Proteasomal chymotrypsin-like peptidase activity is required for essential functions of human monocyte-derived dendritic cells

Abstract

The ubiquitin-proteasome pathway is the principal system for extralysosomal protein degradation in eukaryotic cells, and is essential for the regulation and maintenance of basic cellular processes, including differentiation, proliferation, cell cycling, gene transcription and apoptosis. The 26S proteasome, a large multicatalytic protease complex, constitutes the system's proteolytic core machinery that exhibits different proteolytic activities residing in defined proteasomal subunits. We have identified proteasome inhibitors - bortezomib, epoxomicin and lactacystin - which selectively inhibit the proteasomal beta5 subunit-located chymotrypsin-like peptidase activity in human monocyte-derived dendritic cells (DCs). Inhibition of proteasomal chymotrypsin-like peptidase activity in immature and mature DCs impairs the cell-surface expression of CD40, CD86, CD80, human leucocyte antigen (HLA)-DR, CD206 and CD209, induces apoptosis, and impairs maturation of DCs, as demonstrated by decreased cell-surface expression of CD83 and lack of nuclear translocation of RelA and RelB. Inhibition of chymotrypsin-like peptidase activity abrogates macropinocytosis and receptor-mediated endocytosis of macromolecular antigens in immature DCs, and inhibits the synthesis of interleukin (IL)-12p70 and IL-12p40 in mature DCs. As a functional consequence, DCs fail to stimulate allogeneic CD4(+) and CD8(+) T cells and autologous CD4(+) T cells sufficiently in response to inhibition of chymotrypsin-like peptidase activity. Thus, proteasomal chymotrypsin-like peptidase activity is required for essential functions of human DCs, and inhibition of proteasomal chymotrypsin-like peptidase activity by selective inhibitors, or by targeting beta5 subunit expression, may provide a novel therapeutic strategy for suppression of deregulated and unwanted immune responses.

Figure 1

Inhibitory profiles of the proteasome inhibitors bortezomib, epoxomicin, lactacystin, gliotoxin and epigallocatechin-3-gallate (EGCG), determined in lysates of mature dendritic cells (mDCs). Lysates were incubated for 30 min with the proteasome inhibitors at the indicated concentrations and with the fluorogenic oligopeptidyl substrates Boc-LLE-amc (caspase-like peptidase activity), Boc-LRR-amc (trypsin-like peptidase activity) and Z-GGL-amc (chymotrypsin-like peptidase activity). Catalytic activities were determined as described in the Materials and methods. For each inhibitor, one representative experiment out of four independent experiments is shown.

Figure 2

Inhibition of cell-surface expression of immune receptors in dendritic cells (DCs) exposed to proteasome inhibitors targeting chymotrypsin-like peptidase activity (CPA) at different stages of differentiation. (a) Cells were exposed for 24 hr to the indicated concentrations of bortezomib (Bor), epoxomicin (EPM) or lactacystin (Lac) during the differentiation from monocytes to immature DCs (iDCs). (b) Cells were exposed for 24 hr to the indicated concentrations of bortezomib, EPM or lactacystin during the differentiation from iDCs to mature DCs (mDCs). (c) mDC were exposed for 24 hr to the indicated concentrations of bortezomib, EPM or lactacystin. (d) For measurement of cell-surface receptor expression, a viable and homogeneous population of DCs was gated (right). This gate always contained 95–98% DCs, as determined by staining the cells with anti-CD11c–phycoerythrin (PE) monoclonal antibody and the CD11c-regating procedure (right). Data were measured as mean fluorescence intensity (MFI) and calculated as percentage MFI of inhibitor-incubated cells compared with the MFI of dimethylsulphoxide (DMSO)-incubated cells: (MFI of inhibitor-incubated cells ÷ MFI of DMSO-incubated cells) × 100. Data are given as mean values ± standard error of the mean (SEM) of three independent experiments carried out in triplicate. *P < 0·05, **P < 0·01 (paired Student's t-test) versus DMSO control.

Figure 3

Immunoblot analysis of intracellular expression of CD206 and human leucocyte antigen (HLA)-DR in lysates of cells incubated for 24 hr with the indicated concentrations of bortezomib (Bor). (a) Cells during the differentiation from monocytes to immature dendritic cells (iDCs). (b) Cells during the differentiation from iDCs to mature dendritic cells (mDCs). (c) mDCs. For analysis of expression of housekeeping proteins unrelated to DC function, the amounts of β-actin were determined in whole-cell lysates. DMSO, dimethylsulphoxide.

