Biomedical subjects

Jordan S Pober

Publications and source records attributed to Jordan S Pober.

At least 19 recordsLinked to original sources

PubMed2026

Endothelial MHC expression is required to initiate T cell-mediated rejection of 3D-printed skin grafts.

Vascularized skins were 3D printed using single donor human fibroblasts, pericytes, keratinocytes, and endothelial cells (ECs), the latter either unmodified (WT-ECs) or deleted of MHC molecules (KO-ECs). Adult MISTRG6 immunodeficient mice neonatally inoculated with adult human hematopoietic stem cells (HSCs) received printed skin allogeneic to the HSCs and were boosted 3 weeks after grafting with human PBMCs autologous to the HSCs. HSC inoculation alone produced low levels of circulating human myeloid and lymphoid cells without affecting grafts; PBMC boosting dramatically increased circulating human CD4+ T cells and boosted CD8+ T cells only in mice with WT-EC grafts. These grafts became infiltrated by human macrophages, dendritic cells, CD4+ and CD8+ T cells and showed evidence of rejection. Shared T cell clones were present in skin and spleen. KO-EC grafts had minimal infiltration of graft or spleen without rejection, despite MHC molecule expression on other graft cell types.

Animals↗

PubMed2007

Antibody to human leukocyte antigen triggers endothelial exocytosis.

Although antibodies to HLA play a role in the pathogenesis of diseases processes such as rejection of transplanted organs, the precise mechanisms by which antibodies cause tissue injury are not completely understood. We hypothesized that antibodies to host tissues cause inflammation in part by activating endothelial exocytosis of granules that contain prothrombotic mediators such as von Willebrand Factor (VWF) and proinflammatory mediators such as P-selectin. To test this hypothesis, we treated human endothelial cells with murine monoclonal antibody W6/32 to HLA class I and then measured exocytosis by the release of VWF and the externalization of P-selectin. Antibody to HLA activates endothelial exocytosis in a dose-dependent manner over time. The biologically active complement split product, C5a, adds a slight but significant increase to antibody induction of exocytosis. Antibody to HLA alone or with C5a did not damage the cells. Cross-linking of HLA appears to play a role in the ability of antibody to activate exocytosis, because the W6/32 monovalent Fab fragment did not activate VWF release, but the bivalent Fab'2 was effective in triggering exocytosis. To explore the in vivo effects of antibody upon graft injury, we infused W6/32 Fab'2 antibody to human HLA into severe combined immunodeficient/beige mice that had been transplanted with human skin grafts. Antibody to HLA activated exocytosis and inflammation in human skin grafts. Our data show that antibody to host antigens can activate human endothelial cell exocytosis and leukocyte trafficking. By triggering vascular inflammation, antibody activation of exocytosis may play a role in transplant rejection.

Autoantibodies↗

PubMed2007

Induction of inducible NO synthase in bystander human T cells increases allogeneic responses in the vasculature.

Inducible NO synthase (iNOS) in human T cells is implicated in the pathogenesis of graft arteriosclerosis. Here we analyze the regulation and role of iNOS in human peripheral blood T cells. Allogeneic endothelial cells (EC) or dermal fibroblasts induce iNOS mRNA and protein expression, as well as enzymatic activity in primary human CD8 T cells. Although human EC activate T cells through the presentation of alloantigen, iNOS induction is confined to nonactivated T cells and does not depend on MHC molecules or costimulators. iNOS induction does involve new transcription and depends on NF-kappaB. JAK signaling, initiated during T cell activation, inhibits iNOS expression. Even though iNOS is confined to bystander T cells, inhibition of iNOS activity reduces T cell proliferation in response to allogeneic EC, and addition of low levels of a NO donor rescues T cell responses. Similarly, iNOS is preferentially expressed by nonproliferating T cells within allografted arteries in vivo, and inhibition of iNOS activity reduces the number of activated T cells in these artery segments. These data identify a previously undescribed mechanism for enhanced activation of alloreactive T cells, namely stromal cell-mediated induction of iNOS in bystander T cells.

Bystander Effect↗

PubMed2007

An inflammatory pathway of IFN-gamma production in coronary atherosclerosis.

