Expression of the sialosyl-Tn epitope on CD45 derived from activated peripheral blood T cells

The cell surface protein tyrosine phosphatase CD45 is a major target of IgM anti-T cell autoantibodies in systemic lupus erythematosus (SLE). The autoreactive determinants on CD45 are O-linked glycans expressed on activated T cells and certain T cell lines, rather than linear or conformational polypeptide epitopes or N-linked glycans. To identify oligosaccharide structures that may play a role in the functional interactions of CD45 or are candidate target epitopes of SLE anti-CD45 autoantibodies, autoreactive CD45 purified from Jurkat T cells and non-autoreactive CD45 purified from CLL B cells were tested by ELISA for expression of mucin-type O-glycan structures. Monoclonal antibodies (mAbs) directed against blood group A, type 1 H chains, type 2 H chains, T, Le(a), sialylated-Le(a), Le(b), sialylated-Le(c), Le(x), sialylated-Le(x), multi-fucosylated Le(x), Le(y), and sialylated-extended Le(v) failed to react with CD45 from either B cells or T cells. However, mAbs directed against Tn (galNAcalpha1-->O-ser/thr) or sialosyl-Tn (neuNAcalpha2-6gaINAcalpha1-->O-ser/thr) structures reacted with CD45 derived from Jurkat T cells, but not from CLL B cells. Anti-Tn mAbs also reacted in western blotting procedures with CD45 isolated from Jurkat T cells, but did not react with CD45 isolated from CEM, MOLT-3, or PEER T cells; Daudi, Raji, or CLL B cells; or resting or Con A-activated PBL. However, anti-sialosyl-Tn mAbs stained blots of CD45 isolated from Jurkat and CEM T cells and Con A-activated PBL, a pattern of reactivity similar to that of the anti-CD45 autoantibodies. Flow cytometric analyses demonstrated that the sialosyl-Tn epitopes are expressed on a subpopulation of CD4 +/CD8- T cells.     

The development of anti-CD79 monoclonal antibodies for treatment of B-cell neoplastic disease

The B-cell antigen receptor (BCR) consists of cell surface IgM associated with the CD79 alpha/beta heterodimer. In this paper we describe a panel of monoclonal antibodies (mAbs) recognising the extracellular regions of human CD79 alpha and beta. FACS analysis demonstrated that the mAbs bind to a range of Burkitt's lymphoma lines, a mouse B-cell line (JO-72) transfected with human CD79 alpha and beta, and tumour biopsies from NHL patients. The specificity of the mAbs was confirmed by immunoprecipitation. The Ka for the binding of IgG from the anti-CD79 alpha mAbs to cell surface CD79 alpha on Ramos cells was 3 x 10(8) M-1, and their maximum level of binding, 1.7-2 x 10(5) molecules/cell, matched that obtained with anti-Fc mu and anti-Fd mu mAbs. All four anti-CD79 beta mAbs were of lower affinity. Interestingly, in growth arrest studies, we found that while all anti-Fc mu mAbs caused profound inhibition of proliferation of Ramos cells, a range of other anti-BCR mAbs, which included the anti-CD79, anti-Fab mu, anti-gamma and anti-idiotype reagents, all performed poorly giving a maximum of 25% inhibition. These differences in performance are believed to relate to the ability of anti-BCR mAbs to cross-link neighbouring surface BCR and suggest that, unlike anti-Fc mu which favours cross-linking, most of these mAbs are binding in a monogamous, non-cross-linking, union with the BCR.     

The long and the short isoform of cell-CAM 105 show variant-specific modifications in adult rat organs

The cell adhesion molecule cell-CAM 105 is a member of the carcinoembryonic antigen (CEA)-gene family and expressed on the cell surface at least as a long (L-) and a short (S-) isoform. We have developed anti-peptide antisera to investigate the expression patterns of both cell-CAM 105 variants in rat organs. In immunohistochemistry the S- and the L-isoform were co-localized in every tissue investigated. In contrast, the quantification of both isoforms in rat organs by a sandwich-ELISA revealed several differences in their relative expression ratio if compared to the liver value. In submandibular gland and seminal vesicle a significant increase of the S-isoform was found, whereas in kidney the L-isoform was overexpressed. In immunoblot analysis of kidney and seminal vesicle the two isoforms did not appear with the Mr difference which is deduced from their primary structures, suggesting that the two cell-CAM 105 variants are subjected in these tissues to different post translational modifications.     

