Robert I. Scheinman

NF-κB functions as both a proapoptotic and antiapoptotic regulatory factor within a single cell type

Recently NF-κB has been shown to have both proapoptotic and antiapoptotic functions. In T cell hybridomas, both T cell activators and glucocorticoids induce apoptosis. Here we show that blockade of NF-κB activity, using a dominant negative IκBα, has opposite effects on these two apoptotic signals. Treatment with PMA plus ionomycin (P/I) results in the upregulation of Fas Ligand (FasL) and induction of apoptosis. Inhibition of NF-κB activity inhibits the P/I mediated induction of FasL mRNA and decreases the level of apoptosis in these cultures, thus establishing NF-κB as a proapoptotic factor in this context. Conversely, inhibition of NF-κB confers a tenfold increase in glucocorticoid mediated apoptosis, establishing that NF-κB also functions as an antiapoptotic factor. We conclude that NF-κB is a context-dependent apoptosis regulator. Our data suggests that NF-κB may function as an antiapoptotic factor in thymocytes while functioning as a proapoptotic factor in mature peripheral T cells.

The Human Immunodeficiency Virus-1 Tat Protein Activates Human Umbilical Vein Endothelial Cell E-selectin Expression via an NF-κB-dependent Mechanism

Human immunodeficiency virus infection is associated with inflammation and endothelial cell activation that cannot be ascribed to direct infection by the virus or to the presence of opportunistic infections. Factors related to the virus itself, to the host and/or to environmental exposures probably account for these observations. The HIV protein Tat, a viral regulator required for efficient transcription of the viral genome in host cells is secreted from infected cells and taken up by uninfected by-stander cells. Tat can also act as a general transcriptional activator of key inflammatory molecules. We have examined whether Tat contributes to this endothelial cell activation by activating NF-κB. Human endothelial cells exposed to Tat in the culture medium activated E-selectin expression with delayed kinetics compared with tumor necrosis factor (TNF). Tat-mediated E-selectin up-regulation required the basic domain of Tat and was inhibited by a Tat antibody. Transfection of human E-selectin promoter-luciferase reporter constructs into Tat-bearing cells or into endothelial cells co-transfected with a Tat expression vector resulted in induction of luciferase expression. Either Tat or TNF activated p65 translocation and binding to an oligonucleotide containing the E-selectin κB site 3 sequence. Tat-mediated p65 translocation was also delayed compared with TNF. Neither agent induced new synthesis of p65. A super-repressor adenovirus (AdIκBαSR) that constitutively sequesters IκB in the cytoplasm as well as cycloheximide or actinomycin D inhibited Tat- or TNF-mediated κB translocation and E-selectin up-regulation.

Increased NF‐κB activity in B cells and bone marrow‐derived dendritic cells from NOD mice

Type 1 diabetes results from the breakdown of peripheral tolerance. As regulators of T cell activation, antigen‐presenting cells (APC) modulate peripheral tolerance and hence contribute to theimmune dysregulation characteristic of insulin‐dependent diabetes mellitus (IDDM). We initially observed an increased importance of NOD B cell APC function in a T cell priming assay as compared to non‐autoimmune strains. Consistent with this increased APC function, we found that NF‐κB nuclear translocation is increased in unmanipulated NOD and NOD.B10Sn‐H2b B cells and that, inaddition, NOD B cells are more sensitive to NF‐κB‐activating stimuli. We obtained similar results using NOD bone marrow‐derived dendritic cell (BMDC) cultures. As costimulatory molecules havebeen shown to be NF‐κB responsive, we examined the expression of these markers on NOD APC. Both B cells and BMDC expressed elevated levels of CD80 and CD40. Finally, NOD B cells provided better allostimulation than B cells from non‐autoimmune strains. Therefore, hyperactivation of NF‐κB and increased expression of CD80 and CD40 by NOD B cells and BMDC may be a contributing factor in the selection of effector T cells observed in IDDM.

Functionalized STAT1 siRNA nanoparticles regress rheumatoid arthritis in a mouse model.

