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J S Pober

Publications and source records attributed to J S Pober.

At least 163 records · Page 9Linked to original sources
PubMed1986

Induction and detection of a human endothelial activation antigen in vivo.

We used a murine mAb, H4/18, raised by immunization with IL-1-treated human umbilical vein endothelial cell cultures, to localize an endothelial activation antigen in induced human delayed hypersensitivity reactions (DHR) and in pathological tissues. We used streptococcus varidase to elicit DHR in human skin and we examined sequential skin biopsies with the immunoperoxidase technique. There was no staining for H4/18 binding antigen in normal endothelium of skin and other tissues; strong positive staining, localized to vascular endothelium, was seen at 16 and 23 h but disappeared by 6 d, when the DHR had faded. H4/18 binding antigen, also confined to endothelium, was detected in lymph nodes, skin, and other tissues exhibiting immune/inflammatory reactions. The studies indicate that H4/18 is a useful marker for activated endothelium in vivo and they support the relevance of in vitro studies on inducible endothelial cell functions.

Animals↗
PubMed1986

Two distinct monokines, interleukin 1 and tumor necrosis factor, each independently induce biosynthesis and transient expression of the same antigen on the surface of cultured human vascular endothelial cells.

A murine monoclonal antibody (H4/18) raised against cultured human endothelial cells (HEC) prestimulated by the monokine interleukin 1 (IL 1) recognizes a cell surface molecule inducible by IL 1 or by the distinct monokine tumor necrosis factor (TNF) in primary or serially passaged HEC. H4/18 binding is not basally expressed or inducible by IL 1 in an SV-40 transformed HEC line, in human dermal fibroblasts, or in blood leukocytes. Expression of this molecule by HEC in response to IL 1 can be blocked by protein and RNA synthesis inhibitors but not by cyclooxygenase inhibitors. In addition, H4/18 can immunoprecipitate two biosynthetically labeled polypeptides (Mr 100,000 and 120,000) from HEC stimulated with IL 1 but not from control HEC. Thus, the H4/18 binding site appears to be an inducible surface protein specific for HEC. The majority of HEC in a culture can be induced to express the H4/18 binding protein, but expression is transient (peak 4 to 6 hr) and over the next 24 hr declines to near basal levels either in the continued presence of or upon removal of IL 1. The magnitude of the peak response depends upon IL 1 concentration (peak 5 to 10 U/ml), and the response is optimized by the continued presence of IL 1 during the initial 4- to 6-hr induction period. The time of peak H4/18 binding does not appear to be a function of IL 1 concentration. The decline of H4/18 binding from peak levels is prevented by cycloheximide, a protein synthesis inhibitor. HEC maintained in the presence of IL 1 for 24 hr become refractory to restimulation by IL 1; however, IL 1-stimulated cells rested in the absence of IL 1 for 20 hr can be stimulated by fresh IL 1. HEC expression of the H4/18 binding protein is not induced by interleukin 2 or by interferon-alpha, -beta, or -gamma. Induction of H4/18 binding by TNF is also concentration dependent, transient, and dependent upon protein and RNA synthesis. Several observations suggest that IL1 and TNF act independently on HEC. Our TNF is a recombinant protein, expressed from a cloned cDNA and thus free of IL 1 contamination; it also has no activity in a highly sensitive IL 1 assay. Our standard IL 1 preparation is affinity purified and lacks TNF activity on L929 cells. Thus, our monokine preparations are not cross-contaminated. Most interestingly, HEC incubated with IL 1 and refractory to IL1 restimulation can be restimulated by TNF to express H4/18 binding and vice versa.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1986

Recombinant tumor necrosis factor induces procoagulant activity in cultured human vascular endothelium: characterization and comparison with the actions of interleukin 1.

Human recombinant tumor necrosis factor (rTNF) was found to act directly on cultured human vascular endothelium to induce a tissue factor-like procoagulant activity (PCA). After a 4-hr incubation in rTNF (100 units/ml), serially passaged endothelial cells isolated from umbilical veins, saphenous veins, iliac arteries, and thoracic aortae demonstrated a dramatic increase (4- to 15-fold, 21 experiments) in total cellular PCA as measured with a one-stage clotting assay. rTNF-induced PCA was also expressed at the surface of intact viable endothelial monolayers. Induction of PCA by rTNF was concentration dependent (maximum, 500 units/ml), time dependent, reversible, and blocked by cycloheximide and actinomycin D, and it occurred without detectable endothelial cell damage. Actions of rTNF were compared with those of natural human interleukin 1 (IL-1) derived from stimulated monocytes and two distinct species of recombinant IL-1, each of which also induced endothelial PCA. The use of recombinant polypeptides and specific neutralizing antisera established the distinct natures of the mediators. The kinetics of the endothelial PCA responses to TNF and IL-1 were similar, demonstrating a rapid rise to peak activity at approximately equal to 4 hr, and a decline toward basal levels by 24 hr. This characteristic decline in PCA after prolonged incubation with TNF or IL-1 was accompanied by selective endothelial hyporesponsiveness to the initially stimulating monokine. Interestingly, the effects of TNF and IL-1 were found to be additive even at apparent maximal doses of the individual monokines. Endothelial-directed actions of TNF, alone or in combination with other monokines, may be important in the initiation of coagulation and inflammatory responses in vivo.

