Allogeneic induction of the human T cell-instructed monocyte procoagulant response is rapid and is elicited by HLA-DR.

The recognition of alloantigens by human lymphoid cells initiates a collaborative cellular pathway that rather rapidly induces in adherent cells (monocytes) the synthesis and expression of cell surface tissue factor, the initiating cofactor of the extrinsic coagulation pathway. This response was vigorous, generating tissue factor to a level nearly comparable to the response to endotoxin. However, it was temporally discordant with characterized lymphoid procoagulant responses to endotoxin, virus, and immune complexes in that it reached a maxima at 48 h, well after these other responses but clearly much faster than the well recognized proliferative responses to allogeneic stimulation. Using the Daudi lymphoblastoid B cell line, the allogeneic response could be fully elicited in a dose-dependent fashion within 18 h. The induction of monocyte tissue factor required collaboration with T lymphocytes, in accord with previously described T cell-instructed monocyte responses. HLA-DR was implicated as the allogeneic signal by the ability of two monoclonal antibodies to completely block, in a dose-dependent fashion, the induction of this pathway. Notably, the allogeneic procoagulant response was quantitatively discordant with respect to the allogeneic proliferative response, suggesting differences in specificity. This relatively rapid response may be applicable to typing of determinants in the major histocompatibility complex that are not equivalently identified by alternative analyses, and may be significant in tissue transplantation. The cellular pathway, linking allogeneic recognition with induction of a monocyte response that initiates the coagulation pathway, represents a further example of the linkage between these biologic systems, and is consistent with a pathogenetic role in allograft rejection by the promotion of vascular thrombosis and interstitial fibrin accumulation.

Lymphocyte cooperation is required for amplification of macrophage procoagulant activity.

Murine splenic lymphoid cells have been shown to possess basal procoagulant activity. This activity was localized to most macrophages by assay of cell populations, as well as by a direct plaque assay that permitted identification of expressed procoagulant activity of individual viable cells as well as content. Both content and viable expression of procoagulant activity was markedly increased by exposure to bacterial lipopolysaccharide, reaching a maximum after 6 h. The quantitative increase in procoagulant activity content and viable expression was limited to the macrophage population. Separated populations of lymphocytes or macrophages could not be stimulated by lipopolysaccharide to increase procoagulant activity, whereas the addition of lymphocytes to macrophages at a 3-4:1 ratio maximally reconstituted the amplification of procoagulant activity. Further evidence of cellular collaboration followed from observation that only lymphocytes that had been exposed to lipopolysaccharide were capable of triggering the increase in macrophage procoagulant activity. This appears to represent a new form of lymphocyte-macrophage cooperation in an effector pathway that may participate in some forms of immunologic responses and contribute to the phenotypic features of certain immunologic tissue lesions.

Lipopolysaccharide-mediated transcriptional activation of the human tissue factor gene in THP-1 monocytic cells requires both activator protein 1 and nuclear factor kappa B binding sites.

Lipopolysaccharide (LPS) activation of cells of monocytic lineage leads to rapid and transient expression of a set of inflammatory gene products, including tissue factor (TF). This transmembrane receptor is the major cellular initiator of the blood coagulation cascades, and induced expression of TF is postulated to play a role in inflammation. Functional studies using transfected THP-1 monocytic cells revealed the presence of a 56-bp LPS response element (LRE) within the TF promoter that conferred LPS responsiveness to a heterologous promoter. LPS stimulation of these cells activated proteins that bound to nucleotide sequences within the LRE resembling consensus binding sites for activator protein 1 (AP-1) and nuclear factor kappa B (NF-kappa B). Induction of the TF gene may represent a prototypic example of gene activation in monocytic cells by assembly of transcription factor complexes, and may clarify the role of AP-1 and NF-kappa B in the regulation of other LPS-responsive genes.

Lymphocyte E rosette inhibitory factor: a regulatory serum lipoprotein.

