced T-cell proliferation is MHC-II restricted (mean ± SEM; n = 6). (F) Graph showing IFN-γ levels in the medium at day 8 (n = 6). Asterisks above the straight-line bars represent significant differences compared with T cells plus RBCs day 1; asterisks above the inverted U–shaped spanner bars represent significant differences between the indicated bars (****P < .0001; ***P < .001; **P < .01; *P < .05). Nonsignificant results have not been indicated.

  • Figure 6.

    CD47 blocking on RBCs enhances the induction of TT-specific CD4+T-cell proliferation. (A) RBC phagocytosis by neutrophils was measured by incubating neutrophils ± DiD-labeled plus RBCs or RBC-ops, ± F(ab′)2 CD47 for 45 minutes. Subsequently, RBC were lysed and the percentage of RBC-phagocytosing neutrophils was measured by flow cytometry. Comparing the RBC-ops condition with the RBC-ops- F(ab′)2 CD47 condition, RBC phagocytosis increases from 45% to 73% (mean ± SEM; n = 5). (B) The percentage of proliferation measured for T cells incubated with neutrophils plus RBCs or RBC-ops, ± F(ab′)2 CD47 and ± TT. T cells served as a negative control and T cells plus PBMCs as positive control (mean ± SEM; n = 8). (C) Surface expression of HLA-DR, CD40, and CD80 on nonphagocytosing and phagocytosing neutrophils (mean ± SEM; n = 4). (D) Western blot analysis for HLA-DR expression performed on neutrophils ± RBCs or RBC-ops and ± F(ab′)2 CD47. Fluorescence intensity of the bands was quantified using Odyssey Imaging system and normalized for GAPDH expression. (E) Western blot analysis for CD40 expression performed on neutrophils ± RBCs or RBC-ops and ± F(ab′)2 CD47. Fluorescence intensity of the bands was quantified using Odyssey Imaging system and normalized for actin expression. (D-E) May-Grünwald Giemsa stain; original magnification ×500. Asterisks above the straight-line bars represent significant differences compared with T = 0 control neutrophils; asterisks above the inverted U–shaped spanner bars represent significant differences between the indicated bars (**P < .01; *P < .05).