Carsten Berges

Proteasome inhibition suppresses essential immune functions of human CD4+ T cells

The proteasome constitutes the central proteolytic component of the highly conserved ubiquitin–proteasome system, which is required for the maintenance and regulation of basic cellular processes, including differentiation, proliferation, cell cycling, gene transcription and apoptosis. Here we show that inhibition of proteasomal proteolytic activity by the proteasome inhibitors bortezomib and lactacystin suppresses essential immune functions of human CD4+ T cells activated by allogeneic dendritic cells (DCs). In activated CD4+ T cells, proteasome inhibition induces apoptosis accompanied by rapid accumulation and stabilization of the tumour suppressor protein p53. Activated CD4+ T cells surviving proteasome inhibition undergo inhibition of proliferation by induction of G1 phase cell‐cycle arrest. Induction of G1 arrest is accompanied by the accumulation of cyclin‐dependent kinase inhibitors p21WAF1/CIP1 and p27KIP1 and the disappearance of cyclin A, cyclin D2 and proliferating cell nuclear antigen, proteins known to regulate G1 to S phase cell‐cycle transitions. Expression of the activation‐associated cell surface receptors CD25, CD28, CD120b and CD134 as well as production of interferon‐γ (IFN‐γ), tumour necrosis factor‐α (TNF‐α), interleukin‐4 (IL‐4) and IL‐5 is suppressed in response to proteasome inhibition in CD4+ T cells activated by DCs. Expression of CD25, IFN‐γ, TNF‐α, IL‐4 and IL‐5 is known to be mediated by the transcriptional activity of nuclear factor of activated T cells (NFAT), and we show here that proteasome inhibition suppresses activation and nuclear translocation of NFATc2 in activated CD4+ T cells. Thus, the proteasome is required for essential immune functions of activated CD4+ T cells and can be defined as a molecular target for the suppression of deregulated and unwanted T‐cell‐mediated immune responses.

Increased expression and altered subunit composition of proteasomes induced by continuous proteasome inhibition establish apoptosis resistance and hyperproliferation of Burkitt lymphoma cells

The proteasome is the main protease for extralysosomal protein degradation in eukaryotic cells, and constitutes a sophisticated high molecular mass proteinase complex underlying a tightly coordinated expression and assembly of multiple subunits and subcomplexes. Here we show that continuous inhibition of proteasomal chymotrypsin‐like peptidase activity by the proteasome inhibitor bortezomib induces in human Namalwa Burkitt lymphoma cells increased de novo biogenesis of proteasomes accompanied by increased expression of the proteasome maturation protein POMP, increased expression of 19S‐20S‐19S proteasomes, and abrogation of expression of β1i, β2i and β5i immunosubunits and PA28 in favor of increased expression of constitutive proteolytic β1, β2 and β5 subunits and 19S regulatory complexes. These alterations of proteasome expression and subunit composition are accompanied by an increase in proteasomal caspase‐like, trypsin‐like and chymotrypsin‐like peptidase activities, not inhibitable by high doses of bortezomib. Cells harboring these proteasomal alterations display rapid proliferation and cell cycle progression, and acquire resistance to apoptosis induced by proteasome inhibitors, γ‐irradiation and staurosporine. This acquired apoptosis resistance is accompanied by de novo expression of anti‐apoptotic Hsp27 protein and the loss of ability to accumulate and stabilize pro‐apoptotic p53 protein. Thus, increased expression, altered subunit composition and increased activity of proteasomes constitute a hitherto unknown adaptive and autoregulatory feedback mechanism to allow cells to survive the lethal challenge of proteasome inhibition and to establish a hyperproliferative and apoptosis‐resistant phenotype. J. Cell. Biochem. 103: 270–283, 2008.

Antithymocyte globulins suppress dendritic cell function by multiple mechanisms.

Background. The polyclonal rabbit antithymocyte and anti-T-cell immunoglobulins (ATGs) Thymoglobulin (TG) and ATG-Fresenius S (ATG-F) have been widely used for the prevention and therapy of allograft rejection and graft versus host disease in transplantation. Although immunosuppressive mechanisms of ATGs on T cells are well studied, less is known about their impact on dendritic cells (DCs). Methods. Effects of TG and ATG-F on immune functions and signaling pathways of human monocyte-derived DCs were determined by flow cytometry, enzyme-linked immunosorbent assay, Western blot, apoptosis assays, endocytosis assays, and T cell stimulation assays. Results. TG and ATG-F bind rapidly and with high affinity to CD11c, CD80, CD86, CD40, CD36, CD38, CD206, and human leukocyte antigen-DR on DCs. TG and, to a lesser extent, ATG-F induce apoptosis in immature and mature DCs. Macropinocytotic and receptor-mediated endocytotic antigen uptake in immature DCs is inhibited by TG and ATG-F due to their binding of the C-type lectins CD206 and CD209. TG and ATG-F induce activation of the mitogen-activated protein kinases ERK1/2 and p38 that contributes to the induction of apoptosis. TG and ATG-F also induce cytoplasmic-nuclear translocation of the NF-&kgr;B/Rel transcription factors RelB, RelA, p50, and p52. Production of interleukin-12p70 in mature DCs is suppressed by TG and ATG-F. TG and ATG-F reduce the capacity of mature DCs to stimulate allogeneic and autologous T cells. Conclusions. ATGs interfere with basic DC functions, suggesting that DCs are relevant targets for the immunosuppressive action of ATGs in transplantation.

