Anthrax toxin receptor 1/tumor endothelium marker 8 mediates cell spreading by coupling extracellular ligands to the actin cytoskeleton

Tumor endothelial marker 8 (TEM8) is induced in tumor-associated vasculature and acts as a receptor for Protective Antigen (PA), the cell-binding component of the anthrax toxin determinant for toxin entrance into cells. However, the normal function for TEM8 remains unknown. We show that TEM8 functions as an adhesion molecule mediating cell spreading on immobilized PA and collagen I. The mechanism for TEM8 interaction with collagen I was cell type-specific, because binding to collagen I was abrogated by beta1 integrin function blocking antibody in HEK293 cells, but not in primary synovial rabbit fibroblasts. Binding to PA remained unaffected by the addition of beta1 integrin function blocking antibody. Whereas the extracellular and transmembrane domains of TEM8 were sufficient to provide cell attachment, the intracellular domain was critical for spreading. Fusion of the cytosolic domain of TEM8 to the IL-2 receptor, conferred cell-spreading capability on IL-2 receptor antibody substrates. The cytoplasmic domain mediated linkage with the actin cytoskeleton as it co-precipitated actin and determined partitioning of TEM8 to the actin-containing detergent insoluble cellular fraction. TEM8 anchorage to actin was relevant as spreading was inhibited by the cytoskeleton-disrupting drug cytochalasin D, but persisted in the presence of the microtubule-depolymerizing drug nocodazole, and in cells lacking intermediate filaments. Thus, our results indicate that TEM8 is a new adhesion molecule linking collagen I or PA to the actin cytoskeleton.     

A DC-specific cytolytic T lymphocyte line is OKT8+1

A human cytotoxic T lymphocyte (CTL) line, A9, was generated by limiting dilution and was selected because of its apparent DC specificity. A9 is 100% OKT3+, 90% OKT4+, and 10% OKT8+, but by negative selection the CTL present are entirely OKT8+. These OKT8+ CTL are totally inhibitable by Genox 3.53, an anti-DC1 monoclonal antibody (mAb), and Leu-10, an anti-DC subgroup mAb, but are not inhibitable by a panel of anti-HLA-DR mAb. These CTL are also inhibitable by anti-OKT3 and anti-LFA-2 but not by OKT4 or OKT8 mAb. These findings extend previous studies that showed that OKT8+ CTL recognize HLA-A,B antigens, whereas OKT4+ CTL recognize HLA-DR and SB antigens. It is possible that an as yet undefined T cell surface molecule is involved in DC recognition.     

Selective blockade of tumor angiogenesis

Blocking tumor angiogenesis is an important goal of cancer therapy, but clinically approved anti-angiogenic agents suffer from limited efficacy and adverse side effects, fueling the need to identify alternative angiogenesis regulators. Tumor endothelial marker 8 (TEM8) is a highly conserved cell surface receptor overexpressed on human tumor vasculature. Genetic disruption of Tem8 in mice revealed that TEM8 is important for promoting tumor angiogenesis and tumor growth but dispensable for normal development and wound healing. The induction of TEM8 in cultured endothelial cells by nutrient or growth factor deprivation suggests that TEM8 may be part of a survival response pathway that is activated by tumor microenvironmental stress. In preclinical studies, antibodies targeted against the extracellular domain of TEM8 inhibited tumor angiogenesis and blocked the growth of multiple human tumor xenografts. Anti-TEM8 antibodies augmented the activity of other anti-angiogenic agents, vascular targeting agents and conventional chemotherapeutic agents and displayed no detectable toxicity. Thus, anti-TEM8 antibodies provide a promising new tool for selective blockade of neovascularization associated with cancer and possibly other angiogenesis-dependent diseases.     

