Biomedical subjects

J S Pober

Publications and source records attributed to J S Pober.

At least 37 records · Page 2Linked to original sources

PubMed2000

A phosphatidylinositol 3-kinase/Akt pathway, activated by tumor necrosis factor or interleukin-1, inhibits apoptosis but does not activate NFkappaB in human endothelial cells.

Tumor necrosis factor (TNF) and interleukin-1 (IL-1) activate the transcription of both anti-apoptotic and pro-inflammatory gene products in human endothelial cells (EC) via NFkappaB. Here we report that both TNF and IL-1 activate the anti-apoptotic protein kinase Akt in growth factor and serum-deprived EC, assessed by Western blotting for phospho-Akt. Phosphorylation of Akt is blocked by LY294002 or wortmannin, inhibitors of phosphatidylinositol 3-kinase (PI 3-kinase). Consistent with these biochemical observations, TNF and IL-1 reduce apoptosis caused by growth factor and serum deprivation, and this action is also blocked by LY294002. Although Akt has been reported to activate NFkappaB, LY294002 does not prevent TNF- or IL-1-induced degradation of IkappaBalpha, beta, or epsilon, transcription of NFkappaB-dependent E-selectin or ICAM-1 promoter-reporter genes, or surface expression of E-selectin or ICAM-1 in human EC. LY294002 potentiates the activation of mitogen-activated protein kinases and stress-activated protein kinases by TNF and IL-1, suggesting Akt inhibits these responses. We conclude that TNF and IL-1 activate a PI 3-kinase/Akt anti-apoptotic pathway and that the anti-apoptotic effects of Akt are independent of NFkappaB. Moreover, the PI 3-kinase/Akt pathway does not play a major role in the pro-inflammatory responses of EC to TNF or IL-1.

Androstadienes↗

PubMed2000

Human vascular endothelial cells stimulate memory but not naive CD8+ T cells to differentiate into CTL retaining an early activation phenotype.

Endothelial cell (EC)-selective alloreactive CTL may mediate alloimmune vascular injury. In the present study, EC-selective CTL were generated in cocultures of purified human CD8+ T cells with allogeneic EC and were compared with conventional CTL against corresponding B lymphoblastoid cells (BLC). EC caused activation and expansion of memory but not naive CD8+ T cells, which differentiated into EC-selective CTL that retained high surface expression of CD69, CD25, and CD62L and displayed low intracellular perforin content. In contrast, BLC-stimulated CTL could be generated from naive or memory CD8+ T cells and showed a more mature phenotype (low CD69, CD25, and CD62L with higher levels of perforin). The expansion of alloreactive T cells by EC stimulation was 5- to 20-fold less effective than in corresponding BLC-stimulated cultures, accounting for a reduction in the assayable cytotoxicity of individual microcultures. In these IL-2-supplemented cocultures, no effect on CTL generation or phenotype was observed by mAb blocking of costimulation provided by LFA-3, ICAM-1, or CD40, by addition of comitogenic anti-CD28 mAb, or by preactivation of EC with CD40 ligand. Cyclosporine inhibited CTL expansion and cytotoxicity similarly in both EC- and BLC-stimulated cultures but did not affect the phenotype of those CTL that did emerge. This study extends the characterization of endothelium as an immunoregulatory cell type distinct from conventional APC and may explain why graft rejection within the arterial intima, an anatomic compartment in which EC may be the primary type of APC, is separable from rejection in the graft parenchyma.

B-Lymphocytes↗

PubMed2000

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-kappaB 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.

Apoptosis↗

PubMed2000

Large-scale culture and selective maturation of human Langerhans cells from granulocyte colony-stimulating factor-mobilized CD34+ progenitors.

Dendritic cells (DCs) play a critical role as APCs in the induction of the primary immune response. Their capacity for Ag processing and presentation is tightly regulated, controlled by a terminal developmental sequence accompanied by striking changes in morphology, organization, and function. The maturation process, which converts DCs from cells adapted for Ag accumulation to cells adapted for T cell stimulation, remains poorly understood due in part to difficulties in the culture and manipulation of DCs of defined lineages. To address these issues, we have devised conditions for the culture of a single DC type, Langerhans cells (LCs), using CD34+ cells from G-CSF-mobilized patients. Homogenous populations of LCs, replete with abundant immunocytochemically demonstrable Birbeck granules, could be stably maintained as immature DCs for long periods in culture. Unlike other human DC preparations, the LCs remained fully differentiated after cytokine removal. Following exposure to TNF-alpha, LPS, or CD40 ligand, the LCs could be synchronously induced to mature. Depending on the agent used, distinct types of LCs emerged differing in their capacity for T cell stimulation, IL-12 production, intracellular localization of MHC products, and overall morphology. Most interestingly, the expression of different sets of Toll family receptors is induced or down-regulated according to the maturation stimulus provided. These results strongly suggest that different proinflammatory stimuli might drive distinct developmental events.

