A screen for short-range paracrine interactions.

Conventional methods for studying paracrine signaling in vitro may not be sensitive to short-range effects resulting from signal dilution or decay. We employ a microfabricated culture substrate to maintain two cell populations in microscale proximity. Individual populations can be quickly retrieved for cell-specific readouts by standard high-throughput assays. We show that this platform is sensitive to short-range interactions that are not detectable by common methods such as conditioned media transfer or porous cell culture inserts, as revealed by gene expression changes in a tumor-stromal crosstalk model. In addition, we are able to detect population-specific gene expression changes that would have been masked in mixed co-cultures. We thus demonstrate a tool for investigating an important class of intercellular communication that may be overlooked in conventional biological studies.

Endothelial cell costimulation of T cell activation through CD58-CD2 interactions involves lipid raft aggregation.

Human endothelial cells (EC) costimulate CD4(+) memory T cell activation through CD58-CD2 interactions. In this study we tested the hypothesis that EC activate distinct costimulatory pathways in T cells that target specific transcription factors. AP-1, composed of fos and jun proteins, is a critical effector of TCR signaling and binds several sites in the IL-2 promoter. EC augment c-fos promoter activity in T cells; however, deletion analysis reveals no transcription factor binding sites in the promoter uniquely responsive to EC costimulation. Overexpression of AP-1 proteins in T cells augments the activity of an AP-1-luciferase reporter gene equally in the absence or the presence of EC costimulation. Interestingly, EC stimulate a similar 2- to 3-fold up-regulation of AP-1, NF-AT, NF-kappaB, and NF-IL-2-luciferase reporters. CD2 mAbs completely block EC effects on all of these pathways, as well as costimulation of IL-2 secretion. We conclude that EC costimulation through CD2 does not trigger a single distinct costimulatory pathway in T cells, but rather, it amplifies several pathways downstream of the TCR. Indeed, we find that early EC costimulation acts "upstream" of the TCR by promoting lipid raft aggregation, thus amplifying TCR signaling. Soluble CD2 mAbs block EC-induced raft aggregation, whereas cross-linking CD2 promotes aggregation. These data are consistent with the critical role of CD2 in organizing the T cell-APC contact zone.

The basic helix-loop-helix transcription factor HESR1 regulates endothelial cell tube formation.

Human endothelial cells can be induced to form capillary-like tubular networks in collagen gels. We have used this in vitro model and representational difference analysis to identify genes involved in the formation of new blood vessels. HESR1 (HEY-1/HRT-1/CHF-2/gridlock), a basic helix-loop-helix protein related to the hairy/enhancer of split/HES family, is absent in migrating and proliferating cultures of endothelial cells but is rapidly induced during capillary-like network formation. HESR1 is detectable in all adult tissues and at high levels in well vascularized organs such as heart and brain. Its expression is also enriched in aorta and purified capillaries. Overexpression of HESR1 in endothelial cells down-regulates vascular endothelial cell growth factor receptor-2 (VEGFR2) mRNA levels and blocks proliferation, migration, and network formation. Interestingly, reduction of expression of HESR1 by antisense oligonucleotides also blocks endothelial cell network formation in vitro. Finally, HESR1 expression is altered in several breast, lung, and kidney tumors. These data are consistent with a temporal model for HESR1 action where down-regulation at the initiation of new vessel budding is required to allow VEGFR2-mediated migration and proliferation, but re-expression of HESR1 is necessary for induction of tubular network formation and continued maintenance of the mature, quiescent vessel.

Expression of IFN-inducible T cell alpha chemoattractant by human endothelial cells is cyclosporin A-resistant and promotes T cell adhesion: implications for cyclosporin A-resistant immune inflammation.

IFN-inducible T cell alpha chemoattractant (I-TAC) is a recently discovered member of the CXC chemokine family. It is a potent T cell chemoattractant expressed by IFN-gamma-treated astrocytes, monocytes, keratinocytes, bronchial epithelial cells, and neutrophils. In this study, we show that I-TAC is also expressed by IFN-gamma-treated endothelial cells (EC), both at the mRNA and protein levels. Induction of the I-TAC message is rapid and sustained over 24 h. TNF-alpha does not induce I-TAC mRNA alone, but does act synergistically with IFN-gamma. Blocking Abs to I-TAC, or to its receptor, CXCR3, reduce T cell adhesion to EC monolayers demonstrating that the expressed protein is functional. Finally, the expression of I-TAC by EC is resistant to the immunosuppressive drug cyclosporin A, suggesting that I-TAC may contribute to the chronic immune inflammation characteristic of graft arteriosclerosis.