Selective blockade of tumor angiogenesis

Blocking tumor angiogenesis is an important goal of cancer therapy, but clinically approved anti-angiogenic agents suffer from limited efficacy and adverse side effects, fueling the need to identify alternative angiogenesis regulators. Tumor endothelial marker 8 (TEM8) is a highly conserved cell surface receptor overexpressed on human tumor vasculature. Genetic disruption of Tem8 in mice revealed that TEM8 is important for promoting tumor angiogenesis and tumor growth but dispensable for normal development and wound healing. The induction of TEM8 in cultured endothelial cells by nutrient or growth factor deprivation suggests that TEM8 may be part of a survival response pathway that is activated by tumor microenvironmental stress. In preclinical studies, antibodies targeted against the extracellular domain of TEM8 inhibited tumor angiogenesis and blocked the growth of multiple human tumor xenografts. Anti-TEM8 antibodies augmented the activity of other anti-angiogenic agents, vascular targeting agents and conventional chemotherapeutic agents and displayed no detectable toxicity. Thus, anti-TEM8 antibodies provide a promising new tool for selective blockade of neovascularization associated with cancer and possibly other angiogenesis-dependent diseases.     

Use of human CD3 monoclonal antibody for accurate CD4+ and CD8+ lymphocyte determinations in macaques: phenotypic characterization of the CD3- CD8+ cell subset

Macaque monkeys are frequently used in models for studies of infectious diseases, immunity, transplantation and vaccine development. Such use is largely due to the conservation of functionally important cell surface molecules and the phylogenetic proximity of their immune systems to that of humans. Some monoclonal antibodies (mAb) raised against human leukocyte antigens can be utilized in the monkey. Until recently, many primate centers have utilized the CD2 monoclonal antibody to enumerate T lymphocytes. We have evaluated the anti-human CD3 mAb in macaques and sooty mangabeys. Using this monoclonal antibody, pigtailed macaques were found to have a much higher proportion of CD2+ CD3- CD8+ cells as compared with rhesus macaques and sooty mangabeys. Such cells comprised approximately one-half of all CD8+ cells in the pigtailed macaque, but only one-quarter of CD8+ cells in the rhesus, and one-fifth in the sooty mangabey. Use of the CD2 monoclonal antibody as the T-cell marker resulted in underestimating CD4/CD8 ratios compared with using the CD3 mAb in pigtailed macaques. Phenotypic characterization of this subset of CD3- CD8+ cells indicated that they are CD16+, CD45RA+, CD11b+, CD69+ and CD28-. This would indicate that these cells represent an activated natural killer cell subset.     

Mitogen-like monoclonal anti-actin antibodies

Monoclonal antibodies (IgM kappa) have been produced to actin isolated electrophoretically from L cell extracts. These monoclonal anti-actin antibodies bind to intact L cells and modulate DNA synthesis and cell proliferation, much like affinity-purified polyclonal rabbit antibody to the same Mr 42,000 actin. In addition, monoclonal antibodies specific for actin from Entamoeba histolytica also bound to and modulated the growth of L cells. A monoclonal antibody directed against a neuroblastoma surface antigen did not produce stimulation of L cells, and the binding activity of anti-actin monoclonal antibody to L cells was removed by absorption with actin covalently coupled to Sepharose. These observations demonstrate the specificity of interaction between the anti-actin monoclonal antibodies and the surface of intact L cells. We conclude that a surface actin-like molecule on the L cell, when bound by specific monoclonal antibody, initiates a stimulatory signal which results in enhanced cellular metabolism.     

The human Lyt-3 molecule requires CD8 for cell surface expression.

We have previously identified a monoclonal antibody, T8/2T8-5H7, which clustered serologically with CD8 monoclonal antibodies, but lacked reactivity with L cell transfectants expressing the human CD8 molecule (Lyt-2 homologue). Based on these observations, we postulated that T8/2T8-5H7 might recognize the human Lyt-3 gene product. To test this hypothesis, we have isolated a full-length cDNA encoding the human Lyt-3 molecule and have characterized its product in additional transfection experiments. The results of these studies indicate that the human Lyt-3 cDNA encodes a product recognized by the antibody T8/2T8-5H7. Interestingly, the human Lyt-3 molecule cannot be expressed alone, but requires the human Lyt-2 homologue for efficient cell surface expression. A heterodimer composed of the human Lyt-2 and Lyt-3 molecules may have importance in T cell-target cell interactions.     