CD63 is a component of Weibel-Palade bodies of human endothelial cells

Weibel-Palade bodies are secretory granules of vascular endothelial cells specialized in the storage of von Willebrand factor (vWF) and P-selectin, two adhesion proteins that can be rapidly mobilized to the cell surface by exocytosis in response to thrombin or other agonists. In this study, we attempted to identify additional components of Weibel-Palade bodies by raising monoclonal antibodies to these granules, purified by cell fractionation. One antibody, 2C6, was found to be specific for CD63, a membrane glycoprotein previously described in the lysosomes of platelets and other cell types. The immunopurified 2C6 antigen was recognized by an anti-CD63 reference antibody, 2.28, by Western blotting. Also, the biosynthetic profile of the 2C6 antigen in endothelial cells showed a nascent molecular mass and a glycosylation pattern identical to that of CD63. Immunofluorescence staining with 2C6 showed the lysosomes, and also elongated structures identified as Weibel-Palade bodies by their shape, distribution, and positive staining with anti-vWF antibodies, CD63 was also found by Western blotting of subcellular fractions highly enriched in Weibel-Palade bodies. Our results indicate that CD63 colocalizes with vWF and P-selectin in the Weibel-Palade bodies of endothelial cells, and together with these adhesion proteins it could be rapidly expressed on the cell surface in areas of vascular injury and inflammation.     

Characterization of an adhesion molecule that mediates leukocyte rolling on 24 h cytokine- or lipopolysaccharide-stimulated bovine endothelial cells under flow conditions

Bovine gamma/delta T cells and neutrophils roll on 24 h cytokine- or lipopolysaccharide-stimulated bovine fetal umbilical cord endothelial cells in assays done under physiological flow. An antibody directed against E- and L-selectin has minimal blocking effect on this rolling interaction. mAbs were raised against the stimulated bovine endothelial cells and screened for inhibition of gamma/delta T cell rolling. One mAb (GR113) was identified that recognizes an antigen (GR antigen) selectively expressed by stimulated bovine endothelial cells isolated from fetal umbilical cord, mesenteric lymph nodes, and aorta. GR113 blocked bovine gamma/delta T cell as well as neutrophil rolling on the 24 h-activated endothelial cells. The GR antigen was constitutively expressed at low levels on the cell surface of platelets and its expression was not upregulated after stimulation of these cells with thrombin or phorbol myristate acetate. However, stimulated platelets released a soluble, functionally active form of the molecule that selectively bound in solution to gamma/delta T cells in a mixed lymphocyte preparation. GR113 mAb blocked the binding of the soluble platelet molecule to the gamma/delta T cells. Soluble GR antigen also bound a subset of human lymphocytes. Cutaneous lymphocyte-associated antigen (CLA) bright human lymphocytes exhibited the greatest capacity to bind the GR antigen, though CLA was not required for binding. Subsets of both human CD4 and CD8 T cells bound the GR antigen. Immunoprecipitation experiments showed the GR antigen to be 110-120 kD Mr. The binding of soluble GR antigen was inhibited by EDTA and O-sialoglycoprotease, but not neuraminidase treatment of the target cells.     

Patterns for RANTES secretion and intercellular adhesion molecule 1 expression mediate transepithelial T cell traffic based on analyses in vitro and in vivo