Inflammation is associated with the pathogenesis of coronary atherosclerosis, although the mechanisms remain unclear. We investigated whether cytokine secretion by innate immune responses could contribute to the production of proarteriosclerotic Th1-type cytokines in human coronary atherosclerosis. Cytokines were measured by ELISA in the plasma of patients with coronary atherosclerosis undergoing cardiac catheterization. IL-18 was detected in all subjects, whereas a subset of patients demonstrated a coordinated induction of other IFN-gamma-related cytokines. Specifically, elevated plasma levels of IL-12 correlated with that of IFN-gamma and IFN-gamma-inducible chemokines, defining an IFN-gamma axis that was activated independently of IL-6 or C-reactive protein. Systemic inflammation triggered by cardiopulmonary bypass increased plasma levels of the IFN-gamma axis, but not that of IL-18. Activation of the IFN-gamma axis was not associated with acute coronary syndromes, but portended increased morbidity and mortality after 1-year follow-up. IL-12 and IL-18, but not other monokines, elicited secretion of IFN-gamma and IFN-gamma-inducible chemokines in human atherosclerotic coronary arteries maintained in organ culture. T cells were the principal source of IFN-gamma in response to IL-12/IL-18 within the arterial wall. This inflammatory response did not require, but was synergistic with and primed for TCR signals. IL-12/IL-18-stimulated T cells displayed a cytokine-producing, nonproliferating, and noncytolytic phenotype, consistent with previous descriptions of lymphocytes in stable plaques. In contrast to cognate stimuli, IL-12/IL-18-dependent IFN-gamma secretion was prevented by a p38 MAPK inhibitor and not by cyclosporine. In conclusion, circulating IL-12 may provide a mechanistic link between inflammation and Th1-type cytokine production in coronary atherosclerosis.

Aged↗

PubMed2006

Increased ICAM-1 expression causes endothelial cell leakiness, cytoskeletal reorganization and junctional alterations.

Tumor necrosis factor (TNF)-induced ICAM-1 in endothelial cells (EC) promotes leukocyte adhesion. Here we report that ICAM-1 also effects EC barrier function. Control- or E-selectin-transduced human dermal microvascular EC (HDMEC) form a barrier to flux of proteins and to passage of current (measured as transendothelial electrical resistance or TEER). HDMEC transduced with ICAM-1 at levels comparable to that induced by TNF show reduced TEER, but do so without overtly changing their cell junctions, cell shape, or cytoskeleton organization. Higher levels of ICAM-1 further reduce TEER, increase F/G-actin ratios, rearrange the actin cytoskeleton to cause cell elongation, and alter junctional zona occludens 1 and vascular endothelial-cadherin staining. Transducing with ICAM-1 lacking an intracellular region also reduces TEER. TNF-induced changes in TEER and shape follow a similar time course as ICAM-1 induction; however, the fall in TEER occurs at lower TNF concentrations. Inhibiting NF-kappaB activation blocks ICAM-1 induction; TEER reduction, and shape change. Specific small-interfering RNA knockdown of ICAM-1 partially inhibits TNF-induced shape change. We conclude that moderately elevated ICAM-1 expression reduces EC barrier function and that expressing higher levels of ICAM-1 affects cell junctions and the cytoskeleton. Induction of ICAM-1 may contribute to but does not fully account for TNF-induced vascular leak and EC shape change.

Adherens Junctions↗

PubMed2006

Heparin displaces interferon-gamma-inducible chemokines (IP-10, I-TAC, and Mig) sequestered in the vasculature and inhibits the transendothelial migration and arterial recruitment of T cells.

