Table 1.
Nucleotide sequences of primer pairs used in this study.
Fig 1.
Flow cytometric (A) and confocal laser scan microscopic (B–E) detection of trogocytosis between PKH-26 (red fluorescence)-labeled CD4+ T cells and PKH-67 (green fluorescence)-labeled PMN. (A) The proportion of trogocytosis in PMN (panel R1; double stain in UR) and CD4+ T (panel R2) detected by flow cytometry after PMN-CD4+ T co-culture. (B) Trogocytosis between PMNs (arrows) and CD4+ T cells (arrowheads) leads to the generation of yellow fluorescence (magnification x400). (C) Membrane transfer from PKH-26-labeled CD4+ T cells to PKH-67-labeled PMNs (arrow) leads to the generation of yellow fluorescence at the site of cell-cell close contact (magnification x1000). (D) Trogocytosis between FITC-anti-CD4 antibody stained CD4+ T cell (arrowhead) and PKH-26-labeled PMN (arrow) demonstrating yellow fluorescence. *Indicates FITC-anti-CD4 antibody stained CD4+ T cells with no evidence of trogocytosis. Both cell types were nuclear stained with DAPI (blue fluorescence). (E) Trogocytosis between FITC-anti-CD16 antibody-stained PMN (arrow) and DAPI/PKH-26-labeled CD4+ T cell (arrowhead) demonstrating yellow fluorescence. *Indicates FITC-anti-CD16 antibody stained PMNs with no evidence of trogocytosis. Images are representative from three independent experiments.
Fig 2.
Trogocytosis between PMN and U937 macrophage cells with or without siRNA knockdown of CD11a.
Trogocytosis between PKH-67 (green fluorescence)-labeled PMN and PKH-26 (red fluorescence)-labeled-U937 macrophage cells observed by confocal laser scan microscopy. (A) Direct cell-cell contact of PMNs with U937 cells for 1 h, demonstrating yellow fluorescence in many cells (magnification x400) (B) Transwell co-culture of PKH-67-labeled PMNs with PKH-26 labeled U937 cells for 1 h demonstrating an absence of yellow fluorescence (magnification x400). (C) Magnification of (A) showing yellow fluorescence in a PMN (arrow) and U937 cells (arrowheads) after 1 h co-culture (magnification x1000). (D) Co-culture of PKH-67-labeled PMNs and PKH-26-labeled U937 cells for 24 h; prominent yellow fluorescence is visible on PMNs (arrows) and U937 cells (arrowheads) (magnification x1000). (E) Results of FACS analysis demonstrating transfer of MHC class-II from human monocytes/macrophages to PMNs after co-culture for 2 h. (F) Immunoblot analysis of CD11a expression in U937 cell lysates stably transfected with specific CD11a siRNA or empty vector. (G) Densitometric quantification of CD11a in U937 cell lysates with/without siRNA knockdown by Western blot. (H) Total membrane transfer (expressed as mean fluorescence intensity, MFI) from U937 cells, with or without siRNA knockdown of CD11a, to PMNs after 2 h co-culture.
Fig 3.
Enhanced phagocytic activity and IL-8 production of PMNs after cell-cell contact with autologous T cells.
(A) A representative case from three independent experiments showing PMN phagocytotic activity (%) detected by Flow cytometry. (B) IL-8 concentration (pg/ml) in 6 h co-culture supernatants detected by ELISA (N = 8). *Indicates transwell co-culture.
Table 2.
Effects of different membrane integrity inhibitors on total membrane transfer from PKH26-labeled T cells to PMNs and PMN functions after 1 h pre-incubation detected by flow cytometry
Fig 4.
Co-culture of PMNs with lymphocytes or monocytes decreases PMN apoptosis via the anti-extrinsic pro-apoptotic pathway.
(A) A representative case from four independent experiments showing the effect on PMN apoptosis after cell-cell contact with monocytes, lymphocytes or RBCs for 24 h. UR = late stage apoptosis. LR = early apoptosis. *Transwell chamber co-culture. (B) A representative case from three independent experiments to detect caspase 8 (above) and caspase 9 (below) by FLICA. In each case, the upper panel shows results from PMNs cultured alone, whereas the middle and bottom panels are results from PMNs and MNCs co-cultured or cultured in transwell chambers, respectively. (C) Detection of pro-apoptotic (BAX and MYC) and anti-apoptotic gene (BCL2L1 and BCL2) mRNA expression in PMN by RT-PCR after cell-cell contact co-culture with lymphocytes or monocytes/macrophages for 2 h. Two independent experiments were conducted.
Fig 5.
Detection of intracellular signaling molecule expression in PMN by Western blot after co-culture with lymphocytes (Lym) or monocytes (Mono) for 2 h and 14h.
Fig 6.
Detection of transfer of specific surface-expressed molecules from PMN to T cells and effects on IL-2 production by anti-CD3/anti-CD28 activated MNCs.
Results of FACS analysis demonstrating (A) transfer of adhesion molecules CD11b and LFA-1, (B) transfer of CXCR1, and (C) transfer of HLA class-I from PMN to T cells after co-culture for 2 h. *FITC-antibody-stained PMNs. (D) Effects of autologous PMNs and MAP kinase inhibitor PD98059 on IL-2 production by activated human MNCs.
Table 3.
Specific surface molecule transfer among PMN, T cells and monocytes.
Table 4.
Effects of different inhibitors on PMN-MNC trogocytosis detected by flow cytometry.
Fig 7.
Comparative total membrane transfer from PMNs to MNCs and effects on IL-2 production by MNCs in active SLE versus healthy controls.
Results of FACS analysis of representative cases showing (A) the proportion of total membrane transfer from PMNs to MNCs using cells from a healthy individual and (B) the proportion of total membrane transfer from PMNs to MNCs in a patient with active SLE. (C) Comparative proportion of total membrane transfer from PMNs to MNCs in patients with active SLE versus healthy controls. (D) Comparative IL-2 production in normal and active SLE groups.