Immune cell migration into and through mucosal barrier sites in general and airway sites in particular is a critical feature of immune and inflammatory responses, but the determinants of transepithelial (unlike transendothelial) immune cell traffic are poorly defined. Accordingly, we used primary culture airway epithelial cells and peripheral blood mononuclear cells to develop a cell monolayer system that allows for apical-to-basal and basal-to-apical T cell transmigration that can be monitored with quantitative immunofluorescence flow cytometry. In this system, T cell adhesion and subsequent transmigration were blocked in both directions by monoclonal antibodies (mAbs) against lymphocyte function-associated antigen 1 (LFA-1) or intercellular adhesion molecule 1 (ICAM-1) (induced by interferon gamma [IFN-gamma] treatment of epithelial cells). The total number of adherent plus transmigrated T cells was also similar in both directions, and this pattern fit with uniform presentation of ICAM-1 along the apical and basolateral cell surfaces. However, the relative number of transmigrated to adherent T cells (i.e., the efficiency of transmigration) was increased in the basal-to-apical relative to the apical-to-basal direction, so an additional mechanism was needed to mediate directional movement towards the apical surface. Screening for epithelial-derived beta-chemokines indicated that IFN-gamma treatment caused selective expression of RANTES (regulated upon activation, normal T cell expressed and secreted), and the functional significance of this finding was demonstrated by inhibition of epithelial-T cell adhesion and transepithelial migration by anti-RANTES mAbs. In addition, we found that epithelial (but not endothelial) cells preferentially secreted RANTES through the apical cell surface thereby establishing a chemical gradient for chemotaxis across the epithelium to a site where they may be retained by high levels of RANTES and apical ICAM-1. These patterns for epithelial presentation of ICAM-1 and secretion of RANTES appear preserved in airway epithelial tissue studied either ex vivo with expression induced by IFN-gamma treatment or in vivo with endogenous expression induced by inflammatory disease (i.e., asthma). Taken together, the results define how the patterns for uniform presentation of ICAM-1 along the cell surface and specific apical sorting of RANTES may serve to mediate the level and directionality of T cell traffic through epithelium (distinct from endothelium) and provide a basis for how this process is precisely coordinated to route immune cells to the mucosal surface and maintain them there under normal and stimulated conditions.     

IFN-tau increases PGE2 production and COX-2 gene expression in the bovine endometrium in vitro

Autologous peripheral blood stem cells, obtained by CD34+ stem cell selection, are being used with increasing frequency for transplantation in patients with neuroblastoma. Here, we examined the surface membrane antigens of neuroblastoma cells with a panel of hematopoietic monoclonal antibodies (mAbs), including anti-CD34 mAbs, by flow cytometric analysis. We found stronger binding of anti-CD34 mAbs to clonogenic, less differentiated, non-adherent neuroblastoma cells than to adherent neuroblastoma cells. Moreover, the majority of neuroblastoma cell lines shared hematopoietic-associated antigens with all blood cells. Because of these cross-reactions, especially found with the anti-CD34 mAbs 12.8 and ICH3, we have demonstrated that there is a potential risk of cell harvest contamination by circulating neuroblastoma cells during CD34+ stem cell selection.     

Different immunoreactivity of endothelial markers in well and poorly differentiated areas of angiosarcomas

This study evaluated the immunohistochemical staining of four endothelial cell markers in well differentiated and poorly differentiated areas of angiosarcomas. Formaldehyde-fixed, paraffin-embedded sections from eight angiosarcomas were studied using the antibodies anti-factor VIII-related antigen (FVIII-RA), Ulex europaeus I agglutinin, anti-CD34 (QBEND/10) and anti-CD31 (JC70). The immunostaining of the angiomatous (well differentiated) and solid (poorly differentiated) areas was separately analysed and specificity was evaluated in 20 non-vascular tumours. The antibody anti-CD31 and Ulex europaeus were the most sensitive markers staining well differentiated vasoformative structures and poorly differentiated solid areas. Anti-FVIII-RA and anti-CD34 did not stain undifferentiated malignant endothelial cells from solid areas. Ulex europueus and anti-CD34 showed very low specificity; in contrast, none of the non-vascular tumours expressed CD31 or FVIII-RA. JC70 (anti-CD31) appears to be the most useful marker in elucidating the vascular nature of angiosarcomas. Is important to emphasize the lack of specificity of Ulex europaeus and the low sensitivity of anti-CD34 and anti-FVIII-RA for poorly differentiated lesions.     

Auditory and somatosensory evoked potentials in coma following spontaneous cerebral hemorrhage: early prognosis and outcome

Hereditary hemorrhagic telangiectasia (HHT), or Rendu-Osler-Weber disease, is an autosomal dominant disorder of localized angiodysplasia, although it is sometimes mistakenly identified as a hemostatic disorder due to its associated characteristic bleeding. The vascular lesions that develop consist of direct arteriovenous connections without an intervening capillary bed. Germline mutations in one of two different genes, endoglin or ALK-1, can cause HHT. Both are members of the transforming growth factor (TGF)-beta receptor family of proteins, and are expressed primarily on the surface of endothelial cells. They are associated together in a receptor complex on the cell surface. Biochemical studies suggest that endoglin modulates TGF-beta signaling through ALK-1 and the type I TGF-beta receptor. Most mutations identified in endoglin and ALK-1 create null alleles, which lead to reduced message or protein levels. A model of haploinsufficiency is proposed, in which inheritance of a mutation predisposes an individual to develop HHT-associated vascular lesions. The factors that initiate lesion formation are unknown, but disruption of these genes in mice should provide animal models to address these and other important questions about the pathogenesis of HHT.     

