Polymorphonuclear neutrophils as accessory cells for T-cell activation: major histocompatibility complex class II restricted antigen-dependent induction of T-cell proliferation - PubMed (original) (raw)

Polymorphonuclear neutrophils as accessory cells for T-cell activation: major histocompatibility complex class II restricted antigen-dependent induction of T-cell proliferation

M Radsak et al. Immunology. 2000 Dec.

Abstract

Polymophonuclear cells (PMN) of healthy donors do not express major histocompatibility complex (MHC) class II antigens or the T-cell costimulatory molecules CD80 or CD86. Expression of these receptors, however, is seen in patients with chronic inflammatory diseases. We now report that, by culturing PMN of healthy donors with autologous serum, interferon-gamma (IFN-gamma) and granulocyte-macrophage colony-stimulating factor (GM-CSF), de novo synthesis of MHC class II, CD80 and CD86 could be induced. MHC class II-positive PMN acquired the capacity to present staphylococcus enterotoxin to peripheral T cells, apparent as induction of interleukin-2 (IL-2) synthesis and proliferation of the T cells. Moreover, the PMN also processed tetanus toxoid (TT) and induced proliferation of TT-specific T cells in a MHC class II-restricted manner. Taken together, these data indicate that PMN can be activated to function as accessory cells for T-cell activation.

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Figures

Figure 1

Characterization of cells after isolation with Polymorphprep (PMP) and adherence on plastic plates. Figure 1(a) shows dot-plot analysis (FSC against SSC) of cells directly after PMP isolation (I) and after 2 hr of culture to plastic dishes (II). Cells were not gated and 98% of the cells expressed CD66b as a PMN marker (III) (solid line represents before adherence and dotted line represents after adherence). Two markers (M) were set: M1 to analyse further the CD66b– population and M2 for CD66b+ cells. Figure 1(b) shows characterization of CD66b– cells (cells within M1) (solid lines in Fig. 1b, I, II and III). The majority of cells expressed CD3 (I); only 0·33% of cells were positive for the monocyte marker CD14 (II) and 0·64% were positive for the B-cell marker CD20 (III). No CD83+ or X-11+ cells could be detected (results not shown). Values are corrected for immunoglobulin G1 (IgG1)- and IgG2a-negative controls (dotted lines in Fig. 1b, I, II and III). The data shown are representative of five experiments. FITC, fluorescein isothiocyanate; PE, phycoerythrin.

Figure 2

Expression of major histocompatibility complex (MHC) class II antigens on polymorphonuclear cells (PMN). Freshly isolated PMN (a) and PMN cultured for 48 hr in medium containing interferon-γ (IFN-γ) (100 U/ml) and granulocyte–macrophage colony-stimulating factor (GM-CSF) (50 U/ml) (b) were examined by confocal laser microscopy for the presence of MHC class II antigens (upper panel) using a fluorescein isothiocyanate (FITC)-labelled antibody to MHC class II. By cytofluorometry (lower panel), no MHC class II was seen on untreated PMN (c) (the broken lines show the isotype control, the solid lines represent fluorescence of an FITC-labelled anti-MHC class II). After culturing the PMN for 48 hr in the presence of IFN-γ (100 U/ml) and GM-CSF (50 U/ml), MHC class II antigens were detectable intracellularly in saponin-treated PMN (d) and on the surface of untreated cells (e). HLA, human leucocyte antigen.

Figure 3

Surface expression of costimulatory molecules CD80, CD86, CD58 and CD54 on polymorphonuclear cells (PMN). (a) PMN were treated with interferon-γ (IFN-γ) (100 U/ml) and granulocyte–macrophage colony-stimulating factor (GM-CSF) (50 U/ml) for 48 hr; CD80 and CD86 were analysed by cytofluorometry using PE-labelled antibodies to CD80 or CD86 and a fluorescein isothiocyanate (FITC)-labelled antibody to CD66b. The proportion of double-positive cells (right upper quadrant) was estimated. (b) PMN were cocultured with peripheral T cells for 48 hr. (c) Surface expression of CD58 and CD54 was analysed on cytokine-treated PMN (solid line); the broken line represents the surface expression of untreated PMN. ICAM-1, intracellular adhesion molecule-1; IgG, immunoglobulin G; PE, phycoerythrin.

