Pronounced up-regulation of the PA28alpha/beta proteasome regulator but little increase in the steady-state content of immunoproteasome during dendritic cell maturation.

Dendritic cells (DC) are professional antigen-presenting cells that activate CTL by presenting MHC class I-restricted peptides that are processed through the proteasome pathway. Previously, we reported that upon DC maturation the synthesis is switched towards the exclusive production of immunoproteasomes containing the active site subunits LMP2, LMP7 and MECL-1. In this study we investigated the mechanism by which proteasome assembly is regulated in mature DC. Quantitative analysis of mRNA expression showed very limited transcriptional induction of LMP7, MECL-1 and UMP1 in mature DC and a moderate mRNA increment for LMP2 and PA28alpha and beta. We investigated a role of PA28alpha/beta in regulating proteasome assembly in DC. PA28alpha/beta coprecipitated with 13S/16S proteasome precursor complexes but associated with mature constitutive and immunoproteasomes to the same extent. Furthermore, we determined the steady-state proteasome subunit composition in DC. Replacement of constitutive proteasomes by immunoproteasomes in maturing DC was very slow and occurred only to a minor extent. Our data suggest that the limited turnover of 20S proteasomes in mature DC probably contributes little to recently reported marked differences in antigen presentation between immature and mature DC and that alternative mechanisms may be responsible for this phenomenon.

Immunoproteasomes largely replace constitutive proteasomes during an antiviral and antibacterial immune response in the liver.

The proteasome is critically involved in the production of MHC class I-restricted T cell epitopes. Proteasome activity and epitope production are altered by IFN-gamma treatment, which leads to a gradual replacement of constitutive proteasomes by immunoproteasomes in vitro. However, a quantitative analysis of changes in the steady state subunit composition of proteasomes during an immune response against viruses or bacteria in vivo has not been reported. Here we show that the infection of mice with lymphocytic choriomeningitis virus or Listeria monocytogenes leads to an almost complete replacement of constitutive proteasomes by immunoproteasomes in the liver within 7 days. Proteasome replacements were markedly reduced in IFN-gamma(-/-) mice, but were only slightly affected in IFN-alphaR(-/-) and perforin(-/-) mice. The proteasome regulator PA28alpha/beta was up-regulated, whereas PA28gamma was reduced in the liver of lymphocytic choriomeningitis virus-infected mice. Proteasome replacements in the liver strongly altered proteasome activity and were unexpected to this extent, since an in vivo half-life of 12 days had been previously assigned to constitutive proteasomes in the liver. Our results suggest that during the peak phase of viral and bacterial elimination the antiviral cytotoxic T lymphocyte response is directed mainly to immunoproteasome-dependent T cell epitopes, which would be a novel parameter for the design of vaccines.

Cutting edge: neosynthesis is required for the presentation of a T cell epitope from a long-lived viral protein.

CTLs recognize peptide epitopes which are proteolytically generated by the proteasome and presented on MHC class I molecules. According to the defective ribosomal product (DRiP) hypothesis, epitopes originate from newly synthesized polypeptides which are degraded shortly after their translation. The DRiP hypothesis would explain how epitopes can be generated from long-lived proteins. We examined whether neosynthesis is required for presentation of the immunodominant epitope NP118 of the lymphocytic choriomeningitis virus nucleoprotein, which has a half-life of >3 days. Two days after nucleoprotein biosynthesis was terminated in a tetracycline-regulated transfectant, the presentation of the NP118 epitope ceased. This indicates that NP118 epitopes are generated from newly synthesized nucleoproteins rather than from the long-lived pool of nucleoproteins in the cell. Therefore, the lymphocytic choriomeningitis virus nucleoprotein is the first substrate for which a major prediction of the DRiP hypothesis, namely the requirement for neosynthesis, is shown to hold true.

Structural plasticity of the proteasome and its function in antigen processing.