Blood Coagulation Factors↗
PubMed1986

Recombinant human tumor necrosis factor increases mRNA levels and surface expression of HLA-A,B antigens in vascular endothelial cells and dermal fibroblasts in vitro.

Recombinant human tumor necrosis factor (TNF), purified to greater than 99% homogeneity, increases surface expression of class I major histocompatibility complex (MHC) antigens to a maximum of 9-fold on cultured human endothelial cells (HEC) and human dermal fibroblasts (HDF). The increase is concentration dependent (peak 20-100 units/ml) and time dependent (nearly maximal by 4 days); expression remains elevated in the continued presence of TNF and requires greater than 7 days to return to basal levels upon TNF withdrawal. The increase in surface expression appears to result from increases in steady-state mRNA levels for the class I antigens, although the increase in mRNA is proportionately greater than for surface expression. No surface expression of or mRNA for class II MHC antigens is detectable in either control or TNF-treated HEC or HDF. These effects are similar to those produced by leukocyte or fibroblast (type I) interferons (IFNs). The protein synthesis inhibitor cycloheximide (CHX), when added coincidentally with type I IFNs, leads to superinduction of mRNA for class I MHC antigens and, unexpectedly, leads to the appearance of mRNA for class II MHC antigens. CHX has no effect by itself upon mRNA levels for class I or class II MHC antigens, nor does it modulate the increases in mRNA produced by immune (type II) IFN. Most interesting, CHX blocks the increase in mRNA for class I MHC antigens induced by TNF. Thus TNF appears to act on MHC gene expression through a newly synthesized protein intermediate. Our results provide direct evidence that TNF can modulate gene expression in normal (untransformed) cell types and contribute to understanding the complex nature of MHC gene regulation. Finally, they suggest that TNF may act in vivo as an immunoregulatory molecule.

Cell Membrane↗
PubMed1986

Immunoglobulin M antibodies present in the acute phase of Kawasaki syndrome lyse cultured vascular endothelial cells stimulated by gamma interferon.

Kawasaki syndrome (KS) is characterized by diffuse vasculitis and marked T cell and B cell activation. In this study, sera from 16 patients with acute KS, 15 patients in the convalescent phase of KS, and 19 age-matched controls were assessed for complement dependent cytotoxic activity against 111In-labeled human umbilical vein endothelial (HUVE) cells, Neither sera from patients with KS nor sera from controls had cytotoxic effects on HUVE cells cultivated under standard conditions. Since activated T cells such as those present in acute KS secrete gamma interferon (gamma-IFN), we also examined the effects of sera from acute KS on HUVE cells preincubated with gamma-IFN. We report here that immunoglobulin M (IgM) antibodies in sera from patients with acute KS cause significant (P less than 0.01) killing of gamma-IFN-treated HUVE cells. Pretreatment with interleukin 2, gamma-IFN, or beta-IFN failed to render HUVE susceptible to lysis with acute KS sera. The observed effects were not mediated via immune complexes. The cytotoxic antibodies in acute KS seem to be directed against inducible monomorphic antigenic determinants present on gamma-IFN-treated HUVE cells but not on control or gamma-IFN treated autologous human dermal fibroblasts (HDF). Similarly, acute KS sera also induced lysis of gamma-IFN-treated human saphenous vein endothelial (HSVE) cells but not gamma-IFN treated human saphenous vein smooth muscle (HSVSM) cells. Since gamma-IFN induces the same level of class I and class II major histocompatibility complex (MHC) antigen expression on HDF, HUVE, HSVE, and HSVSM cells, our results suggest that the anti-endothelial cell antibodies in acute KS are directed to gamma-IFN-inducible molecules other than MHC determinants. These observations are further substantiated by the failure of human B cells or monocytes to absorb the anti-endothelial cell activity. Since most vasculitides, including acute KS, are characterized both by marked immune activation and the secretion of lymphokines, antibodies directed to gamma-IFN-inducible endothelial cell antigens may represent a general mechanism for vascular injury.