BACKGROUND: Heparin, used clinically as an anticoagulant, also has antiinflammatory properties and has been described to inhibit interferon (IFN)-gamma responses in endothelial cells. We investigated the effects of heparin on the IFN-gamma-inducible chemokines IP-10/CXCL10, I-TAC/CXCL11, and Mig/CXCL9, which play important roles in the vascular recruitment of IFN-gamma-producing Th1 cells through interactions with their cognate receptor, CXCR3. METHODS AND RESULTS: Patients undergoing coronary artery bypass grafting were studied because coronary atherosclerosis is recognized as a Th1-type inflammatory disease and the subjects required systemic heparinization. Plasma levels of IP-10, I-TAC, and Mig increased immediately after heparin administration and diminished promptly after heparin antagonism with protamine. These effects were independent of detectable circulating IFN-gamma or the IFN-gamma inducer interleukin-12. We confirmed previous reports that heparin inhibits the IFN-gamma-dependent production of CXCR3 chemokine ligands using atherosclerotic coronary arteries in organ culture. In addition to prolonged treatment decreasing chemokine secretion, heparin rapidly displaced membrane-associated IP-10 from cultured endothelial cells that did not express CXCR3 and reduced the IP-10-dependent transendothelial migration of T helper cells under conditions of venular shear stress. Finally, heparin administration to immunodeficient mouse hosts decreased both the recruitment and accumulation of memory T cells within allogeneic human coronary arteries. CONCLUSIONS: Besides inhibiting IFN-gamma responses, heparin has further immunomodulatory effects by competing for binding with IP-10, I-TAC, and Mig on endothelial cells. Disruption of CXCR3+ Th1 cell trafficking to arteriosclerotic arteries may contribute to the therapeutic efficacy of heparin in inflammatory arterial diseases, and nonanticoagulant heparin derivatives may represent a novel antiinflammatory strategy.

Anti-Inflammatory Agents↗

PubMed2006

Endothelial cell-T lymphocyte interactions: IP[corrected]-10 stimulates rapid transendothelial migration of human effector but not central memory CD4+ T cells. Requirements for shear stress and adhesion molecules.

The chemokine interferon (IFN)-gamma-inducible protein of 10 kDa (IP-10; CXCL10) has been implicated in recruitment of T cells into rejecting allografts yet appears ineffective at stimulating human peripheral blood CD4 T cells to transmigrate across tumor necrosis factor (TNF)-treated human endothelial cell (EC) monolayers in vitro. The same cells rapidly (within 15 min) transmigrate across TNF-treated EC monolayers overlaid with stromal cell-derived factor-1 alpha (SDF-1 alpha) and subjected to shear stress. The effector memory subset within the CD4 T cell population, defined as CD45RO, CD62L and CCR7, which constitutes less than 10% of total CD4 T cells, does respond to IP-10 but requires enrichment to be observed in this model. Central memory T cells do not respond to IP-10. Transendothelial migration of effector memory CD4 T cells requires TNF-pretreatment of the EC monolayer and application of venular shear force during the assay. TNF treatment of ECs may be effectively replaced by transduction of vascular cell adhesion molecule-1 or intercellular adhesion molecule-1 but not E-selectin.

CD4-Positive T-Lymphocytes↗

PubMed2006

Vascularization and engraftment of a human skin substitute using circulating progenitor cell-derived endothelial cells.

We seeded tissue engineered human skin substitutes with endothelial cells (EC) differentiated in vitro from progenitors from umbilical cord blood (CB-EC) or adult peripheral blood (AB-EC), comparing the results to previous work using cultured human umbilical vein EC (HUVEC) with or without Bcl-2 transduction. Vascularized skin substitutes were prepared by seeding Bcl-2-transduced or nontransduced HUVEC, CB-EC, or AB-EC on the deep surface of decellularized human dermis following keratinocyte coverage of the epidermal surface. These skin substitutes were transplanted onto C.B-17 SCID/beige mice receiving systemic rapamycin or vehicle control and were analyzed 21 d later. CB-EC and Bcl-2-HUVEC formed more human EC-lined vessels than AB-EC or control HUVEC; CB-EC, Bcl-2-HUVEC, and AB-EC but not control HUVEC promoted ingrowth of mouse EC-lined vessels. Bcl-2 transduction increased the number of human and mouse EC-lined vessels in grafts seeded with HUVEC but not with CB-EC or AB-EC. Both CB-EC and AB-EC-induced microvessels became invested by smooth muscle cell-specific alpha-actin-positive mural cells, indicative of maturation. Rapamycin inhibited ingrowth of mouse EC-lined vessels but did not inhibit formation of human EC-lined vessels. We conclude that EC differentiated from circulating progenitors can be utilized to vascularize human skin substitutes even in the setting of compromised host angiogenesis/vasculogenesis.

Animals↗

PubMed2006

Immune accessory functions of human endothelial cells are modulated by overexpression of B7-H1 (PDL1).