Figure 4

Detection of human leucocyte antigen (HLA)-DR, CD80 and CD86 by reverse transcription–polymerase chain reaction (RT–PCR): RNA derived from Epstein–Barr virus (EBV)-transfected B cells (lane 1) or from polymorphonuclear cells (PMN) cultured in the absence (lane 3) or presence of cytokines interferon-γ (IFN-γ) (100 U/ml) and granulocyte–macrophage colony-stimulating factor (GM-CSF) (50 U/ml) for 16 hr (lane 4) was amplified by RT–PCR with primers specific for HLA-DR (a), CD80 (b) and CD86 (c). In lane 2 the products of a control amplification (using water instead of the RNA) are shown; M is molecular weight marker VI.

Figure 5

Induction of T-cell proliferation by polymorphonuclear cells (PMN) and staphylococcus enterotoxin E (SEE). Proliferation of peripheral T cells (1 × 105) was measured as incorporation of 3H-thymidine and is depicted on the _y_-axes as counts per minute (c.p.m.). All data are expressed as mean±SD (n = 6). Unless indicated otherwise, PMN or monocytes (105) had been precultured with interferon-γ (IFN-γ) (100 U/ml) and granulocyte–macrophage colony-stimulating factor (GM-CSF) (50 U/ml) for 24 hr. (a) T cells were cultured with SEE at different concentrations, with or without PMN (T cells alone). (b) Monocytes in the concentrations indicated and PMN of the same donor were coincubated with T cells and SEE (1 ng) for 5 days. The asterisk indicates values different from that obtained with 105 PMN (P < 0·05). (c) PMN were cocultured with T cells and SEE in the presence of monoclonal antibodies (0·2 µg/well) or mouse immunoglobulin G (IgG) for 5 days. On the very left, data are shown for PMN plus T cells without SEE. The asterisk indicates values different from that of PMN + T cells + SEE + IgG. (d) PMN of the same donor were either precultured with IFN-γ/GM-CSF for 24 hr and then cocultured with T cells for 5 days, or used immediately after isolation. In the absence of PMN no proliferation was seen.

Figure 6

Induction of T-cell proliferation by autologous polymorphonuclear cells (PMN) and tetanus toxoid C fragment (TT). (a) Peripheral, freshly isolated T cells (2 × 104) were cultured in the presence of PMN (2 × 104) + TT (0·2 µg), PMN alone or TT alone. Proliferation was measured after 5 days and expressed as mean±SD (n = 12). The following experiments were carried out with PMN cultured for 24 hr with interferon-γ (IFN-γ) (100 U/ml) and granulocyte–macrophage colony-stimulating factor (GM-CSF) (50 U/ml) or with freshly isolated monocytes and the TT-specific T-cell line (105 cells; all cells obtained from the same donor) and TT (0·2 µg). Proliferation was measured after 5 days and expressed as counts per minute (c.p.m.), mean±SD (n = 12). (b) T cells and TT were cocultured with autologous monocytes or PMN at the numbers indicated. The asterisk indicates values different from those obtained with 5 × 104 PMN. (c) Different numbers of PMN were cocultured with TT-specific T cells and TT. (d) PMN (105) or monocytes (5 × 103) were cultured with T cells and TT in the presence of the monoclonal antibodies (0·2 µg of each). The asterisk indicates values different from those of the respective experiment with immunoglobulin G (IgG). (e) PMN (105) were incubated with TT and T cells for 24 hr. Different amounts of anti-CD86 were then added and proliferation measured after 4 days. The asterisk indicates values different from those obtained with 0·2 µg of IgG. (f) CD4+ or CD8+ TT-specific T cells (5 × 104) were cocultured with PMN (105) and TT with or without antibodies to major histocompatibility complex (MHC) class II or class I antigens, respectively (0·2 µg each). (g) Autologous PMN or PMN of a donor with different DR-type (heterologous) (105 each) were cultured with TT and T cells (105).

Figure 7

Antigen-pulse experiments with tetanus toxoid C fragment (TT)-specific T cells and polymorphonuclear cells (PMN) or monocytes. (a) Freshly isolated PMN or PMN (105 each), precultured for 24 hr in interferon-γ (IFN-γ) (100 U/ml) and granulocyte–macrophage colony-stimulating factor (GM-CSF) (50 U/ml), were incubated with TT (0·2 µg) for 3 hr. The cells were then washed twice and T cells (105) were added with additional TT (+ TT) or without TT. (b) A similar experiment was performed with freshly isolated monocytes (1 × 104) from the same donor or with monocytes that also had been precultured with IFN-γ (100 U/ml) and GM-CSF (50 U/ml) for 24 hr. Proliferation was measured after 5 days. The results are expressed as counts per minute (c.p.m.) mean±SD (n = 12).

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Polymorphonuclear neutrophils as accessory cells for T-cell activation: major histocompatibility complex class II restricted antigen-dependent induction of T-cell proliferation - PubMed (original) (raw)