Adult↗

PubMed2000

IL-11 activates human endothelial cells to resist immune-mediated injury.

IL-11, a gp130-signaling cytokine, is protective in several in vivo models of immune-mediated and inflammatory injury. HUVECs express IL-11 receptor alpha-chain and gp130. Human IL-11 causes rapid (2-10 min) tyrosine phosphorylation of gp130. IL-11 at 0.1 and 10 ng/ml induces tyrosine phosphorylation of STAT3 and STAT1, respectively, although maximal responses require 50 ng/ml. Phospho-STAT3 and phospho-STAT1 levels peak rapidly (2.5 min) and disappear by 60 min. The p42 and p44 mitogen-activated protein kinases (MAPKs) are phosphorylated in response to 0.3 ng/ml IL-11 with maximal activation at 30 ng/ml IL-11. Phosphorylation of p42 and p44 MAPKs, which can be prevented by a mitogen-activated protein/extracellular signal-related kinase kinase-1 inhibitor, peaks by 15-20 min and largely disappears by 40 min. IL-11 does not activate NF-kappaB nor does it inhibit NF-kappaB activation by TNF. Similarly, IL-11 neither induces E-selectin or ICAM-1 nor blocks induction by TNF. Although IL-11 does not alter class I MHC complex molecule expression, pretreatment with 0.5 ng/ml IL-11 partially protects HUVECs against lysis by allospecific class I MHC-restricted cytolytic T lymphocytes or by anti-class I MHC Ab plus heterologous C. IL-11-induced cytoprotection is protein synthesis dependent and may depend on mitogen-activated protein/extracellular signal-related kinase kinase-1. Our results indicate that low (i.e., STAT3- and MAPK-activating) concentrations of IL-11 confer resistance to immune-mediated injury in cultured HUVECs without inhibiting proinflammatory responses.

Antigens, CD↗

PubMed2000

Interferon-gamma elicits arteriosclerosis in the absence of leukocytes.

Atherosclerosis and post-transplant graft arteriosclerosis are both characterized by expansion of the arterial intima as a result of the infiltration of mononuclear leukocytes, the proliferation of vascular smooth muscle cells (VSMCs) and the accumulation of extracellular matrix. They are also associated with the presence of the immunomodulatory cytokine interferon-gamma (IFN-gamma). Moreover, in mouse models of atheroma formation or allogeneic transplantation, the serological neutralization or genetic absence of IFN-gamma markedly reduces the extent of intimal expansion. However, other studies have found that exogenous IFN-gamma inhibits cultured VSMC proliferation and matrix synthesis, and reduces intimal expansion in response to mechanical injury. This discrepancy is generally explained by the idea that IFN-gamma either directly activates macrophages, or, by increasing antigen presentation, indirectly activates T cells within the lesions of atherosclerosis and graft arteriosclerosis. These activated leukocytes are thought to express the VSMC-activating cytokines and cell-surface molecules that cause the observed arteriosclerotic responses. Here we have inserted pig and human arteries into the aorta of immunodeficient mice, and we show that IFN-gamma can induce arteriosclerotic changes in the absence of detectable immunocytes by acting on VSMCs to potentiate growth-factor-induced mitogenesis.

Adult↗

PubMed2000

Control of apoptosis during angiogenesis by survivin expression in endothelial cells.

Mechanisms controlling endothelial cell survival during angiogenesis were investigated. Stimulation of quiescent endothelial cells with mitogens, including vascular endothelial growth factor and basic fibroblast growth factor, induced up to approximately 16-fold up-regulation of the cell cycle-regulated apoptosis inhibitor survivin. Mitogen stimulation rapidly increased survivin RNA expression in endothelial cells, which peaked after 6 to 10 hours in culture and decreased by 24 hours. Inflammatory cytokines, tumor necrosis factor alpha, and interleukin-1 did not induce survivin expression in endothelial cells. Formation of three-dimensional vascular tubes in vitro was associated with strong induction of survivin in endothelial cells, as compared with two-dimensional cultures. By immunohistochemistry, survivin was minimally expressed in endothelium of nonproliferating capillaries of normal skin, whereas it became massively up-regulated in newly formed blood vessels of granulation tissue in vivo. Recombinant expression of green fluorescent protein survivin in endothelial cells reduced caspase-3 activity and counteracted apoptosis induced by tumor necrosis factor alpha/cycloheximide. These findings identify survivin as a novel growth factor-inducible protective gene expressed by endothelial cells during angiogenesis. Therapeutic manipulation of survivin expression and function in endothelium may influence compensatory or pathological (tumor) angiogenesis.