The proteasome is the main provider of peptide ligands for major histocompatibility complex class I molecules. During an immune response to pathogens, the proinflammatory cytokine interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha are released, which induce the proteasome subunits LMP2, LMP7, and MECL-1. These replace the constitutively expressed active site subunits of the proteasome (delta, MB1, and Z) leading to a marked change in the cleavage preference of the proteasome and the production of T-cell epitopes. Proteasome activity is further changed by the IFN-gamma-mediated induction of the proteasome regulator PA28alpha/beta and the downregulation of PA28gamma. Why such an extensive exchange of proteasome active site subunits and regulators occurs is still poorly understood. In this article we discuss recent insights in the structural consequences of proteasome reorganization and their effects on epitope generation and shaping of the cytotoxic immune response. Moreover, we review the latest data on how the ubiquitin pathway targets protein antigens for peptide processing and discuss the potential of proteasome inhibitors for the modulation of antigen presentation.

The selective proteasome inhibitors lactacystin and epoxomicin can be used to either up- or down-regulate antigen presentation at nontoxic doses.

The complete inhibition of proteasome activities interferes with the production of most MHC class I peptide ligands as well as with cellular proliferation and survival. In this study we have investigated how partial and selective inhibition of the chymotrypsin-like activity of the proteasome by the proteasome inhibitors lactacystin or epoxomicin would affect Ag presentation. At 0.5-1 microM lactacystin, the presentation of the lymphocytic choriomeningitis virus-derived epitopes NP118 and GP33 and the mouse CMV epitope pp89-168 were reduced and were further diminished in a dose-dependent manner with increasing concentrations. Presentation of the lymphocytic choriomeningitis virus-derived epitope GP276, in contrast, was markedly enhanced at low, but abrogated at higher, concentrations of either lactacystin or epoxomicin. The inhibitor-mediated effects were thus epitope specific and did not correlate with the degradation rates of the involved viral proteins. Although neither apoptosis induction nor interference with cellular proliferation was observed at 0.5-1 microM lactacystin in vivo, this concentration was sufficient to alter the fragmentation of polypeptides by the 20S proteasome in vitro. Our results indicate that partial and selective inhibition of proteasome activity in vivo is a valid approach to modulate Ag presentation, with potential applications for the treatment of autoimmune diseases and the prevention of transplant rejection.

A ubiquitin-like protein which is synergistically inducible by interferon-gamma and tumor necrosis factor-alpha.

A means of regulating the fate of intracellular proteins is their covalent conjugation to ubiquitin-like proteins. A recently discovered ubiquitin-like protein is called "diubiquitin" because it consists of two ubiquitin-like domains in head-to-tail arrangement. Human diubiquitin is encoded at the telomeric end of the MHC class I locus and was previously found to be expressed in dendritic cells and mature B cells. We have extended the expression analysis of diubiquitin by reverse transcriptase-PCR and Northern blotting in primary endothelial cells and human cancer cell lines derived from nine different tissues. Diubiquitin expression was found to be generally and synergistically inducible with the cytokines IFN-gamma and TNF-alpha but not with IFN-alpha. Diubiquitin mRNA expression was induced within 2 h after cytokine stimulation and was independent of protein neosynthesis but dependent on proteasome activity. The mouse homologue of diubiquitin which is also encoded in the MHC class I locus was likewise induced with IFN-gamma and TNF-alpha. A general and synergistic induction with IFN-gamma and TNF-alpha suggests that diubiquitin may exert its functions in antigen presentation or other cellular processes controlled by these two cytokines.

How an inhibitor of the HIV-I protease modulates proteasome activity.