Rosette inhibitory factor, RIF, previously described in serum from patients with hepatitis B virus infection, has been isolated and identified as a minor species of beta-lipoprotein of the low-density (LDL) class. It is unrelated to hepatitis B virus proteins or particles. Although discrete by reference to charge and density (1.050 +/- 0.004 g/cm3), RIF appears to be a complex macromolecular structure containing apolipoproteins A, B, and C. Greater than 400% recovery is achieved upon 300,000-fold purification from RIF+ sera suggesting activation of a precursor form that is not present in normal serum. RIF inhibits E rosette function of T lymphocytes in vitro with a lag period of approximately 4 h and maximal effect at 24 h consistent with a metabolically-induced event. RIF is functionally active at concentrations of 1 X 10(-12) M or greater, rapidly binds to lymphocytes, and has a functionally effective half-life of approximately 1.5 h. Approximately 2,900 receptors for RIF appear to be present per cell and a high mutual affinity is apparent (k approximately to 9 X 10(10) liters/mol). RIF has no detectable effect on mitogen (PHA) responsiveness of lymphocytes, but inhibits the capacity of lymphocytes to respond to histoincompatible cells in vitro at concentrations greater than 10(-8) M. Equivalent RIF- lipoprotein fractions from normal serum are equally inhibitory in the mixed lymphocyte reaction suggesting that this effect is not directly attributable to RIF activity. These data indicate that RIF is a unique and functionally specific species of LDL that represents either an association complex of lipoproteins or a hybrid molecule of unusual composition. The association of this factor with viral-induced hepatocellular injury underscores the need to elucidate its structure and function in greater detail.

Factor VIII coagulant activity and factor VIII-like antigen: independent molecular entities.

Factor VIII coagulant activity (VIII(C)) and the von Willebrand's disease antigen (Factor VIII-like antigen, vW-Ag) are biologically linked, and it has been suggested that they reside on the same molecule. However, insolubilized human isoantibody to VIII(C) and rabbit antiserum containing antibodies to VIII(C) and vW-Ag differentially bind and remove these entities from plasma, thus physically segregating one from the other. These findings indicate that Factor VIII coagulant activity resides on a molecule distinct from that expressing the von Willebrand's antigen.

Intrahepatic expression of serum hepatitis virus-associated antigens.

SH virus-associated antigens have been identified in the liver and blood by immunofluorescence. This antigenic expression of the hepatitis agent or its genome is found in the cytoplasm of hepatocytes and other cells of the liver. Acute hepatitis is associated with hepatocellular localization of SH antigen, whereas alternative or additional SH antigen in the cytoplasm of nonhepatocytes is observed in chronic or recurrent hepatitis. Persistent intrahepatic SH antigen has also been observed in conversion from acute to chronic hepatitis, and the persistence of this virus-associated antigen in the liver may serve a significant pathogenetic role in mediating direct anti-tissue responses as well as local immune complex reactions. Competitive inhibition studies of SH antigen suggest at least two antigenically and sterically independent determinants on the SH particle.

Immune complex-induced human monocyte procoagulant activity. I. a rapid unidirectional lymphocyte-instructed pathway.

