Stephen E. Maher

Cytoprotection of Human Umbilical Vein Endothelial Cells Against Apoptosis and CTL-Mediated Lysis Provided by Caspase-Resistant Bcl-2 Without Alterations in Growth or Activation Responses

Graft endothelial cells are primary targets of host CTL-mediated injury in acute allograft rejection. As an in vitro trial of gene therapy to reduce CTL-mediated endothelial injury, we stably transduced early passage HUVEC with a caspase-resistant mutant form (D34A) of the anti-apoptotic gene Bcl-2. Bcl-2 transductants were compared with HUVEC transduced in parallel with an enhanced green fluorescent protein (EGFP) gene. Both transduced HUVEC have equivalent growth rates in complete medium and both show contact inhibition of growth. However, compared with EGFP-transduced HUVEC, the Bcl-2-transduced cells are resistant to the apoptotic effects of serum and growth factor withdrawal and are also resistant to the induction of apoptosis by staurosporine or by ceramide, with or without TNF. Transduced Bcl-2 did not reduce TNF-mediated NF-κB activation or constitutive expression of class I MHC molecules. HUVEC expressing D34A Bcl-2 were significantly more resistant to lysis by either class I-restricted alloreactive or PHA-redirected CTL than were HUVEC expressing EGFP. We conclude that transduction of graft endothelial cells with D34A Bcl-2 is a possible approach for reducing allograft rejection.

Ly-6 Superfamily Members Ly-6A/E, Ly-6C, and Ly-6I Recognize Two Potential Ligands Expressed by B Lymphocytes

Most hemopoietic cells express one or more members of the Ly-6 supergene family of small glycosylphosphatidylinositol-linked proteins. Although levels of Ly-6 proteins vary with stages of differentiation and activation, their function largely remains unknown. To ascertain whether ligands for Ly-6 proteins exist, chimeric proteins were constructed in which Ly-6E, Ly-6C, and Ly-6I were fused to the murine IgM heavy chain. These chimeras specifically stained both developing and mature B lymphocytes, as assessed by flow cytometry. Analysis of variants of the CH27 B cell lymphoma revealed that Ly-6A/E and Ly-6I recognized different molecules. CH27 cells with low levels of Ly-6A/E ligand activity also lost expression of CD22, and cells transfected with CD22 gained the ability to bind the Ly-6A/E chimera and, to a lesser extent, the Ly-6C and Ly-6I chimeric proteins. As many mature B cells coexpress Ly-6A/E and CD22, the function of Ly-6 molecules may be to associate with other membrane proteins, possibly concentrating these ligands in lipid rafts, rather than acting directly as cell:cell adhesion molecules.

Ly-6A is required for T cell receptor expression and protein tyrosine kinase fyn activity

To characterize the function of the Ly‐6A antigen in T cell activation, antisense Ly‐6 RNA was expressed in a stably transfected antigen‐specific T cell clone. Reduced Ly‐6A expression results in inhibition of responses to antigen, anti‐TCR (anti‐T cell receptor) crosslinking and concanavalin A plus recombinant interleukin 1 and causes impairment of in vitro fyn tyrosine kinase activity. More substantial reduction of Ly‐6A results in reduction of TCR expression. Analysis of mRNA species indicates that the reduction is specific for the TCR beta chain. These data demonstrate that Ly‐6A may regulate TCR expression and may be involved in early events of T cell activation via regulation of fyn tyrosine kinase activity.

Cell-permeable Foxp3 protein alleviates autoimmune disease associated with inflammatory bowel disease and allergic airway inflammation

Foxp3 is a key transcription factor for differentiation and function of regulatory T (Treg) cells that is critical for maintaining immunological self-tolerance. Therefore, increasing Treg function by Foxp3 transduction to regulate an inflammatory immune response is an important goal for the treatment of autoimmune and allergic diseases. Here we have generated a cell-permeable Foxp3 protein by fusion with the unique human HHph-1-PTD (protein transduction domain), examined its regulatory function in T cells, and characterized its therapeutic effect in autoimmune and allergic disease models. HHph-1-Foxp3 was rapidly and effectively transduced into cells within 30 min and conferred suppressor function to CD4+CD25− T cells as well as directly inhibiting T-cell activation and proliferation. Systemic delivery of HHph-1 Foxp3 remarkably inhibited the autoimmune symptoms of scurfy mice and the development of colitis induced by scurfy or wild-type CD4 T cells. Moreover, intranasal delivery of HHph-1-Foxp3 strongly suppressed ovalbumin-induced allergic airway inflammation. These results demonstrate the clinical potential of the cell-permeable recombinant HHph-1-Foxp3 protein in autoimmune and hypersensitive allergic diseases.