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

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 systems 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 β5 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 β5 subunit expression, may provide a novel therapeutic strategy for suppression of deregulated and unwanted immune responses.

Helenalin suppresses essential immune functions of activated CD4+ T cells by multiple mechanisms.

Helenalin is a naturally occuring sesquiterpene lactone extracted from Arnica montana and Arnica chamissonis ssp. foliosa. Helenalin and its derivatives are known for anti-cancer and anti-inflammatory effects via inhibiting NF-kappaB and telomerase activity and impairing protein and DNA synthesis, suggesting that helenalin is a potential candidate for the treatment of deregulated and unwanted T cell-mediated immune responses. Here we show that helenalin induces apoptosis in activated CD4+ T cells by triggering the mitochondrial pathway of apoptosis. Induction of apoptosis is accompanied by rapid stabilization of p53, nuclear localization of p53 and AIF, and an increase in ROS production that results in loss of mitochondrial membrane potential (DeltaPsim). Activated CD4+ T cells which survive exposure to helenalin undergo inhibition of proliferation by induction of G2/M cell cycle arrest. Cell cycle arrest is accompanied by the accumulation of cell cycle regulator proteins p21(WAF/CIP1), p2(KIP1) and cyclin D2, whereas abundance of cyclin A and B(1) is decreased. Cell surface expression of the activation-associated receptors CD25, CD27, CD28, CD120b as well as production of IL-2 are impaired. Transcriptional activation of genes encoding for CD25, IL-2 and IFN-gamma is mediated by transcription factors of the NFAT family, and we demonstrate that helenalin suppresses nuclear translocation of NFATc2 in activated CD4+ T cells. Thus, helenalin can be defined as a new immunosuppressive compound suited for the treatment of deregulated and unwanted T cell-mediated immune responses.

HMG-CoA reductase inhibitor simvastatin overcomes bortezomib-induced apoptosis resistance by disrupting a geranylgeranyl pyrophosphate-dependent survival pathway.

Simvastatin is a competitive inhibitor of HMG-CoA reductase, the rate-limiting enzyme of the mevalonate pathway required for the biosynthesis of cholesterol and higher isoprenoids such as geranylgeranyl pyrophosphate (GGPP). Apart from its capacity to lower cholesterol plasma levels and to protect against cardiovascular disease, simvastatin induces apoptosis in various cancer cells. We have generated human Namalwa Burkitt lymphoma cells that display general apoptosis resistance and hyperproliferation due to increased expression and proteolytic activity of 26S proteasomes in response to continuous treatment of the cells with the proteasome inhibitor bortezomib. In these cells, simvastatin does not inhibit proteasome activity, but induces apoptosis, G2/M cell cycle arrest and accumulation of p21(Waf1/Cip1), and effectively inhibits hyperproliferation. These effects are reversed by the addition of GGPP. GGPP-dependent plasma membrane localization of the small GTPase RhoA that is required for RhoA-mediated oncogenic signaling is completely inhibited by simvastatin. Finally, bortezomib but not simvastatin induces accumulation and stabilization of the anti-apoptotic protein Mcl-1, which is known to confer resistance to apoptosis in cancer cells. Thus, simvastatin overcomes bortezomib-induced apoptosis resistance by inhibiting synthesis of GGPP and disrupting a GGPP-dependent survival pathway.

Proteasome inhibition activates the mitochondrial pathway of apoptosis in human CD4+ T cells†

We have previously shown that inhibition of the proteolytic activity of the proteasome induces apoptosis and suppresses essential functions of activated human CD4+ T cells, and we report now the detailed mechanisms of apoptosis following proteasome inhibition in these cells. Here we show that proteasome inhibition by bortezomib activates the mitochondrial pathway of apoptosis in activated CD4+ T cells by disrupting the equilibrium of pro‐apoptotic and anti‐apoptotic proteins at the outer mitochondrial membrane (OMM) and by inducing the generation of reactive oxygen species (ROS). Proteasome inhibition leads to accumulation of pro‐apoptotic proteins PUMA, Noxa, Bim and p53 at the OMM. This event provokes mitochondrial translocation of activated Bax and Bak homodimers, which induce loss of mitochondrial membrane potential (ΔΨm). Breakdown of ΔΨm is followed by rapid release of pro‐apoptotic Smac/DIABLO and HtrA2 from mitochondria, whereas release of cytochrome c and AIF is delayed. Cytoplasmic Smac/DIABLO and HtrA2 antagonize IAP‐mediated inhibition of partially activated caspases, leading to premature activation of caspase‐3 followed by activation of caspase‐9. Our data show that proteasome inhibition triggers the mitochondrial pathway of apoptosis by activating mutually independent apoptotic pathways. These results provide novel insights into the mechanisms of apoptosis induced by proteasome inhibition in activated T cells and underscore the future use of proteasome inhibitors for immunosuppression. J. Cell. Biochem. 108: 935–946, 2009.