Integrins alpha v beta 3 and alpha v beta 5 contribute to cell attachment to vitronectin but differentially distribute on the cell surface

We investigated the role of the integrins alpha v beta 3 and alpha v beta 5 in mediating vitronectin adhesion of three phenotypically distinct cell types. M21 human melanoma cells and H2981 lung carcinoma cells use both alpha v-containing integrins in adhering to vitronectin while UCLA-P3 lung carcinoma cells adhere exclusively with alpha v beta 5. Specifically, monoclonal antibodies directed to functional epitopes on both receptors were required to block adhesion of M21 or H2981 cells while adhesion of UCLA-P3 cells to vitronectin could be blocked with a monoclonal antibody to alpha v beta 5. Although both receptors are involved in M21 and H2981 cell adhesion to vitronectin, only alpha v beta 3 can be detected in focal contacts, colocalizing with vinculin, talin, and the ends of actin filaments, while alpha v beta 5 shows a distinct, nonfocal contact, distribution on the cell surface. These results provide the first evidence that two homologous integrins that recognize the same ligand distribute differentially on the cell surface.     

An unusual dependence of human herpesvirus-8 glycoproteins-induced cell-to-cell fusion on heparan sulfate

Human herpesvirus-8 (HHV-8) is known to interact with cell surface heparan sulfate (HS) for entry into a target cell. Here we investigated the role of HS during HHV-8 glycoproteins-induced cell fusion. Interestingly, the observed fusion demonstrated an unusual dependence on HS as evident from following lines of evidence: (1) a significant reduction in cell-to-cell fusion occurred when target cells were treated with heparinase; (2) in a competition assay, when the effector cells expressing HHV-8 glycoproteins were challenged with soluble HS, cell-to-cell fusion was reduced; and, (3) co-expression of HHV-8 glycoproteins gH-gL on target cells resulted in inhibition of cell surface HS expression. Taken together, our results indicate that cell surface HS can play an additional role during HHV-8 pathogenesis.     

Sponge aggregation factor: identification of the specific collagen-binding site by means of a monoclonal antibody

The aggregation factor (AF) from the sponge Geodia cydonium is known to be a complex proteinaceous particle, composed of a series of different (glyco)proteins (Mr lower than 150,000) around a 90S sunburst-like core structure. One of the low-Mr proteins is the 47-KD cell binding fragment. We describe a new monoclonal antibody (mAb), III1E6, raised against purified AF particles, which recognizes in tissue slices structures present both on the plasma membrane and in a network-like manner in the extracellular space. By applying immunoelectron microscopical, immunoblotting, and immunoaffinity chromatographical techniques, the mAb III1E6 was shown to recognize the core structure of the AF particle. Cell adhesion studies revealed that the mAb does not inhibit AF mediated cell-cell adhesion but abolishes AF-caused attachment of cells to collagen. Electron microscopic data show that III1E6 prevents association of AF particles with collagen fibrils. By applying the techniques of immunoblotting and of protein-protein recognition on the solid phase in vitro, we could formulate the following series of events: the AF particle recognizes, with its 47-KD cell binding fragment, the aggregation receptor protein in the plasma membrane and with its core structure the collagen fibrils. These fibrils interact optionally, either via the same route or via the collagen assembly factor, with an adjacent cell surface. These findings demonstrate that the AF particle is not only the key molecule for cell-cell adhesion but also a component of cell-matrix interactions.     

Monoclonal anti-actin antibody recognizes a surface molecule on normal and transformed human B lymphocytes: expression varies with phase of cell cycle

A monoclonal anti-actin antibody, 2C9, was used to study the distribution of an actin-like cell-surface antigen (hereafter termed actin) on a lymphoblastoid cell line LA350 and on human peripheral blood lymphocytes. It was determined that 8-40% of LA350 cells and 3-15% of peripheral blood lymphocytes from healthy donors stain specifically with 2C9, almost exclusively on IgM-positive cells. Treatment of cells with 2C9 prior to incubation caused cell-surface actin to first patch and then to cap. Treatment of cells with nonspecific protease caused a loss of surface actin, with reexpression of the marker after 8-12 hr. The expression of LA350 surface actin also increased with DNA synthesis and was demonstrated to be maximal during late G1/early S phase. Thus, this antigen may be a sensitive marker for activated lymphocytes. These studies contribute to our understanding of the expression and distribution of actin-like membrane proteins that may participate in regulatory signals mediated by anti-actin antibody.     