B7-H1 (PDL1) is a B7-related protein that inhibits T-cell responses. Human endothelial cells (EC), which can support polyclonal stimulation (by anti-CD3 or Phytohemagglutinin (PHA)) or direct alloantigen stimulation of T cells, basally express B7-H1 and increase expression in response to IFN-gamma or coculture with allogeneic T cells. Previous studies have suggested that endogenous B7-H1 on EC reduces T-cell responses. We engineered overexpression of B7-H1 in EC (B7H1-EC) to evaluate whether this manipulation could reduce T-cell responses even further. Compared with green fluorescent protein-transduced EC (GFP-EC), B7H1-EC support less anti-CD3 or PHA-induced proliferation of CD4+ memory T cells; naive CD4+ T-cell or CD8+ T-cell responses were less inhibited. The effect of transduced B7H1-EC was more apparent when the EC were fixed prior to coculture, a manipulation that reduces the strength of costimulation and prevents upregulation of the endogenous B7-H1 molecule. T-cell activation markers, including CD25, CD62L, CD152 (CTLA-4), and CD154 (CD40L), were not altered by EC overexpression of B7-H1, whereas there was a reduction in CD69. B7-H1 reduced secretion of IL-2 and IL-10 by memory T cells. B7H1-EC were less able to stimulate allogeneic proliferation of CD4+ memory T cells than control EC. These data suggest that B7-H1 overexpression may be a useful approach for reducing allogeneic CD4+ memory T-cell responses to EC.

Antigen-Presenting Cells↗

PubMed2006

Human allograft arterial injury is ameliorated by sirolimus and cyclosporine and correlates with suppression of interferon-gamma.

BACKGROUND: Chronic allograft dysfunction may result from arterial injury, manifest as transplant arteriosclerosis (TA). This represents an important factor limiting long-term outcomes after heart and kidney transplantation; a relationship between acute allograft arterial injury and TA has been suggested. We have used SCID/bg mice bearing transplanted human artery, inoculated with allogeneic human PBMC to study arteriopathy in human vessels. Earlier work demonstrated arteriopathy similar to that observed clinically, and identified interferon-gamma as a mediator of the process. This study evaluated whether sirolimus (SRL), with cyclosporine A (CsA) or alone, affects TA, and examined possible mechanisms of action. METHODS: CB17/SCID/bg mice were transplanted with human arteries replacing the abdominal aorta; reconstituted with allogeneic human PBMC. Controls received vehicle alone for comparison with mice given CsA (5 mg/kg/d), SRL (0.1 or 0.5 mg/kg/d), or CsA (5 mg/kg/d) plus SRL (0.1 mg/kg/d). Transplant arteries were examined 28 days later by histology and immunohistochemistry; circulating human interferon-gamma was evaluated by ELISA, and intragraft interferon-gamma mRNA by qRT-PCR. RESULTS: The characteristic TA was modestly reduced by CsA or low-dose SRL, but eliminated by combination CsA plus SRL or higher dose SRL alone. Circulating interferon-gamma was reduced by CsA, but inhibition was dramatic with SRL alone or combined with CsA. Intragraft interferon-gamma and HLA-DR expression were moderately reduced by CsA or SRL, and eliminated with combined CsA plus SRL. CONCLUSIONS: SRL plus CsA prevented allograft arteriopathy, correlating with suppression of intragraft interferon-gamma, suggesting that SRL effects may result from anti-inflammatory consequences from inhibiting interferon-gamma.

Animals↗

PubMed2006

Development of a model system for preliminary evaluation of tissue-engineered vascular conduits.

