Interleukin-15 enhances proliferation and chemokine secretion of human follicular dendritic cells

Abstract

The germinal centre (GC) is a specialized microenvironment where high-affinity antibodies are produced through hypermutation and isotype switching. Follicular dendritic cells (FDCs) are the stromal cells of the GC. The timely expansion and establishment of an FDC network is essential for a protective GC reaction; however, only a few factors modulating FDC development have been recognized. In this study, we report that interleukin-15 (IL-15) enhances human primary FDC proliferation and regulates cytokine secretion. The FDCs express IL-15 receptor complexes for IL-15 signal transduction as well as for specific binding. Moreover, the secretion of chemokines CCL-2, CCL-5, CXCL-5 and CXCL-8 was reduced by blocking IL-15 signalling while the secretion of other cytokines, and the expression of CD14, CD44, CD54 (ICAM-1) and CD106 (VCAM-1) proteins remained unchanged. These results suggest that IL-15 plays a crucial role in the development of FDC networks during GC reaction, offering a new target for immune modulation.

Figure 4

Anti-interleukin-15 (IL-15) monoclonal antibody (mAb) -mediated changes in cytokine secretion levels in media from follicular dendritic cells (FDCs). The FDCs were seeded in 24-well plates (2 × 10⁴ cells/well) and anti-IL-15 mAb, or control immunoglobulin G (IgG), with GC-B cells or tumour necrosis factor-α (TNF-α), was added. GC-B cells (if present) were removed after 12 hr and the supernatants were returned to the original wells. Supernatants were harvested after an additional 24 hr and analysed by LUMINEX assay. (a) Expression levels of IL-6, IL-8, IL-16 and CCL22 in supernatants and CD54 (ICAM-1) on the surface of FDCs cultured with GC-B, and controls. IL-2, IL-4, and sCD40L were added to maintain GC-B-cell survival in the culture system. The cytokine-only (IL-2, IL-4 and sCD40L) control was included to exclude possible cytokine effects on FDCs. The amounts of IL-6 IL-8, IL-16 and CCL22 were measured with the LUMINEX assay. Values represent the concentration of each cytokine (pg/ml) in the culture media. Expression of CD54 (ICAM-1) was measured by fluorescence-activated cell sorting analysis and is represented by the mean fluorescence intensity (MFI). <LOW> values reflect samples below the range measurable by the LUMINEX assay. (b, c) Relative amount of secreted chemokines (b) and cytokines (c) when IL-15 antibody was present, shown as a percentage of the amount when the corresponding control IgG was present. Each experiment with GC-B cells or TNF-α was duplicated. Data are presented at the mean of relative cell numbers ± SD.

Figure 1

Interleukin-15 (IL-15) increases follicular dendritic cell (FDC) recovery. (a) 1 × 10⁴ FDCs were cultured for 4 days in 24-well plates either with or without additional IL-15. Cells were harvested and viable cells were counted by the trypan blue exclusion assay. Numbers of cells are represented as a relative value with the control sample set to 1. Results are expressed as the mean of relative cell numbers ± standard deviation (SD) of two independent experiments. (b) 1 × 10⁴ FDCs were cultured for 3 days with 1, 3, 10 or 30 μg of anti-IL-15 monoclonal antibody (M110, M111, or M112), or control immunoglobulin G. Cells were harvested and counted on day 3. Absolute cell numbers from each antibody treatment are indicated on the graph at each concentration. Each antibody is represented by a different symbol.

Figure 2

Expression of interleukin-15 (IL-15) receptors in primary follicular dendritic cells (FDCs) and reduction in FDC recovery by anti-IL-15 and anti-IL-2Rβ treatment. (a) Expression of three components of the IL-15 receptor (IL-15Rα, IL-2Rβ and IL-2Rγ) was examined by reverse transcription–polymerase chain reaction of messenger RNA from three different human primary FDCs (FDC1, FDC2, FDC3) and freshly isolated GC-B cells. (b) 0·5 × 10⁴ FDCs were cultured for 3 days in 24-well plates with 10 μg/ml of anti-IL-15, anti-IL-2Rβ, or control immunoglobulin G (IgG). Cells were counted on day 3 by the trypan blue exclusion assay. Data are presented as the mean of relative cell numbers ± SD of four independent experiments.

Figure 3

Anti-interleukin-15 (IL-15) monoclonal antibody suppresses the proliferation of follicular dendritic cells (FDCs). (a) The FDCs were labelled with carboxyfluorescein succinimidyl ester (CFSE). After 3 days, the CFSE intensity was measured using a FACSCalibur™ and analysed on flowjo software. (b) FDC recovery was diminished by anti-IL-15 administration when cells were stained with CFSE. (c, d) FDCs cultured with anti-IL-15 or control immunoglobulin G (IgG) were stained with allophycocyanin (APC) -conjugated Annexin V and propidium iodide (PI) (c) or DiOC₆(3) (d). Apoptotic cells were analysed using a FACSCalibur™. There was no significant difference between the control and anti-IL-15-treated groups.

Figure 5

The surface expression levels of CD14, CD44, CD54 (ICAM-1) and CD106 (VCAM-1) are unchanged by anti-interleukin-15 (IL-15) monoclonal antibody (mAb) addition. Follicular dendritic cells (FDCs) were stained with the corresponding phycoerythrin-conjugated antibodies and analysed by fluorescence-activated cell sorting.