John D. Mountz

Regulation of apoptosis proteins in cancer cells by ubiquitin

Ubiquitin inhibitors act at many levels to enhance apoptosis signaling. For TNF-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis signaling, there are at least five mechanisms by which apoptosis are regulated by the ubiquitin–proteasome pathway. First, proteasome inhibitors can decrease Fas-like inhibitor protein (FLIP) protein levels in tumors, resulting in increased apoptosis signaling due to increased caspase-8 activation. This appears to involve the ubiquitin ligase TNF receptor activation factor-2 (TRAF2) and acts indirectly by causing cell-cycle arrest at a stage where there is high degradation of the FLIP–TRAF2 complex. Second, the regulation of the proapoptotic Bcl-2 family member BAX occurs indirectly. Apoptosis signaling and caspase activation results in a confirmation change in the normally monomeric BAX, which exposes the BH3 domain of BAX, leading to dimerization and resistance to ubiquitin degradation. BAX then translocates into the mitochondria, resulting in the release of proapoptotic mitochondrial factors such as cytochrome c and second mitochondria-derived activator of caspase (SMAC). This results in the activation of caspase-9 and formation of the apoptosome and efficient apoptosis signaling. A third mechanism of the regulation of TRAIL signaling in the ubiquitin–proteasome pathway is mediated by the inhibitor of apoptosis proteins (IAP) E3 ligases. These IAPs can directly bind to caspases but also can act as ubiquitin ligases for caspases, resulting in the degradation of these caspases. IAP binding to caspases can be inhibited by SMAC, which exhibits a caspase-9 homology domain. The fourth mechanism for apoptosis activation by proteasome inhibitors is through the stabilization of the inhibitor of the κB (IκB)/NF-κB complex and prevention of nuclear translocation of the antiapoptosis transcription factor NF-κB. During TRAIL-DR4, DR5 signaling, this pathway is activated by interactions of activated Fas-associated death domain with activated receptor-interacting protein (RIP), which in turn activates NF-κB-inducing kinase and phosphorylates IκB. Therefore, the inhibition of IκB degradation blocks this RIP-mediated antiapoptosis signaling event. Last, p53 protein levels, and susceptibility to apoptosis, can be deregulated by the human homolog Hdm2 (Mdm2) E3 ligase. This process is inhibited by p53 phosphorylation and by sequestration of Mdm2 by ARF. Better mechanisms to inhibit the ubiquitin–proteasome pathway targeted at the ubiquitin–proteasome degradation process itself, or more specifically at the E3 ligases known to modulate and downregulate proapoptosis pathways will lead to the enhancement of TRAIL apoptosis signaling and better cancer therapeutic outcomes act through this pathway.

Normal T-cell response and in vivo magnetic resonance imaging of T cells loaded with HIV transactivator-peptide-derived superparamagnetic nanoparticles.

The present study analyzed the feasibility of using magnetic resonance imaging (MRI) to monitor T-cell homing in vivo after loading T cells with superparamagnetic iron oxide (CLIO) nanoparticles derivatized with a peptide sequence from the transactivator protein (Tat) of HIV-1. T cells were isolated from C57BL/6 (B6) mice and loaded with 0, 400, 800, 1600, or 8000 ng/ml of FITC conjugated CLIO-Tat (FITC-CLIO-Tat). There was a dose-dependent uptake of FITC-CLIO-Tat by T cells. Stimulation of FITC-CLIO-Tat loaded T cells with anti-CD3 (0.1 microg/ml) plus IL-2 (5 ng/ml) elicited normal activation and activation-induced cell death (AICD) responses, and normal upregulation of CD69, ICAM-1 (CD54), L-selectin (CD62L), and Fas. The FITC-CLIO-Tat loaded T cells (3 x 10(7)) were transferred intravenously (i.v.) into B6 mice and the in vivo MRI of mice was acquired using a spin-echo pulse sequence at 4.7 T with a Bruker Biospec system. Homing of T cells into the spleen was observed by a decrease in MRI signal intensity within 1 h after the transfer, which remained decreased for 2-24 h after transfer. These homing data were confirmed by FACS analysis and biodistribution analysis using 125I-CLIO-Tat. Thus, T cells can be efficiently loaded with FITC-CLIO-Tat without interfering with their normal activation and AICD, or homing to the spleen, and the biodistribution of FITC-CLIO-Tat loaded T cells can be monitored in vivo over time by MRI.