Heat shock cognate protein 71-associated peptides function as an epitope for Toxoplasma gondii-specific CD4+ CTL

HLA-DR-restricted CD4+ cytotoxic T-lymphocyte (CTL) lines specific for Toxoplasma gondii (T. gondii)-infected melanoma cells have been established from peripheral blood lymphocytes (PBLs) of a patient with chronic toxoplasmosis. The role of heat shock cognate protein (HSC) 71 in antigen (Ag) processing and presentation of T. gondii-infected melanoma cells to these CD4+ CTL lines was investigated. A human melanoma cell line (P36) pulsed with T. gondii-infected P36 cell-derived HSC71 was lysed by a T. gondii-specific CD4+ CTL line (Tx-HSC-1). The Tx-HSC-1 also killed T. gondii-infected P36 cells. The lytic activity of Tx-HSC-1 against P36 cells pulsed with T. gondii-infected P36 cell-derived HSC71 was inhibited by monoclonal antibodies (mAbs) against HSC71. Anti-human leukocyte antigen (HLA)-DR mAb also partially blocked the lytic activity, whereas anti-HLA-A,B,C mAb did not block the lytic activity. In addition, a flow cytometric analysis with these specific mAbs against HSC71 showed HSC71 to be expressed on the cell surface of T. gondii-infected P36 cells as well as uninfected P36 cells. These data indicate that HSC71 molecules are expressed on human melanoma cell line P36, and that HSC71 may play a potential role in Ag presentation and processing of T. gondii-infected P36 cells to CD4+ CTL.     

Analysis of myeloid and lymphoid markers on the surface and in the cytoplasm of mononuclear bone marrow cells in patients with myelodysplastic syndrome

Mononuclear cells of the bone marrow (BM) of patients in various subgroups of the myelodysplastic syndrome (MDS) were studied by flow cytometry for the expression of myeloid and lymphoid markers both on the surface and in the cytoplasm. A significantly higher percentage of the BM cells of MDS patients reacted with monoclonal antibodies (mAbs) to myeloid antigens (CD13, CD15 and CD33) by cytoplasmic staining as compared with cell surface staining. The percentage of BM cells expressing CD34 was markedly elevated in patients with RAEB-T. A distinct finding in MDS patients was the expression of myeloid antigens on mononuclear BM cells. The proportion of individuals whose mononuclear BM cells were positive for surface reactivity with anti-CD13 and anti-CD33 mAbs was highest among RAEB-T patients while none of the patients with RA expressed these surface antigens. Cytoplasmic staining significantly increased the percentage of CD13+ and CD33+ BM cells among RAEB and RAEB-T patients. The proportion of individuals whose BM cells possessed myeloid antigens was increased by cytoplasmic staining in all subgroups of MDS. The BM of a considerable proportion of RAEB-T and RAEB patients showed cells which coexpressed the CD7 and CD3 lymphoid markers along with the CD13 and CD33 myeloid antigens. The present study indicates the importance of comparative surface and cytoplasmic immunophenotyping with CD13 and CD33 mAbs for the diagnosis of subgroups of MDS. The coexpression of CD3 and CD7 with markers of the myeloid lineage may reflect derangement of the differentiation of pluripotent stem cells characteristic for MDS.     

Cell-type specificity of anti-CD45 autoantibodies in systemic lupus erythematosus

OBJECTIVE: To determine the specificity of anti-CD45 autoantibodies in systemic lupus erythematosus (SLE) for native CD45 and for CD45 expressed by T cells and B cells, and at different stages of cellular activation. METHODS: CD45 purified from different types of lymphocytes was examined by immunoblotting with sera from patients with SLE. Indirect immunofluorescence experiments were performed with purified anti-CD45 autoantibodies. RESULTS: IgM anti-CD45 autoantibodies in SLE recognize native CD45 expressed on the surface membrane of viable lymphocytes and CD45 purified from activated peripheral T cells and certain T cell lines, but not CD45 purified from B cells or resting peripheral T cells. The presence or absence of reactivity is independent of the individual isoforms expressed. CONCLUSION: Recognition of CD45 by IgM antilymphocyte autoantibodies in SLE varies with the lineage and state of activation of the lymphocyte target. This pattern of reactivity is consistent with autoantibody specificities involving CD45 glycoforms, rather than CD45 isoforms.     