Apoptosis↗

PubMed2000

The death domain of tumor necrosis factor receptor 1 is necessary but not sufficient for Golgi retention of the receptor and mediates receptor desensitization.

TNF signals are mediated through two different receptors, TNFR1 and TNFR2. In endothelial cells, TNFR1 is predominantly localized in the Golgi apparatus and TNFR2 on the plasma membrane. To investigate structural features responsible for the disparate localization, endothelial cells were transfected with epitope-tagged or green fluorescent protein-fused wild type and mutant receptor molecules. Wild type receptors recapitulated the distribution of endogenous receptors. Deletions of the entire TNFR1 intracellular domain or of the C-terminal death domain (TNFR1(-DD)) allowed expression of the receptor on the plasma membrane. However, addition of the death domain to the C-terminus of TNFR2 (TNFR2(+DD)) did not lead to Golgi-retention of this chimeric receptor. Overexpressed TNFR1, TNFR2, and TNFR2(+DD) increased basal expression of a cotransfected NF-kappaB-dependent promotor-reporter gene. Overexpressed TNFR1(-DD) did not activate NF-kappaB but acted as a ligand-specific dominant negative inhibitor of TNF actions. Unexpectedly, TNF responses were also inhibited by overexpressed TNFR1 and TNFR2(+DD), but not TNFR2. We conclude that the death domain of TNFR1 is required for retention of TNFR1 in the Golgi apparatus but is not sufficient to direct Golgi retention of a TNFR2(+DD) chimera, and that overexpressed receptors that contain the death domain (TNFR1 and TNFR2(+DD)) spontaneously activate NF-kappaB while inhibiting TNF responses.

Animals↗

PubMed1999

Human endothelial cells augment early CD40 ligand expression in activated CD4+ T cells through LFA-3-mediated stabilization of mRNA.

Human endothelial cells (EC) augment CD40 ligand (CD40L) expression on PHA-activated CD4+ T cells at early times (e.g., 4-6 h). Fixed EC, devoid of mRNA, are comparable to living EC in their capacity to augment early CD40L expression on CD4+ T cells. Fixed EC increase T cell mRNA expression of both IL-2 and CD40L compared with PHA alone at 6 h. EC are unable to increase the rate of transcription of CD40L compared with PHA alone as measured with a promoter-reporter gene, although they do increase transcription of an IL-2 promoter-reporter gene. Fixed EC prolong the half-life of CD40L mRNA >2-fold. Inclusion of anti-human LFA-3 (CD58) mAb or pretreatment of EC with an LFA-3 antisense oligonucleotide blocks EC-induced increases in CD40L expression, whereas mAb to ICAM-1 or pretreatment with ICAM-1 antisense oligonucleotide does not. Moreover, mAb to LFA-3 reverses the capacity of EC to prolong the half-life of CD40L mRNA, whereas mAb to ICAM-1, even in combination with mAb to ICAM-2, does not. We conclude that EC use LFA-3 to increase early CD40L protein expression on newly activated CD4+ T cells by stabilizing CD40L mRNA.

Adjuvants, Immunologic↗

PubMed1999

Human vascular endothelial cells favor clonal expansion of unusual alloreactive CTL.

We have shown previously that cultured HUVEC or mixtures of endothelial cells (EC) and B lymphoblastoid cells (BLC) induce the differentiation of purified CD8+ PBL into allospecific, class I MHC-restricted CTL that lyse EC, but not BLC autologous to EC. Furthermore, these EC-selective CTL lines secrete little IFN-gamma after target cell contact. In the present study, we have analyzed these polyclonal populations at a single cell level by cloning at limiting dilution and propagating the resulting CTL clones in the absence of EC. Phenotypically stable, alloreactive EC-selective CTL preferentially emerge from cocultures in which EC or EC + BLC are the initial stimulating cell types compared with cocultures stimulated by BLC alone (p = 0.005). Compared with BLC-stimulated CTL, EC-stimulated CTL clones often fail to secrete IFN-gamma after target cell contact (p = 0.0006) and constitutively express CD40 ligand (CD40L) at rest (p = 0.0006). The absence of IFN-gamma secretion does not result from a switch to IL-4 secretion. The expression of CD40L inversely correlates with the secretion of IFN-gamma after target cell contact (p = 0.0001), but correlations of CD40L expression and failure to secrete IFN-gamma with EC-selective killing did not reach statistical significance. We conclude that in a microenvironment in which allogeneic EC are in close contact with infiltrating CD8+ T cells, such as within a graft arterial intima, CTL subsets may emerge that display EC selectivity or express CD40L and secrete little IFN-gamma after Ag contact.