Regulation of tumor necrosis factor α–mediated apoptosis of rheumatoid arthritis synovial fibroblasts by the protein kinase Akt

OBJECTIVEnTo determine if tumor necrosis factor alpha (TNFalpha)-driven proliferation of rheumatoid arthritis synovial fibroblasts (RASF) is associated with up-regulation of the activity of serine/threonine kinase B/Akt and with survival of RASF.nnnMETHODSnStaining of phosphorylated Akt was done using anti-phosphorylated Thr308 Akt antibody. Levels of phosphorylated Akt were analyzed by Western blot and Akt activity was analyzed using a kinase assay. TUNEL staining was used to analyze the cytotoxicity of TNFalpha treatment or TNFalpha combined with either the Akt activity inhibitor wortmannin, an adenovirus expressing dominant-negative mutant (AdAkt-DN), or an adenovirus expressing phosphatase and tensin homolog deleted on chromosome 10 (AdPTEN).nnnRESULTSnThe levels of phosphorylated Akt were higher in RASF than in osteoarthritis synovial fibroblasts (OASF), as demonstrated by immunohistochemical staining, immunoblot analysis, and an Akt kinase assay. The levels of phosphorylated Akt and Akt kinase activity were increased by stimulation of primary RASF with TNFalpha (10 ng/ml). Treatment of RASF with the phosphatidylinositol 3-kinase inhibitor wortmannin (50 nM) plus TNFalpha resulted in apoptosis of 60 +/- 8% (mean +/- SEM) of RASF within 24 hours. This proapoptosis effect was specific for Akt, since equivalent levels of apoptosis were observed upon TNFalpha treatment of RASF transfected with AdAkt-DN and with AdPTEN, which opposes the action of Akt.nnnCONCLUSIONnThese results indicate that phosphorylated Akt acts as a survival signal in RASF and contributes to the stimulatory effect of TNFalpha on these cells by inhibiting the apoptosis response. This effect was not observed in OASF and may reflect the pathophysiologic changes associated with the proliferating synovium in rheumatoid arthritis.

Overexpression of activation-induced cytidine deaminase in B cells is associated with production of highly pathogenic autoantibodies.

Defective receptor editing or defective B cell checkpoints have been associated with increased frequency of multireactive autoantibodies in autoimmune disease. However, Ig somatic hypermutation and/or class switch recombination may be mechanisms enabling the development of pathogenic multireactive autoantibodies. In this study, we report that, in the BXD2 mouse model of autoimmune disease, elevated expression of activation-induced cytidine deaminase (AID) in recirculating follicular CD86+ subsets of B cells and increased germinal center B cell activity are associated with the production of pathogenic multireactive autoantibodies. CD4 T cells from BXD2 mice that expressed increased levels of CD28 and an increased proliferative response to anti-CD3 and anti-CD28 stimulation are required for this process. Inhibition of the CD28-CD86 interaction in BXD2 mice with AdCTLA4-Ig resulted in normalization of AID in the B cells and suppression of IgG autoantibodies. This treatment also prevented the development of germinal center autoantibody-producing B cells, suggesting that an optimal microenvironment enabling AID function is important for the formation of pathogenic autoantibodies. Taken together, our data indicate that AID expression in B cells is a promising therapeutic target for the treatment of autoimmune diseases and that suppression of this gene may be a molecular target of CTLA4-Ig therapy.