AIM To develop and characterize an RGD peptide functionalized poly(lactide-co-glycolytic) acid (PLGA) nanosystem to deliver a STAT1 siRNA to joint tissues in a mouse model of rheumatoid arthritis. METHODS RGD-PLGA polymer was synthesized and used in preparing functionalized nanoparticles loaded with either tracking material or siRNA. The properties of the nanoparticles and stability of siRNA after encapsulation was assessed. Nanoparticle distribution was determined both noninvasively and based on analysis of dissected organs from arthritic and healthy mice. Arthritic mice were treated with weekly doses of STAT1 siRNA-loaded nanoparticles or controls. Clinical disease was assessed. Paws of arthritic mice were sectioned for histology or processed for RNA. STAT1, Mrc-1, and IL-10 mRNA abundance was determined by quantitative PCR. RESULTS Nanoparticles protected the siRNA from serum degradation. The presence of RGD peptide on the nanoparticles increased paw tissue uptake in arthritic mice. Furthermore, RGD functionalization increased lung delivery of nanoparticles in arthritic mice but not in control mice. Disease regressed in the STAT1 siRNA-treated animals and progressed in all control groups. STAT1 mRNA levels were decreased in paws of treated animals, while Mrc-1 and IL-10 mRNA levels were increased. CONCLUSION RGD functionalized PLGA nanoparticles encapsulating STAT1-targeted siRNAs are efficacious in the treatment of established arthritis, possibly through a selective inhibition of macrophage and dendritic cell activation.

Partial activation precedes apoptotic death in T cells harboring an IAN gene mutation

The Biobreeding diabetes‐prone rat suffers from a profound peripheral lymphopenia and yet succumbs to a T cell‐dependent autoimmune disease. Lymphopenia segregates with a mutated chromosomal locus, termed lyp, recently identified as a frameshift mutation in IAN4. Others have correlated loss of IAN4 function with decreased mitochondrial integrity resulting in T cell apoptosis. Here we report that IAN4–/– T cells enter a state similar to that of partial activation wherein they down‐regulate CD62L and undergo incomplete blasting yet do not progress through mitosis. When given a strong stimulus, this partial activation phenotype can be overcome. This phenotype can be recapitulated in wild‐type T cells through suboptimal stimulation. The phenotype is not simply a reaction to the lymphopenic environment, as spontaneous CD62L down‐regulation occurs in mature single‐positive medullary thymocytes that develop within a non‐lymphopenic environment, and normal T cells do not undergo similar blasting when parked in a lymphopenic environment. Finally, we show that IAN4–/– T cells are more readily triggered via TCR stimulation. Thus, in addition to their role in apoptosis, IAN family members may also play a role in regulating the T cell activation state through modulation of TCR signaling strength.

Pazopanib, a multitargeted tyrosine kinase inhibitor, reduces diabetic retinal vascular leukostasis and leakage

OBJECTIVE To determine the efficacy of pazopanib eye drops in the streptozotocin induced diabetic retinopathy rat model. METHODS A 0.5% w/v pazopanib suspension was prepared in phosphate buffered saline (PBS, pH 7.4) in the presence of 0.5% w/v sodium carboxymethyl cellulose. Brown Norway rats were divided into three groups (n=4) - (1) healthy, (2) diabetic, and (3) diabetic with treatment. The drug suspension was administered twice daily as eye drops to group 3 for 30 days. Efficacy parameters including the number of adherent leukocytes in the retinal vasculature (leukostasis), blood-retinal FITC-dextran leakage, and vitreous-to-plasma protein ratio were measured. RESULTS Pazopanib suspension in the form of eye drops significantly reduced leukostasis (32%), FITC-dextran leakage (39%), and the vitreous-to-plasma protein ratio (64%) in diabetic animals compared to untreated diabetic group. CONCLUSION Pazopanib eye drops can alleviate retinal complications of diabetic retinopathy.

A New Approach for the Treatment of Arthritis in Mice with a Novel Conjugate of an Anti-C5aR1 Antibody and C5 Small Interfering RNA

Rheumatoid arthritis (RA) is an inflammatory autoimmune joint disease in which the complement system plays an important role. Of the several components of complement, current evidence points to C5 as the most important inducer of inflammation. Several groups generated Abs or small interfering RNAs (siRNAs) or small molecule inhibitors against C5 and C5aR1 (CD88) that have showed some efficacy in RA in animal models. However, none of these candidate therapeutics has moved from bench to bedside. In this study, we test in collagen Ab-induced arthritis (CAIA) a new therapeutic strategy using a novel anti–C5ab-C5 siRNA conjugate. We first demonstrate that although C5aR2 or C5L2 (GPR77) plays no role in CAIA, C5aR1 contributes to pathogenesis. We demonstrate that injection of siRNAs blocking C5, C5aR1, or the combination decreased clinical disease activity in mice with CAIA by 45%, 51%, and 58%, respectively. Anti-C5 Ab (BB5.1) has only limited efficacy, but significantly reduced arthritis up to 66%. We then generated a novel anti-C5aR1 Ab–protamine–C5 siRNA conjugate. To our knowledge, we show for the first time that whereas unconjugated Ab plus siRNAs reduce arthritis by 19%, our anti-C5aR1 Ab–protamine–C5 siRNA conjugate was effective in reducing arthritis by 83% along with a parallel decrease in histopathology, C3 deposition, neutrophils, and macrophages in the joints of mice with CAIA. These data suggest that by targeting anti-C5 siRNAs to the receptor for its C5a activation fragment (C5aR1), a striking clinical effect can be realized.

