PingAr Yang

Interleukin 17-producing T helper cells and interleukin 17 orchestrate autoreactive germinal center development in autoimmune BXD2 mice.

Interleukin 17 (IL-17) is a cytokine associated with inflammation, autoimmunity and defense against some bacteria. Here we show that IL-17 can promote autoimmune disease through a mechanism distinct from its proinflammatory effects. As compared with wild-type mice, autoimmune BXD2 mice express more IL-17 and show spontaneous development of germinal centers (GCs) before they increase production of pathogenic autoantibodies. We show that blocking IL-17 signaling disrupts CD4+ T cell and B cell interactions required for the formation of GCs and that mice lacking the IL-17 receptor have reduced GC B cell development and humoral responses. Production of IL-17 correlates with upregulated expression of the genes Rgs13 and Rgs16, which encode regulators of G-protein signaling, and results in suppression of the B cell chemotactic response to the chemokine CXCL12. These findings suggest a mechanism by which IL-17 drives autoimmune responses by promoting the formation of spontaneous GCs.

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.

Bisindolylmaleimide VIII facilitates Fas-mediated apoptosis and inhibits T cell-mediated autoimmune diseases

Fas-mediated apoptosis is essential for the elimination of cells, and impaired apoptosis can have severe detrimental consequences. Bisindolylmaleimide VIII potentiated Fas-mediated apoptosis in human astrocytoma 1321N1 cells and in Molt-4 T cells, both of which were devoid of apoptosis induced by anti-Fas antibody in the absence of bisindolylmaleimide VIII, and in Jurkat and CEM-6 T cells, which showed slight and moderate apoptotic responses, respectively, to low levels of Fas stimulation. Potentiation of Fas-mediated apoptosis by bisindolylmaleimide VIII was selective for activated, rather than non-activated, T cells, and was Fas-dependent, as it was not observed in T cells from Fas-deficient lpr/lpr mice. Administration of bisindolylmaleimide VIII to rats during autoantigen stimulation prevented the development of symptoms of T cell-mediated autoimmune diseases in two models, the Lewis rat model of experimental allergic encephalitis and the Lewis adjuvant arthritis model. Thus, the use of agents such as bisindolylmaleimide VIII may be therapeutically useful for supporting more effective elimination of detrimental cells through enhancement of Fas-dependent apoptosis signaling.

Defective expression of hematopoietic cell protein tyrosine phosphatase (HCP) in lymphoid cells blocks Fas-mediated apoptosis

Protein tyrosine dephosphorylation after Fas cross-linking occurred in Fas apoptosis-sensitive CEM-6 cells but not in Fas apoptosis-resistant MOLT-4 cells, and apoptosis in the CEM-6 cells could be inhibited by the protein tyrosine phosphatase inhibitor, pervanadate. The time course and level of dephosphorylation were correlated with increased hematopoietic cell protein tyrosine phosphatase (HCP) activity, but not with the activity of two other tyrosine phosphatases. The level of expression of HCP was correlated with Fas apoptosis function in eleven human and murine Fas-positive lymphoid cell lines. Expression of recombinant HCP in the MOLT-4 cell line converted this Fas apoptosis-resistant cell line to Fas apoptosis sensitive. HCP-mutant mev/mev mice exhibited increased expression of Fas but decreased Fas-mediated apoptosis function in lymphoid organs after anti-mouse Fas antibody treatment in vivo. Thus, HCP-mediated protein dephosphorylation is involved in the delivery of the Fas apoptosis signal in lymphoid cells.

Adeno-Associated Virus Production of Soluble Tumor Necrosis Factor Receptor Neutralizes Tumor Necrosis Factor α and Reduces Arthritis

The major limitation of adenovirus is its association with induction of an inflammatory response and relatively short-term production of the gene therapy transgene product. Adeno-associated virus (AAV) is a 4.68-kb single-strand DNA virus that contains ITRs for viral replication and a packaging signal, and also has been engineered to contain therapeutic genes up to 5 kb in length. Transduction of recombinant AAV (rAAV) results in low inflammatory response and long-term expression. We have cloned a low-immunogenic form of human sTNFRI (sTNFRI2.6D) into AAV (rAAVsTNFRI). This vector was analyzed for its ability to transfect and neutralize the effect of TNF-alpha on primary rheumatoid arthritis synovial fibroblast (RASFs). The rAAVsTNFRI was transduced into the cells at 1.8 x 10(1), 1.8 x 10(2), and 1.8 x 10(3) viral particles per cell. There was greater than 90% neutralization of TNF-alpha at 1.8 x 10(3) viral particles/cell. There was a significant decrease in the synovial cell hyperplasia and cartilage and bone destruction in human TNF-alpha transgenic mice treated intraarticularly with rAAVsTNFRI. These results indicate that the low-immunogenic and long-term expressing vector, rAAVsTNFRI, can be used to deliver the soluble TNF-alpha in vitro and in vivo and effectively reduce the severity of arthritis.

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

OBJECTIVE To 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. METHODS Staining 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). RESULTS The 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. CONCLUSION These 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.