Antigen-Antibody Complex↗
PubMed1986

Recombinant tumor necrosis factor and immune interferon act singly and in combination to reorganize human vascular endothelial cell monolayers.

Human endothelial cells (HECs) in confluent primary culture reproduce the epithelioid organization of the vascular lining in situ. The addition of greater than or equal to 20 U/ml recombinant tumor necrosis factor (rTNF) or greater than or equal to 16 U/ml recombinant immune interferon (rIFN-gamma) causes HECs 1) to become elongated, 2) to overlap, 3) to rearrange their actin filaments, and 4) to lose their stainable fibronectin matrix. These changes develop over 72-96 hours and are reversible upon withdrawal of the mediator. In serially passaged HECs similar morphologic changes develop. Furthermore, rTNF and rIFN-gamma are each cytostatic for subconfluent passaged cultures. When added in combination, low concentrations of rTNF and rIFN-gamma act synergistically, whereas higher concentrations (eg, 100 U/ml rTNF and 200 U/ml rIFN-gamma) produce unique morphologic changes. Doubly treated primary HECs extend many long, overlapping, spinelike processes and expose the substratum. Doubly treated passaged HECs become extremely elongated and then shed in large numbers. These in vitro changes in endothelial cell morphology and behavior may underlie immunologically mediated vascular responses in vivo.

Actins↗
PubMed1985

Human dermal fibroblasts present tetanus toxoid antigen to antigen-specific T cell clones.

Cultured human dermal fibroblasts treated with immune interferon express HLA-DR antigens. We report here that DR-positive fibroblasts present tetanus toxoid (TT) to autologous TT-specific monoclonal helper T cells vigorously depleted of monocytes by passage over Sephadex G10 columns followed by treatment with the monoclonal antibodies (mAb) OKM1 and Leu M1 plus complement. The extent of T cell proliferation in response to TT presented by DR-positive fibroblasts was similar to that elicited using monocytes as antigen-presenting cells. The proliferative response was TT dependent, antigen specific, depended upon DR expression by fibroblasts, appeared MHC restricted, and was completely blocked by mouse mAb to HLA-DR but not by mAb to HLA-A,B, or DQ. DR-positive fibroblasts pulsed with TT were similarly effective in antigen presentation. In summary, immune interferon-stimulated human dermal fibroblasts can substitute for classical antigen-presenting cells in antigen-specific proliferative responses. Since fibroblasts are a ubiquitous cell type in the body, they may play a significant role in the immunobiology of the host.

Antibodies, Monoclonal↗
PubMed1985

Interleukin 1 acts on cultured human vascular endothelium to increase the adhesion of polymorphonuclear leukocytes, monocytes, and related leukocyte cell lines.

Increased leukocyte adhesion to the endothelial lining of blood vessels is an essential event in inflammation and the pathogenesis of certain vascular diseases. We have studied the effect of interleukin 1 (IL-1), an inflammatory/immune mediator, on endothelial-leukocyte adhesion using quantitative in vitro assays. Selective pretreatment of cultured human umbilical vein endothelial monolayers with IL-1 (5 U/ml, 4 h) resulted in an 18.3 +/- 2.6-fold increase in human peripheral blood polymorphonuclear leukocyte (PMN) adhesion (mean +/- SEM, n = 16) and a 2.6 +/- 0.3-fold increase in monocyte adhesion (n = 7) over basal levels. IL-1-treated endothelial monolayers also supported increased adhesion of the promyelocytic cell line HL-60 and the monocytelike cell line U937 (33.0 +/- 6.0-fold, n = 6 and 4.9 +/- 0.5-fold, n = 15, respectively). In contrast, selective IL-1 pretreatment of leukocytes, or the addition of IL-1 during the adhesion assay, did not alter endothelial-leukocyte adhesion. Conditioned medium from IL-1-treated endothelial cultures also did not promote leukocyte adhesion to untreated monolayers. IL-1 induction of endothelial adhesivity was concentration dependent (maximum, 10 U/ml), time dependent (peak, 4-6 h), and reversible, was blocked by cycloheximide (10 micrograms/ml) or actinomycin D (5 micrograms/ml) but not by acetylsalicylic acid (100 microM), and occurred without detectable endothelial cell damage. IL-1 treatment of SV40-transformed human endothelial cells and dermal fibroblasts did not increase their adhesivity for leukocytes. These data suggest that IL-1 can act selectively on human vascular endothelium to increase its adhesivity for circulating blood leukocytes, and thus to localize leukocyte-vessel wall interactions at sites of inflammation in vivo.