Measurement of IKK activity in primary rat T cells: rapid activation and inactivation.

The translocation of the transcription factor NF-kappaB into the nucleus plays a critical role in many physiological events. In unstimulated cells, NF-kappaB is sequestered in the cytosol, bound to its inhibitor IkappaB. Activation primarily occurs via the IkappaB kinase (IKK) complex which phosphorylates IkappaBalpha at serines 32 and 36, creating a recognition site for IkappaB ubiquitination which then targets IkappaB for degradation. Often it is useful to measure IKK activity to assess upstream signaling events leading to NF-kappaB activation. Current methods of assessing IKK activity are limited to IKK isoforms which are recognized by available IKK antibodies. Here, we describe a procedure to qualitatively assess the overall IKK activity in a cell lysate which can be used on any IKK isoform capable of phosphorylating human IkappaBalpha. This nonradioactive assay is based on measurement of the ability of the cell lysate to phosphorylate GST-IkappaBalpha, as measured by Western blotting, using an anti-phospho-IkappaBalpha antibody. We have used this assay to observe the kinetics of TCR-mediated activation of IKK as compared to PMA/ionomycin in primary rat T cells. PMA/ionomycin induces maximal IKK activity within 1 min of stimulation and this activity remains elevated for over 20 min. In comparison, TCR ligation induces maximal IKK activity after 5 min of stimulation and this activity rapidly diminishes to background levels. These data indicate that different stimuli can activate and inactivate IKK with different kinetics and suggest that TCR-mediated activation of IKK is closely linked to the rapid phosphorylation and dephosphorylation, respectively, of TCR-associated kinases.

The human immunodeficiency virus-1 Tat protein activates endothelial cell E-selectin expression via an NF-κ-dependent mechanism

Human immunodeficiency virus infection is associated with inflammation and endothelial cell activation that cannot be ascribed to direct infection by the virus or to the presence of opportunistic infections. Factors related to the virus itself, to the host and/or to environmental exposures probably account for these observations. The HIV protein Tat, a viral regulator required for efficient transcription of the viral genome in host cells is secreted from infected cells and taken up by uninfected by-stander cells. Tat can also act as a general transcriptional activator of key inflammatory molecules. We have examined whether Tat contributes to this endothelial cell activation by activating NF-κB. Human endothelial cells exposed to Tat in the culture medium activated E-selectin expression with delayed kinetics compared with tumor necrosis factor (TNF). Tat-mediated E-selectin up-regulation required the basic domain of Tat and was inhibited by a Tat antibody. Transfection of human E-selectin promoter-luciferase reporter constructs into Tat-bearing cells or into endothelial cells co-transfected with a Tat expression vector resulted in induction of luciferase expression. Either Tat or TNF activated p65 translocation and binding to an oligonucleotide containing the E-selectin κB site 3 sequence. Tat-mediated p65 translocation was also delayed compared with TNF. Neither agent induced new synthesis of p65. A super-repressor adenovirus (AdIκBαSR) that constitutively sequesters IκB in the cytoplasm as well as cycloheximide or actinomycin D inhibited Tat- or TNF-mediated κB translocation and E-selectin up-regulation.

The Identification of a Novel T Cell Activation State Controlled by a Diabetogenic Gene

The cyclin-dependent kinase inhibitor p27kip regulates the cell cycle at the G1-S phase restriction point. S phase entry and cell cycle commitment in peripheral T cells requires p27kip degradation, normally initiated by the receipt of costimulatory signals such as those provided by B7.1 or IL-2. We have previously reported that T cells from BioBreeding (BB)-diabetes-prone (DP) rats exhibit decreased costimulatory requirements for activation and cell cycle entry. In the present study, we find that peripheral T cell subsets from BB-DP rats demonstrate activation-like characteristics, including significantly reduced levels of p27kip as well as increased levels of proliferating cell nuclear Ag (PCNA). Since our previous studies have established that expression of extracellular activation markers are relatively low in unmanipulated peripheral BB-DP T cells; this p27low PCNAhigh phenotype represents a novel activation state. Analyses of T cell subsets from congenic rats demonstrate that this phenotype segregates with the lyp diabetogenic locus and that the p27low PCNAhigh phenotype is T cell specific. This p27low PCNAhigh phenotype is not seen in medullary thymocytes, but appears abruptly in the recent thymic emigrant population, suggesting that the lyp locus does not act directly on cell cycle regulators but rather alters the interaction between T cells and the peripheral environment. These results provide a biochemical basis for costimulation-independent activation and suggest a mechanism whereby a diabetes susceptibility gene contributes to disease development.