Gene therapy that inhibits nuclear translocation of nuclear factor κB results in tumor necrosis factor α–induced apoptosis of human synovial fibroblasts

OBJECTIVE Tumor necrosis factor alpha (TNFalpha) increases the survival and proliferation of human rheumatoid arthritis (RA) cell lines. These experiments were designed to determine if inhibition of nuclear factor kappaB (NF-kappaB) nuclear translocation leads to increased apoptosis of TNFalpha-treated human RA cell lines. METHODS We constructed an inhibitor of nuclear factor kappaB(IkappaB) dominant-negative adenovirus (AdCMVIkappaB-DN) and an X-linked inhibitor of apoptosis (XIAP) antisense adenovirus (AdCMVXIAP-AS). Primary RA synovial fibroblast (RASF) cell lines were transfected in vitro, and SV40-transformed RA synovial cell lines in SCID mice were transfected in vivo. Cells were treated with TNFalpha and analyzed for apoptosis. RESULTS There was no apoptosis of primary RASF transfected in vitro with AdCMVIkappaB-DN alone. In contrast, there was apoptosis of >85% of cells treated with AdCMVIkappaB-DN plus TNFalpha. Primary RASF in SCID mice also exhibited high levels of apoptosis after in vivo transfection with AdCMVIkappaB-DN followed by treatment with TNFalpha. There was no apoptosis after treatment with AdCMVIkappaB-DN in the absence of TNFalpha. XIAP is an inhibitor of apoptosis which was up-regulated by TNFalpha, and this up-regulation was inhibited by AdCMVIkappaB-DN plus TNFalpha. Transfection of an AdCMVXIAP-AS gene therapy resulted in increased TNFa-induced apoptosis. CONCLUSION AdCMVIkappaB-DN gene therapy greatly enhances apoptosis due to inhibition of an NF-kappaB-mediated antiapoptosis signaling pathway, and XIAP is a TNFalpha-inducible specific inhibitor of apoptosis in RA synovial cell lines. This and other modulators of TNF receptor or the Fas apoptosis pathway may be therapeutically beneficial in facilitating apoptosis of synovial tissue in patients with RA.

CII-DC-AdTRAIL cell gene therapy inhibits infiltration of CII-reactive T cells and CII-induced arthritis

Previously, we described an APC-adenovirus (APC-Ad) FasL cell gene therapy method which could be used to deplete autoreactive T cells in vivo. FasL was toxic, however, and controlled regulation of FasL was not achieved. Here we describe an improved approach to delivering TNF-related apoptosis-inducing ligand (TRAIL) in vivo in which collagen II-induced (CII-induced) arthritis-susceptible (CIA-susceptible) DBA/1j mice were treated with CII-pulsed DCs that had been transfected with a novel Ad system. The Ad was engineered to exhibit inducible TRAIL under the control of the doxycycline-inducible (DOX-inducible) tetracycline response element (TRE). Four groups of mice were treated with CII-DC-AdTRAIL+DOX, CII-DC-AdTRAIL (no DOX), CII-DC-AdGFP+DOX, or DC-AdTRAIL+DOX (no CII), beginning 2 weeks after priming with CII in CFA. The incidence of arthritis and infiltration of T cells in the joint was significantly decreased in CII-DC-AdTRAIL+DOX-treated mice. The in vitro splenic T cell proliferative response and induction of IFN-gamma to bovine CII stimulation were also significantly reduced in mice treated with CII-DC-AdTRAIL+DOX. AdTRAIL+DOX was not toxic to DCs or mice but could induce activated T cells to undergo apoptosis in the spleen. Our results suggest that CII-DC-AdTRAIL+DOX cell gene therapy is a safe and effective method for inhibiting the development of CIA.

Induction of specific T-cell tolerance by adenovirus-transfected, Fas ligand-producing antigen presenting cells.

A major problem associated with adenovirus gene therapy is the T cell-mediated immune response, which is elicited by inoculation of the adenovirus vector and leads to rapid clearance of the virus and loss of transgene expression. In this study, the immune response to adenovirus was prevented by induction of specific T-cell tolerance by pretreatment with adenovirus-infected antigen-presenting cells (APC) that express Fas ligand. Compared with control-treated mice, the tolerized mice showed prolonged expression of lacZ upon administration of AdCMVlacZ 1 week after tolerance induction. In contrast to the control mice, the tolerized mice did not display proliferation of CD3+ T cells in the spleen in response to AdCMVlacZ. Tolerance induction also was indicated by the lower production of interferon-g and interleukin-2 by peripheral T cells isolated from AdCMVlacZ-challenged tolerized mice than by AdCMVlacZ-challenged control-treated mice. The T-cell tolerance was specific for the adenovirus as the T-cell responses to irrelative murine cytomegalovirus remained unimpaired. Our results indicate that adenovirus-specific T-cell tolerance can be induced by APCs that coexpress Fas ligand and adenovirus antigens. We propose that this new strategy can be used to induce tolerance to adenovirus vector gene therapy with resultant prolonged expression of the transgene.

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.