BACKGROUND/PURPOSE: The ability to construct tissue-engineered neovessels for use as arterial or venous grafts holds great promise for the advancement of pediatric surgical disciplines. Although the feasibility of tissue engineering vascular grafts has been demonstrated, the long-term function, safety, and efficacy of these grafts as well as their capacity to grow and adapt remain largely unknown. In an attempt to further characterize and develop this technology, we used severe combined immunodeficiency beige (SCID/bg) mouse recipients, chosen because such animals accept xenogenic human cells, to create a small animal model that would allow a rapid and cost-effective preliminary evaluation of the function of tissue-engineered vascular grafts. METHODS: Eight CB-17 SCID/bg female mice underwent vascular graft placement. Four of these mice received aortic interposition grafts, 1 mouse received an inferior vena cava interposition graft, and 3 mice underwent aortocaval graft insertion. All grafts were fashioned from decellularized ovine arteriole tissue engineering scaffolds. Grafts were evaluated for patency using clinical examination, ultrasound interrogation, and micro-computed tomography. Animals were killed at various time points after implantation, and grafts were harvested and analyzed histologically using standard hematoxylin and eosin staining. RESULTS: All grafts were patent based on clinical examination for up to 35 days. Patency was confirmed in 5 grafts using ultrasound interrogation. Patency was confirmed in 4 grafts using micro-computed tomography. One animal that underwent arteriovenous grafting had to be euthanized secondary to high-output cardiac failure on postoperative day 2. The remaining animals were killed between postoperative days 12 and 35. Histologic evaluation of the specimens demonstrated patent grafts with cellular ingrowth into the tissue engineering scaffold. CONCLUSIONS: From these results, we conclude that the use of the SCID/bg mouse model for preliminary evaluation of new tissue engineering methodologies for construction of vascular conduits is feasible. Use of this model has the added advantage of evaluating nonautologous and even xenograft tissues, including human cells.

Animals↗

PubMed2006

Rat hepatocyte engraftment in severe combined immunodeficient x beige mice using mouse-specific anti-fas antibody.

BACKGROUND: Hepatocyte transplantation holds promise as a treatment for acute and chronic liver failure; however, robust model systems needed to study xenogeneic hepatocyte transfer are lacking. Severe combined immunodeficient x beige (SCID/bg) hybrid mice readily accept foreign tissue. Repopulation of C.B-17 SCID/bg mouse liver with rat hepatocytes was studied following induction of mouse hepatocyte apoptosis using an anti-mouse agonistic fas monoclonal antibody (Jo2 mAb) that does not engage xenogeneic fas. METHODS: SCID/bg mice were transplanted with 1 x 10(6) fresh adult rat hepatocytes intrasplenically and treated with various doses, routes and frequencies of Jo2 mAb. Rat cell repopulation was characterized by quantitative immunofluorescent antibody (q-IFA) staining specific for rat dipeptidyl peptidase type IV (DPP-IV) and leucine amino peptidase, amplification of rat genomic DNA using polymerase chain reaction and histopathological and serum biochemistry analyses. RESULTS: Analysis of liver sections from mice treated twice weekly for 12 weeks with 0.4 mg/kg Jo2 mAb intraperitoneally consistently demonstrated >50% rat hepatocytes in the parenchymal mass by q-IFA. Rat hepatocyte engraftment protected mice from Jo2 mAb-mediated liver hemorrhage and hepatocyte apoptosis. Serum liver enzyme levels did not increase in Jo2 mAb-treated mice that were highly engrafted with rat hepatocytes, in contrast to matched non-engrafted mice. At 12 weeks post-engraftment, minimal fibrosis and inflammation were apparent and liver architecture had returned to near normal. Jo2 mAb did not induce histopathological abnormalities in other tissues known to express fas antigen (i.e. heart, lung). CONCLUSIONS: This novel model represents a simple and robust system of xenogeneic hepatocyte transplantation that could be applied to studies of liver biology, regeneration and hepatocyte transplantation.

Animals↗

PubMed2005

Memory T cells and their costimulators in human allograft injury.

Both CD4(+) and CD8(+) human memory but not naive T cells respond to allogeneic human dermal microvascular endothelial cells (HDMEC) in vitro by secreting cytokines and by proliferating. Several recently identified costimulators, namely, 4-1BB ligand, ICOS ligand, and OX40 ligand, are up-regulated on cultured HDMEC in response to TNF or coculture with allogeneic T cells. Blockade of these costimulators each partially reduces IFN-gamma and IL-2 secretion and proliferation of previously resting memory T cells. The effects of these costimulators are overlapping but not identical. Memory but not naive T cells are the principal effectors of microvascular injury in human skin allografts following adoptive transfer into immunodeficient mice. Furthermore, blocking 4-1BB ligand, ICOS ligand, or OX40 ligand in this model reduces human skin allograft injury and T cell effector molecule expression. These data demonstrate that human memory T cells respond to microvascular endothelial cells and can injure allografts in vivo without priming. Furthermore, several recently described costimulators contribute to these processes.