Cell Differentiation↗

PubMed1999

Apoptosis-inducing agents cause rapid shedding of tumor necrosis factor receptor 1 (TNFR1). A nonpharmacological explanation for inhibition of TNF-mediated activation.

Several chemical compounds not known to interact with tumor necrosis factor (TNF) signal transducing proteins inhibit TNF-mediated activation of vascular endothelial cells (EC). Four structurally diverse agents, arachidonyl trifluoromethylketone, staurosporine, sodium salicylate, and C6-ceramide, were studied. All four agents caused EC apoptosis at concentrations that inhibited TNF-induced IkappaBalpha degradation. However, evidence of apoptosis was not evident until after several (e.g. 3-12) hours of treatment, whereas 2 h of treatment was sufficient to inhibit TNF responses. IL-1-induced IkappaBalpha degradation was unaffected by these treatments. Inhibition of TNF signaling could not be prevented with either of the broad spectrum caspase inhibitors zVADfmk or yVADcmk. The inhibition of p38 kinase with SB203580 prevented the inhibition of TNF signaling by all agents except arachidonyl trifluoromethylketone. No changes in the levels or molecular weights of the adaptor proteins TRADD (TNF receptor-associated death domain), RIP (receptor-interacting protein), or TRAF2 (TNF receptor-associated factor-2) were caused by apoptogenic drugs. However, TNF receptor 1 (TNFR1) surface expression was significantly reduced by all four agents. Furthermore, TNF-dependent recruitment of TRADD to surface TNFR1 was also inhibited. These data suggest that several putative inhibitors of TNF signaling work by triggering apoptosis and that an early event coincident with the initiation of apoptosis, preceding evidence of injury, is loss of TNFR1. Consistent with this hypothesis, cotreatment of EC with the metalloproteinase inhibitor Tapi (TNF-alpha proteinase inhibitor) blocked the reduction in surface TNFR1 by apoptogenic drugs and prevented inhibition of TNF-induced IkappaBalpha degradation without blocking apoptosis. TNFR1 loss could be a mechanism to limit inflammation in response to apoptotic cell death.

Apoptosis↗

PubMed1999

Human allogeneic vascular rejection after arterial transplantation and peripheral lymphoid reconstitution in severe combined immunodeficient mice.

BACKGROUND: Interspecies differences create important shortcomings in existing animal models used to describe in vivo events responsible for allograft rejection. Alloimmune destruction of human dermal microvessels, histologically consistent with rejection, has been demonstrated in human skin-grafted severe combined immunodeficient (SCID) mice receiving allogeneic human peripheral blood mononuclear cells (PBMC). We have now documented human alloimmune injury in a vascularized, SCID-human arterial transplantation model. METHODS: Fresh human artery was used to replace the CB.17 SCID/beige mouse infrarenal aorta. Seven days later, 3x10(8) human PBMC were administered intraperitoneally, and lymphocyte engraftment was considered successful when circulating human CD3+ cells were later identified in peripheral blood. RESULTS: Forty-six of 49 (94%) mice undergoing transplantation survived, including 14 controls with arterial grafts receiving no PBMC. Twenty-eight of 32 mice demonstrated circulating human CD3+ cells, 14 days after PBMC administration. Animals were killed at 14, 21, or 28 days after receiving allogeneic PBMC, and arteries were recovered for histology and immunohistology. All 14 control mice had patent transplanted grafts with normal vascular histology and no lymphoid infiltration. Damage to transplanted arteries among lymphocyte-engrafted mice was apparent by 14 and 21 days in some animals, whereas 16 of 22 exhibited moderate to severe intimal, medial, and/or adventitial lymphocytic infiltration with intimal expansion by day 28. The infiltrate consisted of HLA-A, -B, -C+, and -DR+, human CD3+ cells, approximately equally distributed as CD4+ and CD8+ subsets. Some infiltrating lymphocytes were cytolytic cells as demonstrated by perforin staining. The endothelium of transplanted human arteries exhibited endothelialitis, and the endothelial cells stained intensely with anti-HLA-A, -B, -C and anti-HLA-DR antibodies. The expanded intima was predominantly smooth muscle cells, staining positively for smooth muscle alpha-actin, HLA-A, -B, -C and HLA-DR. Medial necrosis was not observed. CONCLUSION: The results provide evidence of alloimmune-mediated vascular rejection in this human arterial transplantation model.