Regulation of apoptosis in immune cells

Apoptosis is a physiological process of selective cell deletion that occurs during embryogenesis, metamorphosis, tissue atrophy, and tumor regression (1-3). It is also a mechanism for autoreactive T cell deletion in tolerance, in cytotoxic T cell-mediated target cell lysis, and during corticosteroid-induced death of immature lymphocytes (4-9). One of the most distinctive features of apoptotic cell death is the extensive endonuclease degradation of chromosomal DNA into oligomers of about 180 base pairs. In contrast to necrosis, which is the result of nonspecific injury or toxic conditions, apoptosis is initiated by ligand-receptor interactions that are highly regulated or by removal of growth factors necessary to sustain cell proliferation. Apoptosis can be provoked by various stimuli including steroids, interleukin-2 (IL-2) deprivation, T cell receptor (TCR)/CD3 signaling, and ligand binding by the Fas molecule. Apoptosis can be inhibited by ligand binding by CD40 (10-12), Kit (13, 14) receptors, or CD28/CTLA-4 and their counter receptors B7-1/B7-2 (15-18). These stimuli initiate or inhibit a sequence of metabolic events, such as a sustained rise in cytosolic ionized calcium, which, in turn, activates

Age-related increase of tumor susceptibility is associated with myeloid-derived suppressor cell mediated suppression of T cell cytotoxicity in recombinant inbred BXD12 mice.

In this study, our data show that in young BXD12 mice, the implanted TS/A tumor regressed in 4 weeks after implantation, and this regression was associated with extensive T cell infiltration. In contrast, in old BXD12 mice, it was observed that there was rapid tumor growth by 7 weeks. T cell cytotoxicity against TS/A tumor cells exhibited a significant age-related decline, which was correlated with a decline in CD3(+) T cell infiltration of the tumor. Furthermore, the decline of T cell tumor cytotoxicity in aged BXD12 mice was also correlated with the accumulation of CD11b(+)Gr1(+) myeloid-derived suppressor cells in the spleen. Adoptive transfer of these accumulated CD11b(+)Gr1(+)cells from aged mice to 2-month-old BXD12 mice led to the delay of the rejection of implanted tumor cells. The depletion of CD11b(+)Gr1(+)cells from aged BXD12 mice led to the slower growth of tumor. Induction of arginase 1 in myeloid cells isolated from aged mice plays a partial role in immune suppression of T cell cytotoxicity. Thus, the accumulation of immunosuppresssing myeloid cells appears to contribute to the increase of tumor susceptibility as the age of mice increases.

Cellular Mechanism of Thymic Involution

Involution of the thymus and alterations in the development of thymocytes are the most prominent features of age‐related immune senescence. We have carried out a comparative analysis of thymocyte and stroma in rapid thymic involution DBA/2 (D2) strain of mice compared with slow involution C57BL/6 (B6) strain of mice. Analysis of mice at 15 months of age suggested an age‐related decrease in the thymocyte cell count, a block in the development of T cells and cortical involution in D2 mice compared with 3‐month‐old mice. TUNEL (terminal‐deoxynucleotidyl‐transferase‐mediated dUTP–digoxigenin nick end labelling) staining and fluorescence‐activated cell sorter (FACS) analysis showed that there was a significant increase in apoptotic cells in the cortex region of thymus in 15‐month‐old D2 mice compared with the same aged B6 mice. The thymocyte proliferation rate, as assessed by bromodeoxyuridine (BrdU) staining and [3H]‐thymidine incorporation assay, was lower in 3‐month‐old D2 mice compared with the same age B6 mice. Immunohistochemical staining showed that the arrangement of MTS (mouse thymus stromal)‐10+ epithelial cells and MTS‐16+ connective tissue staining pattern had become disorganized in 15‐month‐old D2 mice but remained intact in B6 mice of the same age. These results suggest that, in D2 mice, both the thymocytes and stromal cells exhibit age‐related defects, and that the genetic background of mice plays an important role in determining age‐related alterations in thymic involution.