Interferon α but Not Interleukin 12 Activates STAT4 Signaling in Human Vascular Endothelial Cells

STAT4 signaling, activated by either interleukin 12 (IL12) or interferon α (IFNα), promotes TH1 responses in CD4+ T cells. Vascular endothelial cells (EC) may also become polarized in response to various cytokines, favoring recruitment and activation of TH1 or TH2 effector cells. Here we have investigated the role of the STAT4 pathway in EC. Cultured human umbilical vein EC (HUVEC) express low levels of STAT4, which may be tyrosine-phosphorylated by treatment with IFNα but not IL12. This is because HUVEC lack both subunits of the IL12 receptor (IL12Rβ1 and IL12Rβ2), even following treatment with various cytokines. IL12 phosphorylation of STAT4 can be observed in HUVEC that have been transduced to express the IL12R. To identify STAT4-induced genes we pursued three approaches: analysis by DNA microarray and quantitative RT-PCR (Q-PCR) of the IL12 responses in IL12R-transduced EC; analysis by Q-PCR of IFNα responses in STAT4-overexpressing EC; and analysis of IFNα responses in U3A neuroblastoma cell lines that express either STAT1 or STAT4, but not both. In all three instances we observe STAT4-mediated induction of the chemokine monocyte chemoattractant protein 1 (MCP1) and suppressor of cytokine signaling 3 (SOCS3) mRNA, and we confirm the production of each protein in both IL12R-transduced EC and STAT4-transduced U3A cells. These observations reveal that there is a STAT4 response of EC, activated by IFNα but not IL12, and that it may modulate the pro-inflammatory behavior of EC.

miR-1 mediated suppression of Sorcin regulates myocardial contractility through modulation of Ca2+ signaling.

MicroRNAs are negative gene regulators and play important roles in cardiac development and disease. As evident by cardiomyopathy following cardiac-specific Dicer knockdown they also are required for maintaining normal cardiac contractile function but the specific role of miR-1 in the process is poorly understood. To characterize the role of miR-1 in particular and to identify its specific targets we created a tamoxifen-inducible, cardiac-specific Dicer knockdown mouse and demonstrated that Dicer downregulation results in a dramatic and rapid decline in cardiac function concurrent with significantly reduced levels of miR-1. The importance of miR-1 was established by miR-1 antagomir treatment of wild-type mice, which replicated the cardiac-specific Dicer knockdown phenotype. Down-regulation of miR-1 was associated with up-regulation of its predicted target Sorcin, an established modulator of calcium signaling and excitation-contraction coupling, subsequently verified as a miR-1 target with luciferase constructs. siRNA-mediated knockdown of Sorcin effectively rescued the cardiac phenotypes after Dicer or miR-1 knockdown affirming Sorcin as a critical mediator of the acute cardiomyopathy observed. The regulatory relationship between miR-1 and Sorcin was further confirmed in cultured mouse cardiomyocytes where modulation of miR-1 was associated with discordant Sorcin levels and dysregulation of calcium signaling. Pathological relevance of our findings included decreased miR-1 and increased Sorcin expression in end-stage cardiomyopathy. These findings demonstrate the importance of miR-1 in cardiac function and in the pathogenesis of heart failure via Sorcin-dependent calcium homeostasis.