Adult↗

PubMed1999

TNF recruits TRADD to the plasma membrane but not the trans-Golgi network, the principal subcellular location of TNF-R1.

The subcellular localization of TNF-R1 to the Golgi apparatus, initially observed in endothelial cells, has been confirmed using transfection of bovine aortic endothelial cells with a human TNF-R1 expression plasmid. The subcellular interactions of TNF-R1 and the TRADD (TNFR-associated death domain protein) adaptor protein have been analyzed in the human monocyte cell line U937 and the human endothelial cell line ECV304 by confocal immunofluorescence microscopy and by Western blot analysis of fractionated cell extracts. In untreated cells, in which TNF-R1 is found on the cell surface but principally localizes to the trans-Golgi network, TRADD is concentrated in the cis- or medial-Golgi region, but separates from the Golgi during cell fractionation. Coimmunoprecipitation studies have shown that TRADD binds to TNF-R1 within 1 min of TNF treatment in a cell fraction-containing plasma membrane. This association is followed by a gradual dissociation, which is prevented if receptor-mediated endocytosis is inhibited by hypertonic medium. In contrast, no association is detected between TRADD and TNF-R1 in the Golgi in response to exogenous TNF at any time examined. These results suggest that although TNF-R1 is predominantly a Golgi-associated protein and TRADD also localizes to the Golgi region, exogenous TNF causes TRADD to bind to TNF-R1 only at the plasma membrane.

Animals↗

PubMed1999

Immunobiology of human vascular endothelium.

The author's laboratory studies interactions between human T lymphocytes and vascular endothelial cells (EC). Our work is organized around three hypotheses. First, we propose that vascular EC can initiate secondary (i.e., recall) immune reactions by presenting antigenic peptide-major histocompatibility complex (MHC) complexes to those circulating memory T cells whose cognate antigen is locally present within a peripheral tissue, e.g., as a consequence of infection or allogeneic transplantation. In this way, EC can increase the efficiency of immune surveillance. Second, we propose that T cell signals, both secreted (e.g., cytokines) and contact-dependent (e.g., CD40 ligand), activate new gene expression in EC that induce the capacity to perform new effector functions, such as leukocyte recruitment and activation or initiation of intravascular coagulation. In this way, EC can participate as effector cells for cell-mediated immune reactions. Third, we propose that EC are major targets of immune-mediated injury. Consequently, increasing resistance of endothelium to immune effector mechanisms may protect tissues from damage, e.g., in allograft rejection. These three hypotheses are explored through in vitro experiments, through analyses of human tissue specimens, and through in vivo studies employing novel human-mouse chimeric animals.

Animals↗

PubMed1999

Integrins alpha4beta7 and alphaEbeta7 are expressed on epidermotropic T cells in cutaneous T cell lymphoma and spongiotic dermatitis.

Integrin alpha4beta7 has been associated with tissue-specific homing of malignant and inflammatory lymphocytes to gastrointestinal mucosa, whereas integrin alphaEbeta7 has been associated with intraepithelial lymphocytes in both the gut and the skin. This prompted us to examine the expression of alpha4beta7 on skin-infiltrating lymphocytes in 12 cases of patch/plaque stage cutaneous T cell lymphoma (CTCL) and in 4 cases of spongiotic dermatitis, which also display intraepidermal T cell accumulation. alpha4beta7 was found to be expressed on 64.8+/-7.4% of intraepidermal and 39.1+/-5.0% of intradermal T lymphocytes in CTCL. There was a significant positive correlation (r=0.58) between the degree of epidermotropism and the percentage of intraepidermal T cells expressing alpha4beta7. Similar findings were observed in spongiotic dermatitis, indicating that this result is not unique to malignant T cells. We evaluated staining of T cells in the same specimens for presence of alphaEbeta7 and observed a strong correlation between the expression of both beta7 integrins in each specimen. Staining with antibodies directed against the known ligands of alpha4beta7 was also performed on skin biopsies from CTCL patients. There was significantly increased dermal microvascular endothelial expression of vascular cell adhesion molecule-1 in lesional compared with nonlesional skin, and in nonlesional skin compared with skin of normal control subjects. Dermal and epidermal expression of the CS-1 domain of fibronectin was present but not increased in lesional biopsies compared with nonlesional or normal controls, whereas expression of mucosal addressin cell adhesion molecule-1 was not detectable in any skin biopsy specimens. In summary, alpha4beta7, like alphaEbeta7, is expressed at high levels on epidermotropic T cells and may interact with endothelial cell vascular cell adhesion molecule-1 as part of stepwise recruitment of lymphocytes from the blood to the epidermis.