The human immunodeficiency virus, type I protease inhibitor Ritonavir has been used successfully in AIDS therapy for 4 years. Clinical observations suggested that Ritonavir may exert a direct effect on the immune system unrelated to inhibition of the human immunodeficiency virus, type I protease. In fact, Ritonavir inhibited the major histocompatibility complex class I restricted presentation of several viral antigens at therapeutically relevant concentrations (5 microM). In search of a molecular target we found that Ritonavir inhibited the chymotrypsin-like activity of the proteasome whereas the tryptic activity was enhanced. In this study we kinetically analyzed how Ritonavir modulates proteasome activity and what consequences this has on cellular functions of the proteasome. Ritonavir is a reversible effector of proteasome activity that protected the subunits MB-1 (X) and/or LMP7 from covalent active site modification with the vinyl sulfone inhibitor(125)I-NLVS, suggesting that they are the prime targets for competitive inhibition by Ritonavir. At low concentrations of Ritonavir (5 microM) cells were more sensitive to canavanine but proliferated normally whereas at higher concentrations (50 microM) protein degradation was affected, and the cell cycle was arrested in the G(1)/S phase. Ritonavir thus modulates antigen processing at concentrations at which vital cellular functions of the proteasome are not yet severely impeded. Proteasome modulators may hence qualify as therapeutics for the control of the cytotoxic immune response.

An inhibitor of HIV-1 protease modulates proteasome activity, antigen presentation, and T cell responses.

Inhibitors of the protease of HIV-1 have been used successfully for the treatment of HIV-1-infected patients and AIDS disease. We tested whether these protease inhibitory drugs exerted effects in addition to their antiviral activity. Here, we show in mice infected with lymphocytic choriomeningitis virus and treated with the HIV-1 protease inhibitor ritonavir a marked inhibition of antiviral cytotoxic T lymphocyte (CTL) activity and impaired major histocompatibility complex class I-restricted epitope presentation in the absence of direct effects on lymphocytic choriomeningitis virus replication. A potential molecular target was found: ritonavir selectively inhibited the chymotrypsin-like activity of the 20S proteasome. In view of the possible role of T cell-mediated immunopathology in AIDS pathogenesis, the two mechanisms of action (i.e., reduction of HIV replication and impairment of CTL responses) may complement each other beneficially. Thus, the surprising ability of ritonavir to block the presentation of antigen to CTLs may possibly contribute to therapy of HIV infections but potentially also to the therapy of virally induced immunopathology, autoimmune diseases, and transplantation reactions.

Serum can inhibit reversal of multidrug resistance by chemosensitisers.

The purpose of this study was to evaluate to what extent the ability of various chemosensitisers (CS) to reverse P-glycoprotein-associated multidrug resistance (MDR) is reduced when tested in physiological serum protein concentrations. Utilising drug sensitivity and accumulation assays, the CS were tested in medium containing 10% fetal bovine serum and in 100% horse or human serum. Two RPMI 8226 human myeloma sublines were used which express different levels of P-glycoprotein. The CS were tested at various concentrations, including clinically achievable blood levels. When using the CS at high doses, wide differences were observed in the extent CS activity was diminished by serum. Verapamil, cyclosporin A and quinine were not affected, quinidine and medroxyprogesterone acetate were moderately inhibited, and amiodarone and trifluoperazine were largely inactivated. When the CS were used at concentrations achievable in humans, the activity of all agents except quinine was markedly reduced by serum. With respect to the extent to which CS activity was diminished by serum, good statistical correlation (r > 0.90, P < 0.001) was found between the use of cytotoxicity and drug accumulation assays, horse and human serum or cell lines with high and low levels of P-glycoprotein, respectively. These studies demonstrated that physiological serum protein concentrations can profoundly diminish the MDR reversing activity of particular CS. Some drugs, such as amiodarone and trifluoperazine, are largely inactivated by serum when used at a wide range of concentrations. Other agents, such as verapamil and cyclosporin A, are essentially unaffected when used at high doses but markedly inhibited at concentrations achievable in humans. These data suggest that in vitro studies of CS in medium containing low serum protein concentrations can result in misleading conclusions regarding the potential clinical activity of such agents.

Sensitive and rapid bioassay for analysis of P-glycoprotein-inhibiting activity of chemosensitizers in patient serum.