4-1BB Ligand↗

PubMed2005

The cathepsin B death pathway contributes to TNF plus IFN-gamma-mediated human endothelial injury.

Vascular endothelial cells are primary targets of cytokine-induced cell death leading to tissue injury. We previously reported that TNF in combination with LY294002, a PI3K inhibitor, activates caspase-independent cell death initiated by cathepsin B (Cat B) in HUVEC. We report that TNF in the presence of IFN-gamma activates Cat B as well as a caspase death pathway in both HUVEC and human dermal microvascular endothelial cells, but only activates caspase-mediated death in HeLa cells and human embryonic kidney (HEK)293 cells. Like LY294002, IFN-gamma triggers Cat B release from lysosomes in HUVEC. Cat B-triggered death involves mitochondria, indicated by release of cytochrome c, loss of mitochondrial membrane potential and inhibition of death by overexpressed Bcl-2. Cat B effects on mitochondria do not depend upon Bid cleavage. Unexpectedly, overexpression of a dominant negative mutated form of Fas-associated death domain protein (FADD), which blocks caspase activation by TNF, potentiates TNF activation of Cat B and cell death in HUVEC. Similarly, mutant Jurkat cells lacking FADD also show increased susceptibility to TNF-induced Cat B-dependent cell death. These observations suggest that the Cat B death pathway is cell type-specific and may contribute to cytokine-mediated human tissue injury and to the embryonic lethality of FADD gene disruption in mice.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Caveolae participate in tumor necrosis factor receptor 1 signaling and internalization in a human endothelial cell line.

Caveolae are abundant in endothelial cells (ECs) in situ but markedly diminished in cultured cells, making it difficult to assess their role in cytokine signaling. We report here that the human EC line EA.hy926 retains an abundant caveolar system in culture. Tumor necrosis factor (TNF) receptor 1 (TNFR1/CD120a) was enriched in caveolae and co-immunoprecipitated with caveolin-1 from caveolae isolated from these cells. To further investigate the role(s) of caveolae in TNF signaling in ECs, cells were treated with methyl-beta-cyclodextrin to disrupt caveolae. Methyl-beta-cyclodextrin did not alter total cell surface expression of TNFR1 or TNF-induced degradation of IkappaBalpha, a measure of nuclear factor-kappaB activation, but it did inhibit TNF-induced phosphorylation of Akt, a measure of phosphatidylinositol-3 kinase activation. Serum-induced phosphorylation of AKT was unaffected. Treatment with TNF induced disappearance of TNFR1 from caveolae and dissociation from caveolin-1 within 5 minutes. In contrast to transferrin receptor, internalized TNFR1 did not co-localize with clathrin, except possibly in the Golgi, at any time point examined. By 60 minutes of treatment with TNF, TNFR1 appeared in endosomes. We conclude that caveolae function in ECs to allow TNFR1 to activate phosphatidylinositol-3 kinase and Akt, perhaps through receptor cross talk, and that ligand-induced internalization and trafficking of TNFR1 to endosomes may originate directly from this compartment.

Caveolae↗

PubMed2005

TNFR1- and TNFR2-mediated signaling pathways in human kidney are cell type-specific and differentially contribute to renal injury.

In normal kidney, TNFR1 is expressed in glomerular and peritubular capillary EC, and some tubular cells, and colocalizes with inactive apoptosis signal-regulating kinase-1 (ASK1) phosphorylated at serine 967. Biopsies of rejecting or ischemic renal allografts, which show both tubular cell injury and proliferation, display down-regulation of TNFR1 and activation of ASK1 as well as up-regulation of TNFR2 on tubular cells, where it colocalizes with phosphorylated endothelial/epithelial tyrosine kinase (Etk). We have exploited receptor-selective muteins and evaluated phosphorylation of receptor-specific kinases to study TNF responses in situ. In organ culture, a TNFR1-specific mutein changes phosphorylation of ASK1 to threonine 845, indicative of kinase activation. A TNFR2-specific mutein down-regulates TNFR1 in glomerular EC, up-regulates TNFR2 and Etk in tubular cells, and induces phosphorylation of Etk. Wild-type TNF induces TNFR2 and Etk and activates both ASK1 and Etk but does not down-regulate TNFR1. Wild-type TNF and TNFR1-specific mutein trigger tubular cell apoptosis whereas wild-type TNF and TNFR2-specific mutein induce tubular cells to express proliferating cell nuclear antigen. Differential activation of ASK1 and Etk by regulated TNFRs in patient-derived materials provides an explanation for diverse and opposing responses to TNF at distinct sites, and an in situ bioassay of TNFR signaling.