Development and assessment of a general theory of cervical carcinogenesis utilizing a severe combined immunodeficiency murine-human xenograft model.

OBJECTIVE: Currently, we lack a theoretical explanation for why squamous cell cervical cancer develops predominantly in specific sites (i.e., along the squamocolumnar junction). We therefore implanted human cervical tissues containing the transformation zone in severe combined immunodeficiency (SCID) mice and studied morphology, steroid effects, gene expression, and human papillomavirus (HPV) factors. METHODS: Normal and dysplastic human cervical tissues (3 x 2 mm) were placed subcutaneously in SCID-beige mice and later assessed by in situ hybridization for HPV 16/18 DNA and by immunohistochemistry for expression of CD31, keratin, proliferating-cell nuclear antigen, HPV 16 E6, p53, and Notch-1 (a binary cell fate determination protein). Some normal tissues were implanted with either a 90-day release 1.7-mg 17beta-estradiol pellet or a 5-mg tamoxifen pellet; others were infected prior to implantation with human recombinant adenovirus 5 vector containing a human cytomegalovirus promoter-driven beta-galactosidase gene and later assessed by X-gal staining. RESULTS: Murine and human vessels formed anastomoses by 3 weeks. For at least 11 weeks, normal tissue retained the transformation zone and normal cell-type-specific keratin expression and exhibited normal proliferation; Notch-1 was present only in the basal cell layer. Dysplastic tissues exhibited koilocytosis, increased levels of cellular proliferation, and aberrant keratin, p53, and Notch-1 expression; HPV 16/18 DNA and HPV 16 E6 protein were detected for at least 6 weeks. Squamous metaplasia of normal cervical epithelium resulted from estrogen exposure, and a predominant columnar differentiation pattern was associated with tamoxifen administration. Through stable adenovirus infection, beta-galactosidase was expressed for at least 6 weeks. CONCLUSIONS: This small manipulatable xenograft model maintains normal and dysplastic human cervical epithelium through neovascularization. Neoplastic tissue retains HPV 16/18 DNA and a premalignant phenotype, including elevated levels of cellular proliferation and aberrant keratin, p53, and Notch-1 expression. These attributes constitute essential features of a biologic model through which one may study HPV-mediated human disease and may be superior to cell culture and transgenic murine systems. Furthermore, this may serve as a model for gene therapy. Finally, we suggest that the normal cervical epithelium is maintained through putative interactions between the Notch locus and cell cycle growth regulators such as p53 and pRb. Neoplastic cervical epithelium may arise through disruption of this pathway. This theory may be testable in our animal model.

Single-cell analysis of costimulation by B cells, endothelial cells, and fibroblasts demonstrates heterogeneity in responses of CD4(+) memory T cells.

Human endothelial cells (EC) express MHC class II molecules in vivo and are likely to be involved in presentation of antigens to CD4(+) T cells. We examined, at the single-cell level, EC presentation of superantigens to resting CD4(+) memory T cells. Within 2 h of adherence to class II+ EC early T cell activation is evidenced by translocation of nuclear factor of activated T cells (NFAT), surface expression of CD69, and synthesis of IFN-gamma and IL-2. Naive T cells are not activated. T cell activation is dependent on the prior induction of MHC class II molecules on EC and is blocked by antibodies to LFA-3 (CD58). Our data place EC along a spectrum of antigen-presenting ability. Activated B cells and macrophages trigger more cells to express cytokines than do EC and at lower antigen concentrations; EC are in turn, superior to fibroblasts or smooth muscle cells. Furthermore, the concept of activation thresholds for cytokine synthesis within T cells also extends to earlier activation events: NFAT translocation is relatively easy to trigger, as is CD69 expression; fewer cells can be triggered to express IFN-gamma and fewer still to express IL-2. EC may, therefore, contribute to a graded immune response by inducing qualitatively and quantitatively different responses than professional APC.

Immunohistochemical analysis, human papillomavirus DNA detection, hormonal manipulation, and exogenous gene expression of normal and dysplastic human cervical epithelium in severe combined immunodeficiency mice.

The cervical squamocolumnar junction of normal and dysplastic human xenografts was maintained in SCID-beige mice. Dysplastic tissue maintained a dysplastic morphology, irregular pattern of keratin expression, elevated levels of cellular proliferation, and human papillomavirus type 16 and/or type 18 DNA. Hyperplastic changes of normal xenografts occurred via high-dose estrogen exposure, and through recombinant adenovirus infection, the introduction and stable expression of an exogenous gene was accomplished.