Biopsy↗

PubMed1999

Recent advances in the molecular basis of TNF signal transduction.

Substantial progress has been made in the last decade toward defining the signaling pathways that can be activated by TNF and identifying the relevant intracellular signaling molecules. The in vivo consequences of targeted disruption of many of the genes encoding proteins involved in TNF signaling (as discussed in this review) are quite different from those observed for knockout mutations of TNF and the TNF receptors (Erickson et al, 1994; Marino et al, 1997; Rothe et al, 1993) that use these molecules. This suggests that there is still much to be learned about the mechanisms for determining specificity in signaling. The ability to specifically manipulate the involvement of these molecules in TNF signaling, without affecting other pathways, may provide new therapeutic approaches to the many diseases in which TNF has a crucial role.

Apoptosis↗

PubMed1998

Human endothelial cells induce and regulate cytolytic T cell differentiation.

We compared the capacity of cultured human endothelial cells (EC) vs B lymphoblastoid cells (BLC) from the same donor to stimulate allogeneic CD8+ T cells to differentiate into CTL, assaying for allorestricted cytotoxicity, T cell growth, IFN-gamma secretion, and perforin expression. The input cell number affected specificity and potency of the resulting CTL. At low input (<10(5) cells/ well), anti-EC CTL were rarely detected. At high input (>10(6) cells/well), anti-EC CTL developed that displayed unrestricted, low-titer killing and an unstable phenotype. At intermediate input (1.0-2.5 x 10(5) cells/well), classical class I MHC-restricted, CD8+, and perforin-positive anti-EC CTL developed with reproducible frequencies. However, under all conditions EC were less efficient stimulators than BLC from the same donor. Anti-EC CTL did not kill BLC, whereas anti-BLC CTL killed BLC and EC from the same donor with comparable efficiency. When CD8+ T lymphocytes were grown in the presence of EC and BLC together, the differentiation of anti-BLC CTL was completely suppressed, while the anti-EC response was intact. The inhibition of the allogeneic anti-BLC CTL response was independent of T cell-EC contact, and proliferation of CD8+ T cells was inhibited by EC-conditioned medium. We conclude that EC are competent but less efficient activators of CTL differentiation than are BLC and that EC actively regulate differentiation and/or expansion of allospecific CTL.

Adult↗

PubMed1998

Interferon induction of TAP1: the phosphatase SHP-1 regulates crossover between the IFN-alpha/beta and the IFN-gamma signal-transduction pathways.

Interferon (IFN)-gamma and IFN-alpha/beta induction of the transporter associated with antigen processing-1 (TAP1) promoter was compared in HeLa cells and endothelial cells (ECs). In HeLa cells, IFN-gamma acts through Stat1alpha/Stat1alpha homodimers binding to the gamma activating sequence (GAS) and IFN-alpha/beta acts through Stat1/Stat2/p48 binding to the IFN-stimulated response element (ISRE). In ECs, however, IFN-gamma and IFN-alpha/beta act through both the GAS and ISRE. The basis of the IFN signaling crossover in ECs was investigated. HeLa and ECs contain similar ratios of Stat1alpha to Stat2 proteins, and IFN-alpha/beta also activates the same Janus kinases (JAKs) (Jak1 and tyrosine kinase (Tyk) 2 but not Jak2). However, IFN-alpha/beta activates more Stat1alpha than does IFN-gamma in ECs, whereas the reverse occurs in HeLa, and expression of the IFN-alpha/beta receptor-associated phosphatase SHP-1 is much lower in ECs than HeLa cells. Overexpression of SHP-1 in ECs blocks IFN-alpha/beta signaling through GAS, and expression of a dominan negative SHP-1 in HeLa cells permits IFN-alpha/ss signaling through GAS, demonstrating a role for SHP-1 in regulating crossovers between the IFN-alpha/beta and IFN-gamma signaling pathways.

ATP Binding Cassette Transporter, Subfamily B, Mem↗