Monoclonal antibody to a structure expressed on a subpopulation of rat CD8 T cell subsets.

We have developed a monoclonal antibody, RTS-1, which can divide a rat CD8 (+) peripheral T cell population into two functionally distinct subsets. The cell-surface structure defined by this antibody is a glycoprotein with a molecular weight of 220 kDa found to be a high molecular isoform of rat CD45 antigen. CD4 (+) T cells were not stained by RTS-1 antibody. The cytotoxic T cell-enriched population did not express RTS-1 epitope on the cell surface. CD8 (+) spleen cells as well as RTS-1(+)CD8(+)T cells exhibited strong inhibition on mitogen-induced immunoglobulin G production by rat B cells. Furthermore, RTS-1 antibody, but not the control antibody, abolished CD8(+)T cell-mediated inhibition of immunoglobulin G production by rat B cells. These data suggest that RTS-1 antibody recognizes a unique determinant of rat CD45 antigen that is expressed on a fraction of CD8(+) cells.     

Effector functions of CD8-positive guinea pig T lymphocytes

The response of guinea pig T lymphocytes to different stimuli was analysed with focus on the functions of CD8-positive T cells, which so far had been poorly defined in this animal model. For identification and purification of guinea pig cytotoxic T lymphocytes, three monoclonal antibodies, directed against the CD8 differentiation antigen were characterized and compared with respect to expression pattern and biochemical characteristics of the corresponding cell surface antigen. The antibodies were used for the identification of the cytotoxic T lymphocyte subpopulation within alloreactive T cell lines, and for the depletion of CD8-positive cells in in vitro assays. Purified CD4- and CD8-positive cells were tested for their ability to proliferate in response to antigen, mitogen or anti-guinea pig Thy-1 monoclonal antibodies. Both, CD4- and CD8-positive cells showed IL-2 release and subsequent proliferation after polyclonal stimulation. Cytotoxic activity in CD8-positive alloreactive T cells was expressed in vitro only after repeated stimulation.     

Structure and location of gene product 8 in the bacteriophage T4 baseplate

Many bacteriophages, such as T4, T7, RB49, and phi29, have complex, sometimes multilayered, tails that facilitate an almost 100% success rate for the viral particles to infect host cells. In bacteriophage T4, there is a baseplate, which is a multiprotein assembly, at the distal end of the contractile tail. The baseplate communicates to the tail that the phage fibers have attached to the host cell, thereby initiating the infection process. Gene product 8 (gp8), whose amino acid sequence consists of 334 residues, is one of at least 16 different structural proteins that constitute the T4 baseplate and is the sixth baseplate protein whose structure has been determined. A 2.0A resolution X-ray structure of gp8 shows that the two-domain protein forms a dimer, in which each monomer consists of a three-layered beta-sandwich with two loops, each containing an alpha-helix at the opposite sides of the sandwich. The crystals of gp8 were produced in the presence of concentrated chloride and bromide ions, resulting in at least 11 halide-binding sites per monomer. Five halide sites, situated at the N termini of alpha-helices, have a protein environment observed in other halide-containing protein crystal structures. The computer programs EMfit and SITUS were used to determine the positions of six gp8 dimers within the 12A resolution cryo-electron microscopy image reconstruction of the baseplate-tail tube complex. The gp8 dimers were found to be located in the upper part of the baseplate outer rim. About 20% of the gp8 surface is involved in contacts with other baseplate proteins, presumed to be gp6, gp7, and gp10. With the structure determination of gp8, a total of 53% of the volume of the baseplate has now been interpreted in terms of its atomic structure.     

Occurrence of alpha 2-8 linked polysialosyl units in a neural cell adhesion molecule

A brain cell surface protein (BSP-2) was isolated from mice of different ages by affinity chromatography using a monoclonal antibody. Analysis of glycopeptides obtained after pronase digestion revealed that the embryonal and neonatal forms of the antigen contained an unusually high proportion of sialic acid, which decreased during development. Methylation analysis of native and neuraminidase treated glycopeptides indicated that the sialic acid occurred as alpha 2-8 bound polysialosyl units, similar to those of the recently described developmentally regulated polysialosyl glycopeptides of rat brain. Furthermore, the carbohydrate and amino acid composition, and electrophoretic mobility of BSP-2 antigen correspond to those reported for a neural cell adhesion molecule (N-CAM).     