Interferon-γ Induces X-linked Inhibitor of Apoptosis-associated Factor-1 and Noxa Expression and Potentiates Human Vascular Smooth Muscle Cell Apoptosis by STAT3 Activation

Interferon (IFN)-γ actions on the vessel wall play an important role in the pathogenesis of arteriosclerosis, yet the contribution of different IFN-γ signaling pathways to the phenotypic modulation of vascular smooth muscle cells (VSMCs) are poorly understood. We investigated the effects of IFN-γ on VSMCs and arteries through interactions involving signal transducer and activator of transcription (STAT) proteins. In addition to STAT1 activation, IFN-γ consistently phosphorylated STAT3 in human VSMCs but weakly or not at all in human endothelial cells or mouse VSMCs. STAT3 activation resulted in nuclear translocation of this transcription factor. By selectively inhibiting STAT3 and not STAT1 signaling, we identified a number of candidate IFN-γ-inducible, STAT3-dependent gene products by microarray analysis. Results for selected genes, including the pro-apoptotic molecules X-linked inhibitor of apoptosis associated factor-1 (XAF1) and Noxa, were verified by real time quantitative reverse transcription-PCR and immunoblot analyses. IFN-γ-induced STAT3 and STAT1 signaling in VSMCs demonstrated reciprocal inhibition. STAT3 activation by IFN-γ sensitized VSMCs to apoptosis triggered by both death receptor- and mitochondrial-mediated pathways. Knock down of XAF1 and Noxa expression inhibited the priming of VSMCs to apoptotic stimuli by IFN-γ. Finally, we confirmed the in vivo relevance of our observations using a chimeric animal model of immunodeficient mice bearing human coronary artery grafts in which the expression of XAF1 and Noxa as well as the pro-apoptotic effects induced by IFN-γ were dependent on STAT3. The data suggest STAT1-independent signaling by IFN-γ via STAT3 that promotes the death of human VSMCs.

Mutation of POLB Causes Lupus in Mice

A replication study of a previous genome-wide association study (GWAS) suggested that a SNP linked to the POLB gene is associated with systemic lupus erythematosus (SLE). This SNP is correlated with decreased expression of Pol β, a key enzyme in the base excision repair (BER) pathway. To determine whether decreased Pol β activity results in SLE, we constructed a mouse model of POLB that encodes an enzyme with slow DNA polymerase activity. We show that mice expressing this hypomorphic POLB allele develop an autoimmune pathology that strongly resembles SLE. Of note, the mutant mice have shorter immunoglobulin heavy-chain junctions and somatic hypermutation is dramatically increased. These results demonstrate that decreased Pol β activity during the generation of immune diversity leads to lupus-like disease in mice, and suggest that decreased expression of Pol β in humans is an underlying cause of SLE.

PEC‐A: An immortalized porcine aortic endothelial cell

Abstract: Cultured porcine aortic endothelium was transfected with sequences that encode the SV40 T antigen, resulting in an immortalized cell line that retains the differentiated properties of normal aortic endothelial cells. Specifically, these cells form cobblestone monolayers, synthesize von Willebrand factor, and endocytose acetylated LDL. These cells can be readily propagated in culture and have been passaged over a year in culture. The cells form tubular structures when grown on Matrigel. The cells in culture express class I but do not express MHC class II antigens. Both class I and class II expression can be induced by treatment with recombinant swine interferon‐γ. Expression of CD44 and several other cell surface antigens have been observed. Co‐culture of these cells with purified human CD4+ T cells resulted in a significant T‐cell proliferative response similar to that observed for primary porcine EC. Finally, the cells are readily susceptible to transfection and express exogenous genes. These cells should be valuable for study of human anti‐porcine endothelial responses.

A Humanized Mouse Model of Autoimmune Insulitis

Many mechanisms of and treatments for type 1 diabetes studied in the NOD mouse model have not been replicated in human disease models. Thus, the field of diabetes research remains hindered by the lack of an in vivo system in which to study the development and onset of autoimmune diabetes. To this end, we characterized a system using human CD4+ T cells pulsed with autoantigen-derived peptides. Six weeks after injection of as few as 0.5 × 106 antigen-pulsed cells into the NOD-Scid Il2rg−/− mouse expressing the human HLA-DR4 transgene, infiltration of mouse islets by human T cells was seen. Although islet infiltration occurred with both healthy and diabetic donor antigen-pulsed CD4+ T cells, diabetic donor injections yielded significantly greater levels of insulitis. Additionally, significantly reduced insulin staining was observed in mice injected with CD4+ T-cell lines from diabetic donors. Increased levels of demethylated β-cell–derived DNA in the bloodstream accompanied this loss of insulin staining. Together, these data show that injection of small numbers of autoantigen-reactive CD4+ T cells can cause a targeted, destructive infiltration of pancreatic β-cells. This model may be valuable for understanding mechanisms of induction of human diabetes.