Activation of the CD2 pathway in lamina propria T cells up-regulates functionally active AP-1 binding to the IL-2 promoter, resulting in messenger RNA transcription and IL-2 secretion.

The aim of this study was to identify molecular mechanisms involved in transcriptional regulation of IL-2 expression following CD2 and CD3 activation in lamina propria (LP) T cells. Studies used T cells from normal, ulcerative colitis, and Crohn's disease mucosa and freshly isolated PBMC, PBMC stimulated with IL-2 alone, and PBMC stimulated with IL-2 and cocultured with B cell lines (LP-like T cells). Electrophoretic mobility shift assays were performed with nuclear extracts from cells activated with either anti-CD2 or anti-CD3 Abs. CD2 signaling in LPMC and LP-like T cells led to a pattern of sustained up-regulation of AP-1-binding complexes, whereas CD3 activation resulted in only transient up-regulation. While the pattern of regulation of AP-1 binding observed in normal, uninflamed, or inflamed Crohn's disease LPMC is similar, differences in intensity of AP-1 binding were observed. Activation of LP-like T cells mimics the up-regulation of AP-1 with a kinetic profile similar to that observed with freshly isolated LPMC from Crohn's disease-inflamed tissue. The AP-1 complex formed following CD2 activation is composed of jun/fos heterodimers. The CD2-enhanced responsiveness is reflected in functional analysis experiments utilizing transfection of both multimeric-TRE or IL-2 promoter-luciferase constructs directly into normal, ulcerative colitis, or Crohn's disease LPMC. Our data suggest that activation of LP T cells from normal, ulcerative colitis, or Crohn's disease mucosa through the CD2 pathway leads to induction of AP-1 complexes that bind to the IL-2 promoter, and may play a pivotal role in modulating IL-2 production in the gut.

c-Rel is a target of pentoxifylline-mediated inhibition of T lymphocyte activation.

The possible clinical use of the methyl xanthine derivative, pentoxifylline (PF), for the treatment of T cell-dependent diseases is being noted with increasing interest. In this paper, we studied the molecular consequences of PF treatment during lymphocyte activation. We found that in T cells, anti-CD3-induced c-Rel expression was blocked by PF, whereas the induction of other NF-kappaB family members was not significantly affected. However, induction of NF-AT, which has the same signaling requirements as c-Rel induction, was not inhibited by PF. Among genes that respond to these transcription factors, IL-2 mRNA induction was suppressed by PF, whereas IL-2R(alpha) chain mRNA induction was not affected. These observations implicated c-Rel as an IL-2 promoter factor, for which experimental support was obtained from transient transfection experiments. In contrast with the observation in T cells, c-Rel induction was not blocked by PF in B cells. The greater selectivity of PF, compared with FK506, at both the molecular and cellular levels may prove advantageous in manipulating T cell responses in vivo.

Porcine endothelial CD86 is a major costimulator of xenogeneic human T cells: cloning, sequencing, and functional expression in human endothelial cells.

Porcine aortic endothelial cells (PAECs), unlike human endothelial cells, express a surface protein recognized by human CTLA4Ig fusion protein that costimulates human T cells through CD28. We have cloned porcine CD86 (pCD86) from an immortalized porcine endothelial cell line, PEC-A, that expresses high levels of this CTLA-4-binding protein. pCD86 mRNA is expressed in PEC-A and PAECs but not in human endothelial cells. Expression of stably transfected pCD86 in CHO cells modestly costimulates human T cell proliferation and IL-2 secretion. Expression of transiently transfected pCD86 in human umbilical vein endothelial cells strongly costimulates IL-2 production by human T cells, comparable to costimulation by PAECs. Costimulation of human T cells by pCD86 in both systems is as effective as costimulation by human CD80 or CD86, and can be blocked by human CTLA4Ig. We conclude that pCD86 contributes to the strong xenoreactivity of porcine endothelium.

IL-4 and IL-13 activate the JAK2 tyrosine kinase and Stat6 in cultured human vascular endothelial cells through a common pathway that does not involve the gamma c chain.