TEM8 expression stimulates endothelial cell adhesion and migration by regulating cell-matrix interactions on collagen

The TEM8 gene is selectively expressed in tumor versus normal blood vessels, though its function in endothelial cell biology is not known. Towards the goal of clarifying this function, we tested whether TEM8 overexpression, or blocking TEM8's function with a dominant negative protein, would modulate endothelial cell activities. We found that TEM8-expressing endothelial cells migrated at a rate 3-fold greater than control cells in a monolayer denudation assay. Also, the addition of recombinant TEM8 extracellular domain (TEM8-ED) specifically inhibited both chemokinetic and chemotactic migration on collagen in the denudation and Boyden chamber assays, respectively. The TEM8-ED binds preferentially to collagen, and TEM8 expression enhanced endothelial adhesion to collagen 3-fold; the latter response was antagonized by the TEM8-ED. Consistent with the TEM8-ED acting as a dominant negative inhibitor of endogenously expressed protein were data showing that the TEM8-ED had no effect on the activation of beta1 integrin. TEM8 protein is present in human umbilical vein in situ and is expressed in low passage HUVEC in vitro. TEM8 protein expression in HUVEC was increased 5-fold by the initiation of tube formation, correlating expression of TEM8 with the angiogenic response. Taken together, these results indicate that TEM8 plays a positive role in endothelial cell activities related to angiogenesis.     

Hepatocyte-derived cultured cells with unusual cytoplasmic keratin-rich spheroid bodies

Cytoplasmic inclusions are found in a variety of diseases that are characteristic morphological features of several hepatic, muscular and neurodegenerative disorders. They display a predominantly filamentous ultrastructure that is also observed in malignant rhabdoid tumor (MRT). A cellular clone containing an intracytoplasmic body was isolated from hepatocyte cell culture, and in the present study we examined whether this body might be related or not to Mallory-Denk body (MDB), a well characterized intracytoplasmic inclusion, or whether this cellular clone was constituted by malignant rhabdoid tumor cells. The intracytoplasmic body was observed in electron microscopy (EM), confocal immunofluorescence microscopy and several proteins involved in the formation of its structure were identified. Using light microscopy, a spheroid body (SB) described as a single regular-shaped cytoplasmic body was observed in cells. During cytokinesis, the SB was disassembled and reassembled in a way to reconstitute a unique SB in each progeny cell. EM examination revealed that the SB was not surrounded by a limiting membrane. However, cytoplasmic filaments were concentrated in a whorled array. These proteins were identified as keratins 8 and 18 (K8/K18), which formed the central core of the SB surrounded by a vimentin cage-like structure. This structure was not related to Mallory-Denk body or aggresome since no aggregated proteins were located in SB. Moreover, the structure of SB was not due to mutations in the primary sequence of K8/K18 and vimentin since no difference was observed in the mRNA sequence of their genes, isolated from Huh-7 and Huh-7w7.3 cells. These data suggested that cellular factor(s) could be responsible for the SB formation process. Aggregates of K18 were relocated in the SB when a mutant of K18 inducing disruption of K8/K18 IF network was expressed in the cellular clone. Furthermore, the INI1 protein, a remodeling-chromatin factor deficient in rhabdoid cells, which contain a spheroid perinuclear inclusion body, was found in our cellular clone. In conclusion, our data suggest that Huh-7w7.3 cells constitute an excellent model for determining the cellular factor(s) involved in the process of spheroid perinuclear body formation.     