Cell Adhesion↗
PubMed1985

Interleukin-1 activation of vascular endothelium. Effects on procoagulant activity and leukocyte adhesion.

Interleukin-1 (IL-1), an inflammatory/immune mediator, acts directly and selectively on cultured human vascular endothelial cells to alter two important functional properties. First, IL-1 induces endothelial cell biosynthesis and surface expression of a tissue factor-like procoagulant activity. Second, IL-1 dramatically increases the adhesiveness of the endothelial cell surface for human peripheral blood polymorphonuclear leukocytes (6-42-fold increase) and monocytes (2-5-fold increase), as well as the related leukocyte cell lines HL-60 and U937. These IL-1 effects are concentration-dependent (maximum, 5-10 U/ml), time-dependent (peak 4-6 hours), and reversible. Cycloheximide and actinomycin D block these IL-1 actions on endothelium, which suggests the requirement for de novo protein synthesis. Human-monocyte-derived IL-1, cell-line--derived IL-1, and recombinant IL-1 exhibited comparable biologic activities in our assays, whereas two other mediators, IL-2 and immune interferon, were without effect. IL-1 stimulated procoagulant activity and leukocyte adhesion in human endothelial cells cultured from both umbilical veins and adult saphenous veins but not in other cultured cell types, including SV-40-transformed human endothelial cells and human dermal fibroblasts. Similar actions of IL-1 on vascular endothelium in vivo may contribute to the development of intravascular coagulation and enhanced leukocyte--vessel wall adhesion at sites of inflammation.

Adhesiveness↗
PubMed1984

Interleukin 1 (IL-1) induces biosynthesis and cell surface expression of procoagulant activity in human vascular endothelial cells.

Human monocyte-derived interleukin 1 (IL-1) was found to be a potent inducer of procoagulant activity in cultured human vascular endothelium. IL-1-induced human umbilical vein endothelial cell procoagulant activity (HEC-PCA) was transiently expressed, manifest in intact cell monolayers, and required protein synthesis. Data obtained with coagulation factor-deficient plasma and a goat anti-human apoprotein III antiserum suggested that most, if not all, of IL-1-induced endothelial cell procoagulant activity is tissue factor-like. IL-1 induction of HEC-PCA may be important in the pathogenesis of intravascular coagulation in a variety of immunological and inflammatory conditions.

Blood Coagulation↗
PubMed1984

Interactions of T lymphocytes with human vascular endothelial cells: role of endothelial cells surface antigens.

We have studied the interactions of peripheral blood T lymphocytes with cultured human vascular endothelial cells, focusing upon endothelial cell surface antigens important for T cell recognition. Under standard culture conditions endothelial cells express class I but not class II major histocompatibility complex (MHC) antigens. However, class II antigens may be induced by activated T cells or T cell products, including the lymphokine immune interferon. Immune interferon concomitantly increases class I antigen expression and causes a change in cell shape. In addition to vascular endothelial cells, we have found that vascular smooth muscle cells and human dermal fibroblasts may also be induced by immune interferon to express class II antigens. All known human class II antigens are induced (i.e. HLA-DR, DC and SB) as is the associated invariant chain. Induced antigen expression in these cells is stable over several days, although mRNA levels decline rapidly upon withdrawal of interferon. Vascular and stromal cell class II antigens are functional, in that they can be recognized by cytolytic and helper T cell clones. Several non-MHC antigens are also involved in the recognition of endothelial and stromal cells by T cells. We propose a model for the role of inducible class II molecules on endothelium and stromal cells in vivo: The induction of class II MHC antigens on endothelial cells, locally mediated by activated T cells, enables endothelium to present an immunogenic cell surface structure, comprised of antigen plus self class II polymorphic determinants, which in turn, serves to recruit additional antigen-specific T cells from the circulation into the site of a developing cell mediated immune response. Class II molecules on stromal cells, also induced locally at the site of a developing response, confers immune accessory function on these cells and may serve to augment and sustain a T cell response.

Antigen-Presenting Cells↗
PubMed1984

Immune interferon activates multiple class II major histocompatibility complex genes and the associated invariant chain gene in human endothelial cells and dermal fibroblasts.