It has previously been described that soluble antigen:antibody complexes in antigen excess can induce an increase in the procoagulant activity of human peripheral blood mononuclear cells. It has been proposed that this response may explain the presence of fibrin in immune complex-mediated tissue lesions. In the present study we define cellular participants and their roles in the procoagulant response to soluble immune complexes. Monocytes were shown by cell fractionation and by a direct cytologic assay to be the cell of origin of the procoagulant activity; and virtually all monocytes were able to participate in the response. Monocytes, however, required the presence of lymphocytes to respond. The procoagulant response required cell cooperation, and this collaborative interaction between lymphocytes and monocytes appeared to be unidirectional. Lymphocytes once triggered by immune complexes induced monocytes to synthesize the procoagulant product. Intact viable lymphocytes were required to present instructions to monocytes; no soluble mediator could be found to subserve this function. Indeed, all that appeared necessary to induce monocytes to produce procoagulant activity was an encounter with lymphocytes that had previously been in contact with soluble immune complexes. The optimum cellular ratio for this interaction was four lymphocytes per monocyte, about half the ratio in peripheral blood. The procoagulant response was rapid, reaching a maximum within 6 h after exposure to antigen:antibody complexes. The procoagulant activity was consistent with tissue factor because Factors VII and X and prothrombin were required for clotting of fibrinogen. WE propose that this pathway differs from a number of others involving cells of the immune system. Elucidation of the pathway may clarify the role of this lymphocyte-instructed monocyte response in the Shwartzman phenomenon and other thrombohemorrhagic events associated with immune cell function and the formation of immune complexes.

Human T lymphocyte "E" rosette function. I. A process modulated by intracellular cyclic AMP.

The capacity of normal human T lymphocytes to form rosettes with sheep red blood cells can be inhibited by drugs or agents which induce elevations in intracellular levels of cyclic AMP. The effect is early in the presence of agents which elicit rapid elevations in intracellular cyclic AMP (isoproterenol, aminophylline) and occurs later in the presence of cholera toxin which induces a dalayed increase in endogenous cyclic AMP. Dibutyryl cyclic AMP is inhibitory, and the effects of dibutyryl cyclic AMP and the adenyl cyclase stimulators are potentiated by inhibition of phosphodiesterase. These data provide substantial evidence that elevation of intracellular cyclic AMP diminishes E rosette function of lymphocytes.

Induction of monocyte procoagulant activity by murine hepatitis virus type 3 parallels disease susceptibility in mice.

The in vitro induction of procoagulant activity (PCA) in murine peripheral blood mononuclear cells (PBM) by murine hepatitis virus type 3 (MHV-3) correlates with the disease susceptibility in three strains of mice. PBM from BALB/c mice, a strain in which MHV-3 infection results in fatal acute fulminant hepatitis, responds to the virus with a robust PCA response, whereas PBM from C3H/St mice, a strain which develops mild acute hepatitis followed by chronic hepatitis, only exhibit a modest PCA response. In contrast, PBM from A/J mice, a strain fully resistant to MHV-3, generate no increase in PCA above control levels. The induction phase of MHV-3 PCA is rapid, with an increase within 1-1.5 h, with maximum activity at 18h, and it precedes MHV-3 replication in either 17 CL1 cells, a fully permissive cell line, or in monocytes from these strains of mice. The PCA response of BALB/c PBM exceeds the response to any other known stimulus. No induction occurs upon direct stimulation of monocytes by MHV-3, but in the presence of lymphocyte collaboration, the PCA response is observed first at a lymphocyte:monocyte ratio of 2:1 and reaches a maximum as the lymphocyte:monocyte ratio approaches 4:1. This response appears to provide a functional marker for susceptibility to MHV-3 infection in inbred strains of mice and could be important in the pathogenesis of MHV-3-induced disease.

Autologous immune complex nephritis induced with renal tubular antigen. I. Identification and isolation of the pathogenetic antigen.

The nephritogenic antigen, responsible for the immunogenic stimulus in experimental allergic glomerulonephritis induced with tubular antigen, has been identified as a renal tubular epithelial (RTE)-specific antigen and has been isolated in a relatively purified form. This antigen, RTE-alpha(5), is a distinct and antigenically specific lipoprotein of high density which is derived primarily from the brush border of proximal convoluted tubular epthelium of the rat kidney. It has been suggested that this molecule may be a plasma membrane subunit. Immunization of rats with as little as 3 microg N of RTE-alpha(5) in complete Freund's adjuvant has effectively induced this form of membranous glomerulonephritis. RTE-alpha(5) is not a constituent of normal rat glomeruli; however, with the onset of autologous immune complex nephritis it is deposited in a granular fashion along glomerular capillary walls indistinguishable from the deposits of gamma-globulin and complement. The antigenic specificity of this antigen and its tissue derivation has been explored, and the observations support the autologous immune complex pathogenesis of the glomerulonephritis induced in rats by immunization with renal tubular antigen.

