Generation of the vesicular stomatitis virus nucleoprotein cytotoxic T lymphocyte epitope requires proteasome-dependent and -independent proteolytic activities

The proteasome is involved in the generation of most of the MHC class I antigenic epitopes. However, it is not known if the proteasome generates the exact cytotoxic T lymphocyte (CTL) epitope or only epitope precursors which require further modification by additional proteases. Digestion of the extended vesicular stomatitis virus nucleoprotein epitope 52 – 59 (RGYVYQGL) by the 20S proteasome in vitro shows that the proteasome is capable of generating the correct C terminus but not the exact N terminus of the CTL epitope. This finding suggests that proteolytic activity in addition to the proteasome is required for generation of the CTL epitope. By using the proteasome inhibitor lactacystin we were able to confirm this finding in vivo. Lactacystin prevented the processing of N- and C-terminally extended epitopes, whereas the processing of only N-terminally extended epitopes was unaffected. Thus, the proteasome is necessary and sufficient for the generation of the exact C terminus of this CTL epitope, whereas the exact N terminus seems to be generated by a different protease.     

Fas (CD95)/Fas ligand interactions regulate antigen-specific,major histocompatibility complex-restricted T/B cell proliferative responses

The effect of Fas ligand (FasL) cytotoxicity on T/B collaboration was examined in vitro using cloned T helper 1 cells and antigen-pulsed, activated B cells. We compared antigen-pulsed B cells that had been activated through different membrane receptors (IgM, CD14 and CD40) for their ability to induce T cell proliferation and to respond to T cell help. We also used a Fas-Ig fusion protein, an inhibitor of FasL-mediated cytotoxicity, to determine the effect of FasL cytotoxicity on the T and B cell proliferative responses. The data show that the extent of both T and B cell proliferative responses correlate with the relative resistance of activated B cell populations to FasL cytotoxicity. Moreover, both T and B cell proliferation could be enhanced by Fas-Ig. Our results demonstrate that FasL cytotoxicity is a negative regulatory mechanism for both T and B cell proliferative responses and that Fas-Ig can be an immunopotentiating agent for both T and B cell immunity.     

Regulation of FasL expression in natural killer cells

Fas ligand (FasL)-mediated cytotoxicity is initiated in natural killer (NK) cells through ligation of their activating receptors. The CD16 receptor has been shown to induce FasL expression and cytotoxicity in NK cells. In this study, we made the novel observation that FasL expression was upregulated in NKL cells stimulated through 2B4 and LFA-1 activating receptors, implying a role for FasL-mediated cytotoxicity early in the immune response. Coligation with CD94/NKG2A human leukocyte antigen (HLA) class I inhibitory receptor did not block the induced FasL expression; therefore, these opposing pathways appear to function independently. We also showed, however, that FasL-mediated cytotoxicity was downregulated in CD94/NKG2A-expressing LAK cells in response to the HLA-E ligand, suggesting a mechanism by which aberrant cells expressing class I may evade FasL-mediated cytotoxicity. Thus we show for the first time that 2B4, LFA-1, and CD94/NKG2A receptors are involved in modulating FasL expression and, therefore, cytotoxicity mediated by NK cells.     

Inhibition of proteasome activity blocks cell cycle progression at specific phase boundaries in African trypanosomes

Proteasomes are one of the cellular complexes controlling protein degradation from archaebacteria to mammalian cells. We recently purified and characterized the catalytic core of the proteasome, the 20S form, from Trypanosoma brucei, a flagellated protozoa which causes African trypanosomiasis. To identify the role of proteasomes in African trypanosomes, we used lactacystin, a specific inhibitor of proteasome activity. Lactacystin showed potent inhibition of the activity of 20S proteasomes purified from both bloodstream and procyclic (insect) forms of T. brucei (IC₅₀=1 μM). It also inhibited proliferation of T. brucei cells in culture assays, with 1 μM inhibiting growth of bloodstream forms, whereas 5 μM was required to block proliferation of procyclic forms. Analysis of the DNA content of these cells by flow cytometry showed that 5 μM lactacystin arrested procyclic cells in the G2+M phases of the cell cycle. Fluorescence microscopy revealed that most of the cells had one nucleus and one kinetoplast each, indicating that the cells had replicated their DNA, but failed to undergo mitosis. This suggests that transition from G2 to M phase was blocked. On the other hand, incubation of bloodstream forms with 1 μM lactacystin led to arrest of 30–35% of the cell population in G1 and 55–60% of the cells in G2, indicating that both transition from G1 to S and from G2 to M were blocked. These observations were also confirmed by using another inhibitor of proteasome, N-carbobenzoxy- -leucyl- -leucyl- -norvalinal (LLnV), which arrested procyclic forms in G2, and bloodstream forms in both G1 and G2. These results suggest that proteasome activity is essential for driving cell cycle progression in T. brucei, and that proteasomes may control cellular functions differently in bloodstream and procyclic forms of T. brucei.     