IL-17 Activates the Canonical NF-κB Signaling Pathway in Autoimmune B Cells of BXD2 Mice To Upregulate the Expression of Regulators of G-Protein Signaling 16

We previously identified that autoreactive B cells from BXD2 mice can be targeted by IL-17, leading to upregulation of the expression of regulators of G-protein signaling (Rgs) genes that facilitated the development of spontaneous germinal centers. Little is known about the signaling pathway used by IL-17 to upregulate RGS. In the current study, we found that IL-17 rapidly activates the canonical NF-κB signaling pathway and that BXD2 B cells exhibit higher basal and activated phosphorylated p65 levels than B6 or BXD2-Il17ra−/− B cells. Inhibition of p65 phosphorylation downregulated RGS16 expression and abrogated the IL-17‑induced chemotactic arrest of B cells in response to CXCL12. Knockdown of TNFR-associated factor 6 or NF-κB activator 1 in 70Z/3 pre-B cells led to decreased Rgs16 expression, indicating that both of these two genes are involved in IL-17‑mediated activation of NF-κB signaling in B cells. These findings identify the signaling pathway regulated by IL-17 to contribute to the development of spontaneous germinal centers in autoimmune BXD2 mice.

Genetic segregation of spontaneous erosive arthritis and generalized autoimmune disease in the BXD2 recombinant inbred strain of mice.

The BXD2 strain of mice is one of approximately 80 BXD recombinant inbred (RI) mouse strains derived from an intercross between C57BL/6J (B6) and DBA/2J (D2) strains. We have discovered that adult BXD2 mice spontaneously develop generalized autoimmune disease, including glomerulonephritis (GN), increased serum titres of rheumatoid factor (RF) and anti‐DNA antibody, and a spontaneous erosive arthritis characterized by mononuclear cell infiltration, synovial hyperplasia, and bone and cartilage erosion. The features of lupus and arthritis developed by the BXD2 mice segregate in F2 mice generated by crossing BXD2 mice with the parental B6 and D2 strains. Genetic linkage analysis of the serum levels of anti‐DNA and RF by using the BXD RI strains shows that the serum titers of anti‐DNA and RF were influenced by a genetic locus on mouse chromosome (Chr) 2 near the marker D2Mit412 (78u2003cm, 163u2003Mb) and on Chr 4 near D4Mit146 (53.6u2003cm, 109u2003Mb), respectively. Both loci are close to the B‐cell hyperactivity, lupus or GN susceptibility loci that have been identified previously. The results of our study suggest that the BXD2 strain of mice is a novel model for complex autoimmune disease that will be useful in identifying the mechanisms critical for the immunopathogenesis and genetic segregation of lupus and erosive arthritis.

Cleavage of p53-Vimentin Complex Enhances Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand- Mediated Apoptosis of Rheumatoid Arthritis Synovial Fibroblasts

Rheumatoid arthritis synovial fibroblasts (RASFs) contribute to arthritic cartilage degradation. Although RASFs are normally resistant to apoptosis, Apo2L/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-based gene therapy has been successfully used in a mouse model of arthritis. We investigated this further by treating human RASFs with nontoxic doses of the proteasome inhibitor lactacystin. Treatment induced cytosolic accumulation of p53 and enhanced the susceptibility of RASFs to apoptosis mediated by TRAIL-R2 (DR5) but not Fas. A specific role for p53 in TRAIL-R2-mediated apoptosis was indicated by the ability of p53 siRNA to significantly reduce RASF apoptosis and by the reduced apoptosis of RASFs bearing p53 mutations on treatment with anti-DR5 antibody or anti-DR5 antibody plus lactacystin. p53 immunoprecipitation followed by mass spectrometry identified a vimentin-p53 complex, an interaction that was confirmed by reciprocal vimentin-p53 immunoprecipitation and by co-immunofluorescence. Interestingly, human caspase-4 cleaved human vimentin, and blockade of caspase-4 with a chemical inhibitor or with specific siRNA significantly inhibited TRAIL-R2-mediated apoptosis of RASFs. Furthermore, blockade of caspase-4 was paralleled by persistence of a cytosolic pattern of p53 and absence of p53 translocation to the nucleus. Taken together, our findings suggest a unique role for caspase-4 in cleaving vimentin and releasing cytosolic p53 for nuclear translocation, events that may regulate the sensitivity of RASFs to receptor-mediated apoptosis.