Autologous immune complex nephritis induced with renal tubular antigen. II. The pathogenetic mechanism.

The pathogenetic mechanism involved in a form of experimental allergic glomerulonephritis induced by immunization of rats with renal tubular antigen has been investigated. A single immunization with less than a milligram of a crude renal tubular preparation, probably containing less than 25 microg of the specific nephritogenic antigen, is effective in the induction of this form of chronic membranous glomerulonephritis. In the nephritic kidney autologous nephritogenic tubular antigen is found in the glomerular deposits along with gamma-globulin and complement. When large amounts of antigen are injected during induction of the disease the exogenous immunizing antigen can also be detected in the glomerular deposits. It appears that this disease results from the formation of circulating antibodies capable of reacting with autologous renal tubular antigen(s) and the deposition of these antibodies and antigen(s) plus complement apparently as immune complexes in the glomeruli. This pathogenetic system has been termed an autologous immune complex disease and the resultant glomerulonephritis has been similarly designated.

Murine lymphoid procoagulant activity induced by bacterial lipopolysaccharide and immune complexes is a monocyte prothrombinase.

Murine lymphoid cells respond rapidly to bacterial lipopolysaccharide or antigen-antibody complexes to initiate or accelerate the blood coagulation pathways. The monocyte or macrophage has been identified as the cellular source, although lymphocyte collaboration is required for the rapid induction of the procoagulant response. This procoagulant activity is identified in the present study as a direct prothrombin activator, i.e., a prothrombinase. Studies with plasmas deficient in single coagulation factors demonstrate that the induced murine procoagulant activity effector molecule does not require factors XII, VIII, VII, X, or V, but does require prothrombin to transform fibrinogen to fibrin. This enzyme(s) produces limited proteolysis of prothrombin to yield thrombin or thrombinlike products that are functionally capable of converting fibrinogen to fibrin. The prothrombinase is undetectable in freshly isolated Murine lymphoid cells respond rapidly to bacterial lipopolysaccharide or antigen-antibody complexes to initiate or accelerate the blood coagulation pathways. The monocyte or macrophage has been identified as the cellular source, although lymphocyte collaboration is required for the rapid induction of the procoagulant response. This procoagulant activity is identified in the present study as a direct prothrombin activator, i.e., a prothrombinase. Studies with plasmas deficient in single coagulation factors demonstrate that the induced murine procoagulant activity effector molecule does not require factors XII, VIII, VII, X, or V, but does require prothrombin to transform fibrinogen to fibrin. This enzyme(s) produces limited proteolysis of prothrombin to yield thrombin or thrombinlike products that are functionally capable of converting fibrinogen to fibrin. The prothrombinase is undetectable in freshly isolated

Monocytes can be induced by lipopolysaccharide-triggered T lymphocytes to express functional factor VII/VIIa protease activity.