Tripeptidyl peptidase II promotes maturation of caspase-1 in Shigella flexneri-induced macrophage apoptosis

The invasive enteropathogenic bacterium Shigella flexneri activates apoptosis in macrophages. Shigella-induced apoptosis requires caspase-1. We demonstrate here that tripeptidyl peptidase II (TPPII), a cytoplasmic, high-molecular-weight protease, participates in the apoptotic pathway triggered by Shigella. The TPPII inhibitor Ala-Ala-Phe-chloromethylketone (AAF-cmk) and clasto-lactacystin beta-lactone (lactacystin), an inhibitor of both TPPII and the proteasome, protected macrophages from Shigella-induced apoptosis. AAF-cmk was more potent than lactacystin and irreversibly blocked Shigella-induced apoptosis by 95% at a concentration of 1 microM. Conversely, peptide aldehyde and peptide vinylsulfone proteasome inhibitors had little effect on Shigella-mediated cytotoxicity. Both AAF-cmk and lactacystin prevented the maturation of pro-caspase-1 and its substrate pro-interleukin 1beta in Shigella-infected macrophages, indicating that TPPII is upstream of caspase-1. Neither of these compounds directly inhibited caspase-1. AAF-cmk and lactacystin did not impair macrophage phagocytosis or the ability of Shigella to escape the macrophage phagosome. TPPII was also found to be involved in apoptosis induced by ATP and the protein kinase inhibitor staurosporine. We propose that TPPII participates in apoptotic pathways.     

Effect of proteasome inhibitors on the growth,encystation, and excystation of <Emphasis Type="Italic">Entamoeba histolytica and Entamoeba invadens</Emphasis>

The effect of three proteasome inhibitors, lactacystin, clasto-lactacystin beta-lactone, and MG-132, on the growth, encystation, and excystation of Entamoeba histolytica and Entamoeba invadens was examined. All of these drugs blocked E. histolytica growth in a concentration-dependent manner; lactacystin was most potent for the inhibition and MG-132 showed the inhibitory effect only at higher concentrations. E. invadens was more resistant to these drugs than E. histolytica. Encystation of E. invadens was also inhibited and was more sensitive to the drugs than was growth. Beta-lactone was the most potent encystation inhibitor. The inhibitory effect of lactacystin and the beta-lactone on encystation was slightly and little abrogated by the removal of the drug, respectively. Multinucleation occurred in E. histolytica trophozoites treated with these drugs, being most marked with lactacystin. In contrast, no multinucleation was observed in E. invadens treated with the drugs. Electron microscopy revealed that the treatment of E. histolytica trophozoites with lactacystin led to an increase in the number of cells with many glycogen granules in the cytoplasm. Lactacystin, beta-lactone and MG-132 had no or little effect on the excystation and metacystic development of E. invadens. These results suggest that proteasome function plays an important role for Entamoeba growth and encystation, but has no obvious effect on excystation or metacystic development.     

Suppression of Lung Inflammation in Rats by Prevention of NF-κB Activation in the Liver

Activation of NF-B and production of NF-B-dependent chemokines are thought to be involved in the pathogenesis of neutrophilic lung inflammation. Calpain-1 inhibitor (Cl-1) blocks activation of NF-B by preventing proteolysis of the inhibitory protein IB- by the ubiquitin/proteasome pathway. We hypothesized that inhibition of proteasome function with CI-1 would block NF-B activation in vivo after intraperitoneal (IP) treatment with bacterial lipopolysaccharide (LPS), and that NF-B inhibition would be associated with suppression of chemokine gene expression and attenuation of neutrophilic alveolitis. We treated rats with a single IP injection of CI-1 (10 mg/kg) two hours prior to IP LPS (7 mg/kg). Treatment with CI-1 prevented degradation of IB- and activation of NF-B in the liver in response to LPS; however, CI-1 treatment had no detected effect on NF-B activation in lung tissue. CI-1 treatment prior to LPS resulted in 40% lower MIP-2 concentration in lung lavage fluid compared to rats treated with vehicle prior to LPS (502 +/– 112 pg/ml vs. 859 +/– 144 pg/ml, P < 0.05). In addition, CI-1 treatment substantially inhibited LPS-induced neutrophilic alveolitis (2.7 +/– 1.2 × 10⁵ vs. 43.7 +/– 12.2 × 10⁵ lung lavage neutrophils, P < 0.01). These data indicate that NF-B inhibition in the liver can alter lung inflammation induced by systemic LPS treatment and suggest that a liver-lung interaction contributes to the inflammatory response of the lung.     