Apoptosis↗

PubMed2005

Recruitment of CXCR3+ and CCR5+ T cells and production of interferon-gamma-inducible chemokines in rejecting human arteries.

Chemokine receptors preferentially expressed by Th1 cells and their IFN-gamma-inducible ligands predominate in experimental and clinical allograft rejection. Previous chemokine-related transplantation studies have focused on parenchymal and microvascular inflammation which are of importance in acute rejection, but are not necessarily relevant in immune-mediated injury of conduit arteries. We have recently described a model of progressive human T cell-mediated infiltration and injury of allogeneic coronary artery segments using immunodeficient mouse hosts. In the present study, we investigated if recruitment of allogeneic T cells to different vascular compartments correlated with the expression of chemokines and their receptors. Transcripts were quantified by laser capture microdissection/real-time RT-PCR and their distribution was correlated to the corresponding protein expression detected by immunohistochemistry. Infiltrating T cells, confined to the adventitia and intima, expressed CXCR3 and CCR5, but were not recruited into the media despite production by vascular smooth muscle cells of IP-10, Mig, I-TAC, RANTES and MIP-1beta. Chemokine mRNA was detected primarily in vascular cells, although chemokine protein largely localized to infiltrating leukocytes which uniquely expressed their cognate receptors. These data explain the recruitment of IFN-gamma-secreting T cells to the vessel wall, and reinforce the suggestion that the arterial media may be a site of immunological privilege.

Antineoplastic Agents↗

PubMed2005

Human endothelial cells enhance human immunodeficiency virus type 1 replication in CD4+ T cells in a Nef-dependent manner in vitro and in vivo.

Infected CD4+ T cells are the primary sites of human immunodeficiency virus type 1 (HIV-1) replication in vivo. However, signals from professional antigen-presenting cells (APCs), such as dendritic cells and macrophages, greatly enhance HIV-1 replication in T cells. Here, we report that in cocultures, vascular endothelial cells (ECs), which in humans can also serve as APCs, can enhance HIV-1 production of both CCR5- and CXCR4-utilizing strains approximately 50,000-fold. The observed HIV-1 replication enhancement conferred by ECs occurred only in memory CD4+ T cells, required expression of major histocompatibility complex class II (MHC-II) molecules by the ECs, and could not be conferred by fixed ECs, all of which are consistent with a requirement for EC-mediated T-cell activation via T-cell receptor (TCR) signaling. Deletion of nef (Nef-) decreased HIV-1 production by approximately 100-fold in T cells cocultured with ECs but had no effect on virus production in T cells cocultured with professional APCs or fibroblasts induced to express MHC-II. Human ECs do not express B7 costimulators, but Nef- replication in CD4(+)-T-cell and EC cocultures could not be rescued by anti-CD28 antibody. ECs act in trans to enhance wild-type but not Nef- replication and facilitate enhanced wild-type replication in naive T cells when added to T-cell or B-lymphoblastoid cell cocultures, suggesting that ECs also provide a TCR-independent signal to infected T cells. Consistent with these in vitro observations, wild-type HIV-1 replicated 30- to 50-fold more than Nef- in human T cells infiltrating allogeneic human skin grafts on human huPBL-SCID/bg mice, an in vivo model of T-cell activation by ECs. Our studies suggest that ECs, which line the entire cardiovascular system and are, per force, in frequent contact with memory CD4+ T cells, provide signals to HIV-1-infected CD4+ T cells to greatly enhance HIV-1 production in a Nef-dependent manner, a mechanism that could contribute to the development of AIDS.

Animals↗