In the present study we demonstrate that human monocytes can be induced by the model stimulus, lipopolysaccharide (LPS), to produce and assemble on their surface functional Factor VII/VIIa. This protease was not induced in relatively purified monocytes alone following exposure to LPS; but was induced in the presence of Leu-3a positive helper/inducer T cells. The Factor VII/VIIa protease activity represented 35-40% of the potential initiating activity for the extrinsic coagulation pathway and was demonstrated using functional coagulation assays, as well as in amidolytic assays for the activation of Factor X. This activity of cell-bound Factor VII/VIIa appeared to involve a tight adduct of calcium. The identity of the Factor X-activating protease as Factor VII/VIIa was confirmed by the capacity of antibody specific for Factor VII/VIIa to neutralize the cell-bound protease. Further propagation of the extrinsic pathway following generation of Factor Xa required addition of exogenous Factor Va. These results expand the repertoire of proteases that have been identified with appropriately triggered cells of the monocyte/macrophage series, and suggest that initiation and propagation of the extrinsic coagulation protease network on induced monocytes involves not only expression of the initiating cofactor molecule, tissue factor, but also production of Factor VII and its organization into the molecular assembly. Thus, in the absence of exogenous Factor VII/VIIa a directly proteolytic effector cell can be generated. Further molecular assembly of the extrinsic pathway on the monocyte surface sequentially expands the proteolytic capacity of this response. The synthesis and assembly of the extrinsic activation complex by the monocyte and its derived progeny, the macrophage, provides a mechanism by which coagulation is initiated under T cell instruction at sites of immunologic responses.

Oligospecificity of the cellular adhesion receptor Mac-1 encompasses an inducible recognition specificity for fibrinogen.

Mitogenesis, cellular aggregation, and motility follow upon the interaction of fibrinogen with certain defined cell surface receptors. In addition to circulating platelets and vascular endothelium, monocytes express what appears to be a receptor for fibrinogen. Evidence is presented here that the leukocyte adhesion receptor Mac-1 can be specifically induced to bind fibrinogen with characteristics immunochemically and functionally distinct from the established Arg-Gly-Asp-directed fibrinogen receptors. The competence of Mac-1 as a fibrinogen receptor is a general property of cells of monocyte and myeloid lineage acquired after maturational changes of some regions of the alpha subunit of Mac-1 during the process of cell differentiation. This ligand recognition specificity of Mac-1 is lacking for the resting cell. Rather, induction of fibrinogen binding capacity of Mac-1 is due to a cellular response to selected agonists characterized by inducing rapid transients of cytosolic Ca2+. Although different in activation pathways and recognition specificity, Mac-1 exhibits an oligospecific ligand versatility characteristic of other homologous Arg-Gly-Asp-directed adhesion receptors.

Functional tissue factor is entirely cell surface expressed on lipopolysaccharide-stimulated human blood monocytes and a constitutively tissue factor-producing neoplastic cell line.

Tissue factor (TF) is an integral membrane glycoprotein which, as the receptor and essential cofactor for coagulation factors VII and VIIa (FVII and FVIIa, respectively), is the primary cellular activator of the coagulation protease cascade. Previous studies on the procoagulant activity of a variety of cell types (either lysed or in the intact state) have variously been interpreted as showing that TF is either stored intracellularly or is present in a cryptic form in the surface membrane. Using mAbs to TF, we have directly investigated the subcellular localization and functional activity of TF in lipopolysaccharide-stimulated blood monocytes and J82 bladder carcinoma cells. Blocking of surface TF of viable cells with inhibitory anti-TF mAbs abolished greater than 90% of TF activity of the intact cells as well as of lysed cells. Furthermore, quantitative analysis of the binding of FVII and anti-TF mAb to J82 cells demonstrated that all surface-expressed TF molecules were capable of binding the ligand, FVII. By immunoelectron microscopy, TF was present only in the surface membrane of monocytes and J82 cells, although the latter also contained apparently inactive TF antigen in multivesicular bodies. On the intact cell surface the catalytic activity of the TF-FVIIa complex was investigated and found to be markedly less relative to cell lysates. Membrane alterations that affect the cofactor activity of TF may be a means of regulating the extent of initiation of the coagulation protease cascade in various cellular settings.

Autologous immune-complex pathogenesis of experimental allergic glomerulonephritis.

A renal tubular epithelial antigen is deposited in association with gamma globulin and complement in glomeruli from rats with experimental allergic glomerulonephritis induced by immunization with renal tubular antigens. Apparently, in normal kidneys this antigen is concentrated in the distal segment of the proximal convoluted tubular epithelium, and the principal source of this antigen in the glomerular deposits is autologous. This form of glomerulonephritis provides an experimental prototype for what may be termed "autologous immunecomplex" diseases.