Figure 4

Inhibition of nuclear translocation and accumulation of RelB and RelA in immature dendritic cells (iDCs) exposed to bortezomib (Bor). (a) iDCs were incubated with 10 nm bortezomib for 1 hr before the induction of maturation with lipopolysaccharide (LPS) and interferon-γ (IFN-γ). The amounts of RelB and RelA proteins were determined in the nuclear and cytoplasmic fractions of the cells by immunoblotting at the indicated time points. For analysis of expression of housekeeping proteins unrelated to DC function, the amounts of β-actin were determined in the cytoplasmic fractions. Immunoblots were performed in triplicate, with similar results obtained on each occasion. Similar results were obtained with epoxomicin (EPM) and lactacystin. (b) Inhibition of maturation of iDCs exposed to proteasome inhibitors targeting chymotrypsin-like peptidase activity (CPA). iDCs were incubated with bortezomib, EPM or lactacystin for 1 hr prior to induction of maturation. Cell-surface expression of CD83, the general marker for mature dendritic cells (mDCs), was analysed after 24 hr by flow cytometry, as described in the Materials and methods. Only viable and non-apoptotic cells were analysed. Data are given as mean values ± standard error of the mean (SEM) of three independent experiments carried out in triplicate. *P < 0·05 (paired Student's t-test) versus the dimethylsulphoxide (DMSO) control.

Figure 5

Induction of apoptosis in dendritic cells (DCs) exposed to bortezomib (Bor). (a) Monocytes, (b) immature dendritic cells (iDCs) and (c) mature dendritic cells (mDCs) were incubated for 24 hr with the indicated concentrations of bortezomib. Apoptosis was analysed by flow cytometry. CD11c⁺ cells were gated and analysed for annexin V bindings, as described in the Materials and methods. Similar results were obtained using epoxomicin (EPM) and lactacystin. Data are given as the mean values ± standard error of the mean (SEM) of four independent experiments carried out in triplicate. **P < 0·01, ***P < 0·0005 (paired Student's t-test) versus the dimethylsulphoxide (DMSO) control.

Figure 6

Inhibition of receptor-mediated endocytosis and macropinocytosis in immature dendritic cells (iDCs) exposed to bortezomib (Bor). (a) Inhibition of receptor-mediated endocytosis of fluorescein isothiocyanate (FITC)-labelled 40 000 molecuar weight (MW) dextran in iDCs exposed for 24 hr to the indicated concentrations of bortezomib. Intracellular accumulation of FITC-labelled 40 000 MW dextran was analysed at the indicated time points by flow cytometry, as described in the Materials and methods. (b) Inhibition macropinocytosis of Lucifer Yellow in iDCs exposed for 24 hr to the indicated concentrations of bortezomib. Intracellular accumulation of Lucifer Yellow was analysed at the indicated time points by flow cytometry, as described in the Materials and methods. Measurement of receptor-mediated endocytosis and macropinocytosis was measured as mean fluorescence intensity (MFI) and by using a CD11c-regating procedure, as described in Fig. 2(d). Similar results were obtained using epoxomicin (EPM) and lactacystin. Data given represent the mean values ± standard error of the mean (SEM) of four independent experiments carried out in triplicate. *P < 0·05, **P < 0·01 (paired Student's t-test) versus dimethylsulphoxide (DMSO) control.

Figure 7

Inhibition of production of interleukin (IL)-12 in dendritic cells (DCs) exposed to bortezomib (Bor). (a) Production of agonistic (bioactive) IL-12p70 heterodimers, (b) production of antagonistic IL-12p40 homodimers and (c) the resulting IL-12p70/IL-12p40 ratio were quantified during the maturation of iDCs into mature dendritic cells (mDCs) and simultaneous exposure of the cells for 24 hr to the indicated concentrations of bortezomib. The concentrations of IL-12p70 and IL-12p40 proteins were quantified in the cell culture supernatants by enzyme-linked immunosorbent assay (ELISA), as described in the Materials and methods. Data given represent the mean values ± standard error of the mean (SEM) of four independent experiments in triplicate.

Figure 8

Inhibition of mature dendritic cell (mDC)-mediated allogeneic and autologous T-cell stimulation by bortezomib (Bor). Stimulation of (a) allogeneic CD4⁺ T cells, (b) allogeneic CD8⁺ T cells and (c) autologous CD4⁺ T cells by mDCs after exposure of mDCs for 24 hr to the indicated concentrations of bortezomib. Stimulation experiments were carried out at DC:T-cell ratios of 1 : 10, 1 : 20, 1 : 50 and 1 : 100. For stimulation of autologous CD4⁺ T cells, DCs were pulsed with ovalbumin, as described in the Materials and methods. Only viable and non-apoptotic DCs were counted and used for T-cell stimulation. Data given represent the mean values ± standard error of the mean (SEM) of five independent experiments. *P < 0·05, **P < 0·01 (paired Student's t-test) versus the dimethylsulphoxide (DMSO) control. E:T, effector:target ratio.