Macrophages express membrane bound form of APRIL that can generate immunomodulatory signals

Abstract

Members of the tumour necrosis factor superfamily play an essential role in inducing various biological responses including proliferation, differentiation, survival and cell death. A proliferation-inducing ligand (APRIL), first identified as a stimulant of tumour proliferation, is now known as a regulator of B-cell-mediated immune responses through the modulation of B-cell survival and activation. However, the role of APRIL in macrophage function has not been explored. High level expression of APRIL was detected on the surface of cells of the monocytic lineage including the human macrophage-like cell line, THP-1. To identify the role of APRIL in macrophage functions, THP-1 cells were stimulated with either its counterpart (TACI : Fc fusion protein) or a monoclonal antibody that is specific to APRIL. Stimulation of APRIL resulted in the expression of pro-inflammatory mediators such as interleukin-8 and matrix metalloproteinase-9 through the activation of mitogen-activated protein kinase and nuclear factor-κB. In contrast, stimulation of APRIL had an inhibitory effect on processes that require cytoskeletal movement such as phagocytosis of opsonized zymosan and chemotaxis through an inhibition of phosphatidylinositol 3-kinase activity. These observations demonstrate that macrophages express a membrane-bound form of APRIL which, upon stimulation, modulates the activities of macrophages through stimulation or inhibition of processes associated with inflammation.

Figure 1

Human monocytic cell lines express APRIL on the cell surface. THP-1 (a) and U937 (b) cells were stained with anti-APRIL monoclonal antibody. Histograms from specific staining (open area) and background staining (filled area, stained with mouse immunoglobulin G1) are compared. (c) TF-1A cells were incubated in the presence or absence of 10 nm of phorbol 12-myristate 13-acetate for 2 days and then stained for the expression of APRIL. (d) THP-1 cells were transfected with control small interfering (si) RNA or siRNAs specific for APRIL or BAFF. Transfectants were then tested for the expression of BAFF or APRIL. Each transfectant was analysed for the message level of APRIL, BAFF and GAPDH using reverse transcription–polymerase chain reaction (inset). A, APRIL (394 base pairs; bp); B, BAFF (370 bp); G, GAPDH (391 bp). Results are representative for three independent experiments.

Figure 2

The stimulation of APRIL induced the expression of matrix metalloproteinase 9 (MMP-9) and interleukin-8 (IL-8) in THP-1 cells. THP-1 cells were stimulated with 1, 3, or 10 μg/ml concentrations of TACI : Fc or anti-APRIL monoclonal antibody (mAb) for 24 hr and the culture supernatant was collected. Gelatin zymogram was performed to analyse the expression of MMP-9 (a) and enzyme-linked immunosorbent assay was performed to measure the concentrations of IL-8 (b). (c) No treatment control; L, lipopolysaccharide at 1 μg/ml; H, human immunoglobulin G (IgG; 10 μg/ml), M, mouse IgG (10 μg/ml). (c, d) THP-1 cells transfected with small interfering RNA were stimulated with 1 μg/ml of anti-BAFF mAb (b), anti-APRIL mAb (a), or isotype-matching mouse IgG (M) for 24 hr. Culture supernatants were then collected for the measurement of MMP-9 and IL-8. Results are representative for three independent experiments.

Figure 3

The APRIL-induced expression of interleukin-8 (IL-8) requires the activity of mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB). (a) THP-1 cells were pre-treated with 10 μm of U0126 (U), SB203580 (SB), Jun N-terminal kinase (JNK) inhibitor (JI), or negative control of JNK inhibitor (J–) for 30 min and the cells were stimulated with anti-APRIL monoclonal antibody (mAb; 1 μg/ml). Culture supernatants were collected after 24 hr to measure IL-8 concentrations using enzyme-linked immunosorbent assay. C, no treatment control; L, 1 μg/ml lipopolysaccharide as a positive control; D, 0·1% dimethyl sulphoxide as a vehicle control. D, THP-1 cells were pre-treated for 30 min with 20 and 40 μm TPCK, 5 and 10 mm ethyl pyruvate (EP), or 0·5 and 1 mm of sulfasalzine (SSZ) and stimulated with anti-APRIL mAb (1 μg/ml). Culture supernatants were collected after 24 hr to measure IL-8 concentrations. Results are representative for three independent experiments.

Figure 4

APRIL-induced expression of matrix metalloproteinase 9 (MMP-9) and interleukin-8 (IL-8) is independent of the expression TACI or BCMA in THP-1 cells. (a) THP-1 cells were either transfected with control small interfering (si) RNA or siRNAs specific for BCMA and TACI. Cell surface expression levels of APRIL, TACI and BCMA were measured after 5 days. Histograms from specific staining (open area) and background staining (filled area, stained with mouse immunoglobulin G1; IgG1) were compared. (b) Cells transfected with TACI and BCMA specific siRNAs were stimulated with 1 μg/ml lipopolysaccharide, anti-APRIL monoclonal antibody (mAb), mouse IgG, TACI : Fc, or TACI : Fc plus 1 μg/ml of anti-TACI mAb for 24 hr and the culture supernatant was collected for the analysis of MMP-9 activity and IL-8 measurement. Transfection experiments were independently repeated twice with essentially the same results. ***P < 0·001 when compared with the control.

Figure 5

The stimulation of APRIL inhibited phagocytosis of opsonized zymosan and transmigration in THP-1 cells through suppressing the activities of phosphatidyl inositol 3-kinase (PI3K). (a) THP-1 cells were pre-treated for 30 min with 10 μg/ml of anti-APRIL monoclonal antibody (mAb) or mouse immunoglobulin G (mIgG). Cells were then incubated with 30 μg/ml of fluorescence-labelled opsonized zymosan. Three hours later, fluorescence levels in each sample were measured using flow cytometry. (b) The assay in (a) was repeated three times and the flow cytometry results were subjected to a statistical analysis after setting the control measurement as 100%. Values indicate mean + SEM. **P < 0·01, when compared with the control. C, no treatment control; M, mIgG; A, anti-APRIL. (c) Transmigration potential of THP-1 cells pre-treated with 20 μg/ml of mIgG (M) or 10 and 20 μg/ml of anti-APRIL mAb (A) was measured with 10 μg/ml of fibronectin as a chemoattractant. Experiments were repeated three times for statistical analysis. Numbers represent mean + SEM of migrated cell numbers. **P < 0·01, when compared with the no treatment control (N). ^#P < 0·05, ^##P < 0·01, when compared with the control (C). (d) THP-1 cells were stimulated with 30 μg/ml of opsonized zymosan in the presence or absence of 10 μg/ml of anti-APRIL mAb (A) or mouse IgG (M) for 3 hr. Cell lysates were obtained after 2 hr for the Western blot analysis of AKT and phospho-AKT (inset). Phospho-AKT band intensities were then measured with densitometer and normalized with band intensities of corresponding AKT. Results are representative for three independent experiments.