Tumor infarction in mice by antibody-directed targeting of tissue factor to tumor vasculature.

Selective occlusion of tumor vasculature was tested as a therapy for solid tumors in a mouse model. The formation of blood clots (thrombosis) within the tumor vessels was initiated by targeting the cell surface domain of human tissue factor, by means of a bispecific antibody, to an experimentally induced marker on tumor vascular endothelial cells. This truncated form of tissue factor (tTF) had limited ability to initiate thrombosis when free in the circulation, but became an effective and selective thrombogen when targeted to tumor endothelial cells. Intravenous administration of the antibody-tTF complex to mice with large neuroblastomas resulted in complete tumor regressions in 38 percent of the mice.

Structurally homologous ligand binding of integrin Mac-1 and viral glycoprotein C receptors.

Three spatially distant surface loops were found to mediate the interaction of the coagulation protein factor X with the leukocyte integrin Mac-1. This interacting region, which by computational modeling defines a three-dimensional macromotif in the catalytic domain, was also recognized by glycoprotein C (gC), a factor X receptor expressed on herpes simplex virus (HSV)-infected endothelial cells. Peptidyl mimicry of each loop inhibited factor X binding to Mac-1 and gC, blocked monocyte generation of thrombin, and prevented monocyte adhesion to HSV-infected endothelium. These data link the ligand recognition of Mac-1 to established mechanisms of receptor-mediated vascular injury.

A large noncoding RNA is a marker for murine hepatocellular carcinomas and a spectrum of human carcinomas.

Tumor markers can facilitate understanding molecular cell biology of neoplasia and provide potential targets for the diagnosis and insight for intervention. We here identify a novel murine gene, hepcarcin (hcn), encoding a 7-kb mRNA-like transcript. The gene appears to be the murine ortholog of the human alpha gene, that is, MALAT-1. The gene and homologs lack credible open reading frames, consistent with a highly conserved large noncoding RNA (ncRNA). In all nodules of procarcinogen-induced murine hepatocellular carcinomas (HCCs) and human HCCs, expression was markedly elevated compared to the uninvolved liver. Quantitative analyses indicated a 6-7-fold increased RNA level in HCCs versus uninvolved liver, advancing this as a molecule of interest. This ncRNA was overexpressed in all five non-hepatic human carcinomas analysed, consistent with a potential marker for neoplastic cells and potential participant in the molecular cell biology of neoplasia.

Coupling of the adhesive receptor CD11b/CD18 to functional enhancement of effector macrophage tissue factor response.

Initiation and regulation of localized selective proteolysis is an important effector property of cells of macrophage (Mo) lineage. Among such effector responses is the induced expression of tissue factor (TF) by cells of Mo lineage. In characterizing the regulation of the Mo responses that may influence the magnitude of the effector phase of the cellular immune response, we have identified a role for the cell surface adhesive receptor CD11b/CD18 (Mac-1, CR3) to amplify the induced TF response. Occupancy of CD11b/CD18 by MAb as surrogate ligands does not directly initiate a TF response. In contrast, after either T cell-derived cytokine or LPS as initial signals, engagement of CD11b/CD18 by MAb induces a two- to eight-fold functional enhancement of the TF response in murine and human Mo. This pathway of CD11b/CD18 enhancement of this Mo effector response was also confirmed with recognized ligands for CD11b/CD18 by exposure of Mo to immobilized fibrinogen. A quantitative increase of Mo surface expression of TF was validated by flow cytometry. We suggest that engagement of CD11b/CD18 by complementary ligands including adherence to extracellular matrix, and possibly in antigen-driven TH:Mo collaborative responses, results in the transduction of cellular signals that quantitatively enhance the expression of TF per se and thereby enhance the inflammatory component of Mo mediated response.