Myelin oligodendrocyte glycoprotein-induced autoimmune encephalomyelitis is chronic/relapsing in perforin knockout mice,but monophasic in Fas- and Fas ligand-deficient lpr and gld mice

The expression and action of Fas/Fas ligand (FasL) in multiple sclerosis has been postulated as a major pathway leading to inflammatory demyelination. To formally test this hypothesis, C57BL/6-lpr and -gld mice, which due to gene mutation express Fas and FasL in an inactive form, were immunized with myelin oligodendrocyte glycoprotein peptide_35–55. Whereas in wild-type C57BL/6 mice, experimental autoimmune encephalomyelitis (EAE), was chronic/relapsing, EAE in lpr and gld mice was characterized by a lower incidence of disease and a monophasic course. This contrasts with C57BL/6 perforin knockout mice, which showed the most severe form of EAE of all mouse strains tested, the course being chronic relapsing. The difference noted cannot be attributed to an involvement of FasL in oligodendrocyte damage since oligodendrocytes are insensitive to FasL-mediated cytotoxicity in vitro, and since in the acute phase of EAE gld mice also show CD4⁺T cell infiltrates with associated demyelination in brain and spinal cord. Unlike oligodendrocytes, astrocytes were killed by FasL in vitro. It remains to be established whether this latter finding explains the different disease course of lpr and gld mice compared to wild-type and perforin knockout mice.     

Intracellular B7-H4 suppresses bile duct epithelial cell apoptosis in human primary biliary cirrhosis

The expression and function of B7-H4, a recently identified co-inhibitory molecule of the B7 superfamily, in the pathogenesis of primary biliary cirrhosis (PBC) is still unclear. Here the expression of B7-H4 in sections from PBC patients (n = 16) was examined by immunohistochemistry and it was detected in primary bile duct epithelial cells (BECs) which were isolated from PBC patients by flow cytometry (FACs). Moreover, we also analyzed BECs-associated B7-H4 function through knock-down of its expression via RNA interference (RNAi) in vitro. Immunohistochemistry and FACs evidenced that the expression of B7-H4 was restricted in the cytoplasm of BECs from PBC patients, while it was completely absent in normal liver tissues. The cytoplasmic B7-H4 gene was cloned, and sequenced analysis showed it was encoded by the same gene to the membrane B7-H4. Interesting, silencing B7-H4 by specific RNAi resulted in enhanced FasL expression and BEC apoptosis. Conversely, interruption of Fas\FasL interaction with using FasL blocking antibodies (clone 4H9) reversed cell apoptosis. Our results suggested that the intracellular B7-H4 appears to prevent Fas/FasL-mediated BEC apoptosis during the progression of PBC, and indicates B7-H4 is a possible target for therapeutic intervention of this disease.     

Stimulation of FasL Induces Production of Proinflammatory Mediators Through Activation of Mitogen-Activated Protein Kinases and Nuclear Factor-κB in THP-1 Cells

FasL is a member of the tumor necrosis factor (TNF) superfamily involved in the various immune reactions such as activation-induced cell death, cytotoxic effector function, and establishment of immune privileged sites through its interaction with Fas. On the other hand, FasL is known to transmit a reverse signal that serves as a T cell co-stimulatory signal. However, the role of FasL-mediated reverse signaling in macrophage function has not been investigated. In order to investigate the presence of FasL-mediated signaling in macrophages, the human macrophage-like cell line THP-1 was analyzed after treatment with FasL ligating agents such as recombinant Fas:Fc fusion protein or anti-FasL monoclonal antibody. Stimulation of FasL induced the expression of proinflammatory mediators such as matrix metalloproteinase-9, TNF-α, and IL-8. The specificity of the reaction was confirmed by the transfection of the FasL-specific siRNAs, which suppressed FasL expression as well as the production of proinflammatory mediators. Utilization of various inhibitors of signaling adaptors and ELISA-base nuclear factor (NF)-κB binding assay demonstrated that the signaling initiated from FasL is mediated by mitogen-activated protein kinases including extracellular signal-regulated kinase, p38, and c-Jun N-terminal kinase which induce subsequent activation of NF-κB. These data indicate that membrane expression of FasL and its interaction with its counterpart may contribute to the inflammatory activation of macrophages during immune reactions or pathogenesis of chronic inflammatory diseases.