IL-4 and IL-13 each act on human endothelial cells (ECs) to induce expression of vascular cell adhesion molecule-1. On hematopoietic cells. IL-4 responses may be mediated either through a pathway involving gc, the common signaling subunit of the IL-2, IL-4, IL-7, IL-9, and IL-15 receptors, or through a gc-independent pathway that may be alternatively activated by IL-13. We find that human ECs do not express gc, as detected by indirect immunofluorescence and FACS analysis or by a reverse transcription-PCR method. Like IL-4, IL-13 activates a protein tyrosine kinase that phosphorylates the IL-4R binding protein. In addition, we find that IL-4 and IL-13 each induce tyrosine phosphorylation of the JAK2 tyrosine kinase. Furthermore, both IL-4 and IL-13 induce binding of the Stat6 transcription factor to a consensus sequence oligonucleotide. We conclude that the IL-4 response of human ECs involves the IL-13 shared pathway that is independent of gc, and uses JAK2-Stat6 signaling.

Endothelial cells augment the expression of CD40 ligand on newly activated human CD4+ T cells through a CD2/LFA-3 signaling pathway.

Cultured human endothelial cells (EC) increase CD40 ligand expression on polyclonally activated human peripheral blood CD4+ helper T cells compared to T cells activated in the absence of accessory cells or in the presence of peripheral blood adherent cells or B cells. Induction of CD40 ligand expression appears to be biphasic with early induction observable at 6 h and later induction at 24 h. EC cause T cells to increase CD40 ligand expression during the early phase at 6 h after activation. CD40 ligand expression is restricted to the CD4+ helper T cell subset of the peripheral blood T cells, even when EC is present. Blocking monoclonal antibodies to co-stimulatory molecules on EC and T cells indicate that the CD2/LFA-3 pathway, which also contributes to induction of augmented interleukin-2 (IL-2) secretion is involved in EC-induced up-regulation of CD40 ligand. Exogenous IL-2 can also increase CD40 ligand expression. However, increased IL-2 secretion in the presence of EC can not fully account for endothelial-induced CD40 ligand up-regulation as (1) the effect of exogenous IL-2 is greater at 24 h than at 6 h, whereas the opposite is true for EC; (2) the effect of saturating levels of IL-2 is considerably smaller than that of EC; and (3) blocking of IL-2 receptors does not fully inhibit endothelial effects on CD40 ligand expression. We conclude that EC provide unique co-stimulatory signals that effect the phenotype of activated CD4+ T cells.

Transcriptional regulation of the interleukin-2 gene in normal human peripheral blood T cells. Convergence of costimulatory signals and differences from transformed T cells.

To study transcriptional regulation in normal human T cells, we have optimized conditions for transient transfection. Interleukin-2 (IL-2) promoter-reporter gene behavior closely parallels the endogenous gene in response to T cell receptor and costimulatory signals. As assessed with mutagenized promoters, the most important IL-2 cis-regulatory elements in normal T cells are the proximal AP-1 site and the NF- kappaB site. Both primary activation, with phytohemagglutinin or antibodies to CD3, and costimulation, provided by pairs of CD2 antibodies or B7-positive (B cells) or B7-negative (endothelial) accessory cells, are mediated through the same cis-elements. Interestingly, the nuclear factor of activated T cell sites are much less important in normal T cells than in Jurkat T cells. We conclude that IL-2 transcriptional regulation differs in tumor cell lines compared with normal T cells and that different costimulatory signals converge on the same cis-elements in the IL-2 promoter.

CD40 on human endothelial cells: inducibility by cytokines and functional regulation of adhesion molecule expression.

Cultured human umbilical vein endothelial cells (EC) constitutively express a low level of CD40 antigen as detected by monoclonal antibody binding and fluorescence flow cytometric quantitation. The level of expression on EC is increased about 3-fold following 24 h treatment with optimal concentrations of tumor necrosis factor, interleukin 1, interferon beta, or interferon gamma; both interferons show greater than additive induction of CD40 when combined with tumor necrosis factor or interleukin 1. Expression of CD40 increases within 8 h of cytokine treatment and continues to increase through 72 h. A trimeric form of recombinant murine CD40 ligand acts on human EC to increase expression of leukocyte adhesion molecules, including E-selectin, vascular cell adhesion molecule 1, and intercellular adhesion molecule 1. CD40 may be detected immunocytochemically on human microvascular EC in normal skin. We conclude that endothelial CD40 may play a role as a signaling receptor in the development of T-cell-mediated inflammatory reactions.

Costimulation of peripheral blood T cell activation by human endothelial cells. Enhanced IL-2 transcription correlates with increased c-fos synthesis and increased Fos content of AP-1.