Mouse Siglec-F and human Siglec-8 are functionally convergent paralogs that are selectively expressed on eosinophils and recognize 6'-sulfo-sialyl Lewis X as a preferred glycan ligand

Mouse sialic acid-binding immunoglobulin-like lectin F (Siglec-F) is an eosinophil surface receptor, which contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain, implicating it as a regulator of cell signaling as documented for other siglecs. Here, we show that the sialoside sequence 6'-sulfo-sLe(X) (Neu5Acalpha2-3[6-SO4] Galbeta1-4[Fucalpha1-3]GlcNAc) is a preferred ligand for Siglec-F. In glycan array analysis of 172 glycans, recombinant Siglec-F-Fc chimeras bound with the highest avidity to 6'-sulfo-sLe X. Secondary analysis showed that related structures, sialyl-Lewis X (sLe X) and 6-sulfo-sLe X containing 6-GlcNAc-SO4 showed much lower binding avidity, indicating significant contribution of 6-Gal-SO4 on Siglec-F binding to 6'-sulfo-sLe x. The lectin activity of Siglec-F on mouse eosinophils was "masked" by endogenous cis ligands and could be unmasked by treatment with sialidase. Unmasked Siglec-F mediated mouse eosinophil binding and adhesion to multivalent 6'-sulfo-sLe X structure, and these interactions were inhibited by anti-Siglec-F monoclonal antibody (mAb). Although there is no clear-cut human ortholog of Siglec-F, Siglec-8 is encoded by a paralogous gene that is expressed selectively by human eosinophils and has recently been found to recognize 6'-sulfo-sLe X. These observations suggest that mouse Siglec-F and human Siglec-8 have undergone functional convergence during evolution and implicate a role for the interaction of these siglecs with their preferred 6'-sulfo-sLe X ligand in eosinophil biology.     

In vivo treatment with anti-CD8 and anti-CD5 monoclonal antibodies alters induced tolerance to adjuvant arthritis

Resistance (low dose tolerance) to adjuvant arthritis was induced by intradermal immunization with 10 micrograms Mycobacterium tuberculosis administered 5 and 3 weeks before induction of arthritis. With the purpose of determining phenotypes of cells which participate in the maintenance of the induced resistance to adjuvant arthritis, tolerized rats were treated with two different anti-T-cell monoclonal antibodies. In tolerized rats, it was shown that anti-CD8 (OX8) antibodies, which caused an elimination of CD8+ lymphoid cells as determined by immunofluorescence analysis, made the rats responsive to an arthritogenic challenge with mycobacteria. Nine of 19 (47.4%) rats developed the disease as compared with 2 of 18 (11.1%) (P less than 0.05) in the control antibody-treated group. Also, in vivo treatment with anti-CD5 (OX19) monoclonal antibodies made the rats responsive to an arthritogenic challenge with mycobacteria. Nine of 15 (60%) anti-CD5-treated rats developed the disease as compared with 2 of 18 (11.1%) (P less than 0.01) rats in the control group. Immunofluorescence analysis performed after anti-CD5 treatment showed a reduction of staining of CD5+ cells as well as a down-regulation of the staining intensity of CD5 cell surface receptors on the remaining CD5+ cells. These data indicate that CD8+- as well as CD5+ cells participate in the maintenance of low dose tolerance to adjuvant arthritis.     

Impairment of protein trafficking by direct interaction of gliadin peptides with actin

Intestinal celiac disease (CD) is triggered by peptic-tryptic digest of gluten, known as Frazer's Fraction (FF), in genetically predisposed individuals. Here, we investigate the immediate effects of FF on the actin cytoskeleton and the subsequent trafficking of actin-dependent and actin-independent proteins in COS-1 cells. Morphological alterations in the actin filaments were revealed concomitant with a drastic reduction in immunoprecipitated actin from cells incubated with FF. These alterations elicit impaired protein trafficking of intestinal sucrase–isomaltase, a glycoprotein that follows an actin-dependent vesicular transport to the cell surface. However, the actin-independent transport of intestinal lactase phlorizin hydrolase remains unaffected. Moreover, the morphological alteration in actin is induced by direct interaction of this protein with gliadin peptides carrying the QQQPFP epitope revealed by co-immunoprecipitation utilizing a monoclonal anti-gliadin antibody. Finally, stimulation of cells with FF directly influences the binding of actin to Arp2. Altogether, our data demonstrate that FF directly interacts with actin and alters the integrity of the actin cytoskeleton thus leading to an impaired trafficking of intestinal proteins that depend on an intact actin network. This direct interaction could be related to the endocytic segregation of gliadin peptides as well as the delayed endocytic vesicle trafficking and maturation in gliadin-positive intestinal epithelial cells and opens new insights into the pathogenesis of CD.