Immune interferon (IFN-gamma) increases the surface expression of HLA-A,B antigens and induces the surface expression of HLA-DR antigens on vascular endothelial cells and dermal fibroblasts. Here we report that IFN-gamma induces parallel expression of two other class II major histocompatibility complex (MHC) antigens, SB and DC. Maximal surface expression of all three antigens is reached in 4-6 days, and HLA-DR and -SB are induced to a higher level of expression than HLA-DC. For all three class II antigens, induction is marked by the de novo appearance of detectable transcripts of class II heavy and light chains and of the non-MHC-encoded invariant chain, suggestive of the transcription of multiple previously silent genes. Class I message levels and antigen expression are also increased by IFN-gamma at similar rates but from initial levels that are 50% of maximal. After removal of IFN-gamma, class II antigen expression persists for at least 4 days, while mRNA levels decrease rapidly. The parallel induction and persistence of the several class II MHC antigens may be important in conferring immune accessory function on vascular and stromal cells.

Endothelium↗
PubMed1984

Human cytolytic T lymphocyte interactions with vascular endothelium and fibroblasts: role of effector and target cell molecules.

Monoclonal antibodies (mAb) against cell surface structures have been used to identify several molecules involved in the interaction of human cytolytic T lymphocytes (CTL) with lymphoid and other bone marrow-derived targets. In allograft rejection or in graft-vs-host disease, however, major cellular targets are vascular and stromal cells, especially endothelium. Yet little is known about whether the same cell surface molecules are involved in the interactions of CTL with these cell types. We assessed the ability of mAb against effector or target cell structures to inhibit cytolysis of susceptible, cultured human vascular endothelium or dermal fibroblasts by a cloned human CTL line. Using mAb reactive with T3, T4, LFA-1, LFA-2, LFA-3, and HLA-DR, we found a qualitatively similar but quantitatively different pattern of inhibition of cytolysis as previously established for lymphoid targets by using the same CTL clone. These results have two implications: 1) the target cell structures recognized by CTL molecules such as T4, LFA-1 and LFA-2 are present on diverse cell types; and 2) the relative importance of such interactions may vary with target cell type. Furthermore, our studies provide several insights into the mechanisms of the interacting molecules. Our model system, and the use of pathophysiologically important target cells, may be useful for further analysis of CTL-mediated immune injury.

Antibodies, Monoclonal↗
PubMed1983

Ia expression by vascular endothelium is inducible by activated T cells and by human gamma interferon.

We have used monoclonal antibody binding, measured by radioimmunoassay, fluorescence flow cytometry, and ultrastructural immunocytochemistry, to measure expression of Ia antigens on cultured human umbilical vein endothelial (HUVE) cells. Under standard culture conditions, HUVE cells do not express Ia antigens. However, treatment of primary HUVE cultures with phytohemagglutinin induces the expression of Ia antigens. Every endothelial cell in the culture becomes Ia-positive and endothelial cells appear to synthesize Ia. HLA-A,B is concomitantly increased. The expression of Ia appears to be mediated by T cells because (a) pretreatment of primary HUVE cultures with OKT3 plus complement blocks the action of the lectins but not of medium conditioned by lectin-activated peripheral blood mononuclear cells; (b) co-culture of endothelial cells with allogeneic T cells, in the absence of lectin, also induces endothelial Ia; and (c) human immune (gamma) interferon, produced by Chinese hamster ovary cells transfected with the human gamma interferon gene, directly induces endothelial Ia. During co-culture with lymphocytes, about one-third of the endothelial cells are Ia-positive after 24 h and all of the endothelial cells are Ia-positive by 72 h. Proliferation of allogeneic T cells starts by 96 h and peaks at 144 h. Thus, endothelial Ia appears sufficiently early to be a determinant for the proliferation of allogeneic T cells. Inducible expression of Ia by endothelium may be important both for allograft rejection and for recruitment of circulating T cells into the site of an immune response.

Cells, Cultured↗
PubMed1982

Expression of Ia-like antigens by human vascular endothelial cells is inducible in vitro: demonstration by monoclonal antibody binding and immunoprecipitation.

The expression of Ia-like antigens by cultured human endothelial cells has been investigated by means of monoclonal antibody binding to intact cells and by immunoprecipitation of radioiodinated membrane proteins. Primary growing and confluent cultures of human umbilical vein endothelium express little, if any, detectable Ia-like antigens under standard culture conditions. However, treatment of primary cultures with the lectin phytohemagglutinin induces the expression of Ia-like antigens. This action of the lectin uniformly affects all the endothelial cells in a culture, does not depend on cell division, and is associated with a cell shape change. The data presented in this report provide unequivocal serological and biochemical demonstration of Ia-like antigens on human vascular endothelial cells. The fact that the expression of Ia-like antigens by endothelium can be induced may have important implications for organ transplantation and for regulation of the immunological response.

Antibodies, Monoclonal↗