Clinical studies of agents capable of reversing P-glycoprotein (Pgp)-mediated multidrug resistance have attracted much attention in recent years. One question of interest in such studies is whether the concentrations achieved by chemosensitizers are sufficient to inhibit Pgp function. The goal of the present study was to develop a reliable ex vivo bioassay for analysis of the Pgp-inhibiting activity of chemosensitizer-containing patient serum. The fluorescent Pgp substrates daunorubicin (DNR) and rhodamine 123 (R123) were used as probes for Pgp function. The 8226/DOX6 human myeloma cell line, which expresses Pgp at levels that can be detected in clinical cancers, was used as a model system. The index chemosensitizers tested were dexverapamil (DVPM) and cyclosporin A, with particular focus on DVPM. Using flow cytometry, chemosensitizer effects on 1-h drug accumulation and on drug retention at 30 min were evaluated. In the studies using pooled human serum spiked in vitro with graded chemosensitizer concentrations, the order of assay sensitivity was R123 retention >>> R123 accumulation > DNR retention equal to DNR accumulation. Keeping serum spiked with DVPM for several hours at room temperature or 4 degreesC or for several months at -80 degreesC had no effect on Pgp-blocking activity. Sixteen blood samples from patients with metastatic breast cancer receiving DVPM to overcome epirubicin resistance were analyzed for Pgp-inhibiting activity and for levels of DVPM and nor-DVPM, the major metabolite of verapamil. Each patient sample was found capable of increasing R123 retention in the 8226/DOX6 cells, with activity factors of 3- to 8-fold and good agreement between DVPM blood levels and bioassay activity (r = 0.7168; two-sided P = 0.0018). The R123 retention assay developed and validated in this study seems to be a sensitive, reproducible, and easy-to-use method for analysis of Pgp-inhibiting activity of chemosensitizer-containing human serum. The assay seems capable of estimating DVPM blood levels and could prove to be a valuable tool for monitoring chemosensitizer treatment in cancer patients.

In vitro evaluation of chemosensitizers for clinical reversal of P-glycoprotein-associated Taxol resistance.

The purpose of this study was to estimate the potential usefulness of currently available chemosensitizers (CS) for clinical reversal of P-glycoprotein-mediated Taxol resistance. The 8226/DOX6 human myeloma cells were used to evaluate CS effects in serum-rich medium by means of the human tumor cloning assay. The 8226/DOX6 cells express low levels of P-glycoprotein as typically found in clinical cancer specimens and are approximately 40-fold resistant to Taxol when continuously exposed to the drug in serum-rich medium. The CS were used at maximum tolerated plasma levels (Cmax) and at the concentrations achieved in human plasma by oral administration (Coral). Of nine CS tested, cyclosporine A (CSA), verapamil (VER), quinidine (QD), and quinine (Q) were able to overcome Taxol resistance. QD and Q were effective at Coral, whereas CSA and VER required Cmax to significantly enhance Taxol cytotoxicity. The most potent agent was CSA, which increased sensitivity to Taxol by 8-, 12-, and 18-fold when used at concentrations of 1.0, 2.0, and 4.0 microM, respectively. At 5.0 microM, CSA was capable of fully normalizing Taxol sensitivity. On the basis of these data, infusional CSA might prove useful for clinical reversal of P-glycoprotein-associated Taxol resistance. It remains to be seen, however, whether the CSA concentrations needed to reverse Taxol resistance effectively can be safely achieved in human plasma when CSA is coadministered with Taxol.

Immunological changes following posttraumatic splenectomy.

In 31 splenectomized individuals blood lymphocytes and serum immunoglobulins were examined 1-11 years after splenectomy. The results demonstrate: 1. Increase of the surface immunoglobulin bearing lymphocytes. 2. Reduction of the E-rosette forming lymphocytes, especially in a combined test assay. 3. Reduction of serum IgM. A surface-restoring effect of the RES of the spleen is discussed as a reason for the diminished E-rosette formation in splenectomized individuals.