Endothelial cells (EC) act as APC for resting PBL in vitro, and may have important roles in vivo in the pathogenesis of allograft rejection and delayed hypersensitivity. We previously reported that human umbilical vein EC provide costimulatory signals to PHA-stimulated PBL via CD2:lymphocyte function-associated Ag-3 and an unidentified ligand pair, resulting in a three- to eight-fold enhancement of IL-2 production. The physiologic relevance of this increase was demonstrated by the proliferative advantage provided by EC to PBL suboptimally stimulated with mAb OKT3. We now report that EC costimulation causes increased levels of IL-2 mRNA as a result of increased IL-2 transcription in PBL. We therefore examined the effects of EC on T cell nuclear factors known to regulate IL-2 transcription, including c-jun and c-fos-two components of the transcription factor AP-1, NFAT, and others. PBL constitutively express c-jun transcripts, and the level of c-jun mRNA is not altered by PHA activation in the absence or presence of EC. In contrast, c-fos mRNA is absent from resting T cells and is induced on PHA activation. EC alone do not induce c-fos mRNA but augment the level of c-fos mRNA in PHA-activated T cells by 3- to 10-fold. This effect is largely independent of the CD2:lymphocyte function-associated Ag-3 pathway. Gel-shift analysis reveals the constitutive presence of nuclear factors in resting PBL that bind to the proximal AP-1 site of the IL-2 promoter and that contain immunoreactive c-Jun but not c-Fos protein. In contrast, AP-1 from PHA-activated cells contains c-Jun and low levels of c-Fos. Strikingly, costimulation with EC results in a dramatic increase (up to 15-fold) in the c-Fos content of AP-1. Levels of other nuclear factors involved in IL-2 regulation were not altered by EC, although NFAT-DNA complexes migrated at a slightly different mobility. In summary, our data suggest that changes in the composition of transcription factor AP-1 is a key molecular mechanism for increasing IL-2 transcription and may underlie the phenomenon of costimulation by EC.

Antigen-specific damage to brain vascular endothelial cells mediated by encephalitogenic and nonencephalitogenic CD4+ T cell lines in vitro.

Experimentally induced and naturally occurring inflammatory diseases of the central nervous system (CNS) are often associated with a breakdown of the blood-brain barrier and edema within the CNS itself. CD4+ T cells are now clearly implicated in the pathogenesis of the induced CNS disease, experimental autoimmune encephalomyelitis, and previous in vivo experiments had indicated that these cells may be capable of directly damaging the CNS vasculature. To assess the capacity of CD4+ T cells to damage brain vascular endothelial cells (EC) in vitro, two lines with specificity for myelin basic protein and OVA were prepared and added to cultures of EC. We show here that both lines, when added in a resting state, severely disrupt the EC monolayers in an Ag-specific manner. The interaction is dependent on the recognition of Ag in the context of MHC class II and is blocked in the presence of mAb specific for CD4. Addition of T cell lines preactivated on irradiated thymocyte APC caused a high level of Ag nonspecific damage to the EC, which was not blocked by the addition of anti-CD4 mAb. Supernatants derived from these latter cells did not alone damage the EC monolayers despite the presence of TNF activity suggesting that T cell-EC contact may be required for these cell lines to mediate their effector function. Both resting and preactivated lines adhered strongly to the EC in the absence of Ag. The capacity of CD4+ T cells to strongly adhere to, and disrupt the integrity of, brain vascular EC may be important in the early stages of CNS disease mediated by this cell type.

Adhesion of lymphocytes to cerebral microvascular cells: effects of interferon-gamma, tumour necrosis factor and interleukin-1.

Although lymphocyte traffic through the brain is normally low, this can increase dramatically in response to infection or an autoimmune reaction. We have studied the adhesion of lymphocytes to cerebral endothelium in vitro in an attempt to model the first step of the infiltration process--that is, the initial interaction between the lymphocytes and endothelial cells--by brief co-culture of lymph node cells with monolayers of cultured cerebral endothelium. In this system we find that the basal level of adhesion can be increased in a dose-dependent manner by pre-treatment of the endothelial cells with interferon-gamma (IFN-gamma) and/or tumour necrosis factor (TNF) but not with interleukin-1 (IL-1). This increased adhesion can be blocked by incubating the IFN-gamma-treated cells with an antibody that is thought to bind to the common beta-chain of the lymphocyte functional antigen-1 (LFA-1) family of molecules. This suggests that endothelial cells express either LFA-1 or a molecule sharing the beta-subunit (of which several have been described) and furthermore that this molecule is involved in the regulation of lymphocyte traffic into the brain.