Stimulation through CD50 (ICAM-3) induces both activation and programmed cell death of human thymocytes

CD50 (ICAM-3) has been identified as the third CD11a/CD18 (LFA-1) counter receptor. We investigated the expression and possible role of this molecule in the induction of early and late activation events in human thymocytes. We observed that CD50 expression is acquired by early T cell progenitors (CD34+) and maintained during thymic development, reaching the highest levels in the most mature population of thymocytes (CD3high). Neither basal nor cytokine-induced expression of CD50 was observed on untransformed human thymic epithelial cell lines. Cross-linking of CD50 expressed on the surface of human thymocytes, by using mAbs recognizing epitopes not related to the CD11a binding site, transduced transmembrane signals leading to an increase of intracellular calcium concentration. This calcium mobilization was inhibited when CD50 was co-cross-linked with CD45, suggesting that tyrosine phosphorylation is also involved in CD50 signaling. The same anti-CD50 mAbs that were able to affect intracellular calcium levels were shown to induce CD69 but not CD25 expression on human thymocytes. This effect was preferentially observed on CD3low/CD3high thymocyte subpopulations. Cross-linking of CD50 also significantly increased activation-induced cell death of human thymocytes. These results support the idea that CD50 molecule can play a role in developing functionally mature T lymphocytes.     

Tissue factor expression by endothelial cells in sickle cell anemia

The role of the vascular endothelium in activation of the coagulation system, a fundamental homeostatic mechanism of mammalian biology, is uncertain because there is little evidence indicating that endothelial cells in vivo express tissue factor (TF), the system's triggering mechanism. As a surrogate for vessel wall endothelium, we examined circulating endothelial cells (CEC) from normals and patients with sickle cell anemia, a disease associated with activation of coagulation. We find that sickle CEC abnormally express TF antigen (expressed as percent CEC that are TF-positive), with 66+/-13% positive in sickle patients in steady-state, 83+/-19% positive in sickle patients presenting with acute vasoocclusive episodes, and only 10+/-13% positive in normal controls. Repeated samplings confirmed this impression that TF expression is greater when sickle patients develop acute vasoocclusive episodes. Sickle CEC are also positive for TF mRNA, with excellent concurrence between antigen and mRNA expression. The TF expressed on the antigen-positive CEC is functional, as demonstrated by a binding assay for Factor VIIa and a chromogenic assay sensitive to generation of Factor Xa. By establishing that endothelial cells in vivo can express TF, these data imply that the vast endothelial surface area does provide an important pathophysiologic trigger for coagulation activation.     

ICAM-1 upregulation in distant tissues after hepatic ischemia/reperfusion: a clue to the mechanism of multiple organ failure

BACKGROUND/PURPOSE: Endothelial cell adhesion molecules (ECAMs) are felt to play an important role in ischemia/reperfusion (I/R) injury by causing adhesion of leukocytes to endothelial cells. It is possible that ECAMs play a role in multiple organ system failure. ICAM-1 is one of the adhesion molecules that has been shown to be upregulated in response to cytokines. This upregulation leads to leukocyte endothelial cell interaction (adhesion) and to neutrophil infiltration of the affected tissue. The purpose of our study was to measure ICAM-1 expression in the liver and other organs after hepatic ischemia/reperfusion (I/R). METHODS: A laparotomy was performed on 14 Sprague-Dawley rats; 45 minutes of occlusive ischemia to the left lateral lobe was followed by 5 hours of reperfusion. The rat was injected with I125-labeled ICAM-1 MAb and I131-labeled nonbinding MAb (to control for nonspecific accumulation of ICAM-1 MAb). Entire organs were harvested and accumulated activity was measured in each organ. ICAM-1 levels were expressed as percent injected dose per gram of tissue. Control animals underwent sham laparotomy. RESULTS: ICAM-1 was upregulated in the ischemic lobe of the liver, nonischemic lobe of the liver, heart, kidney, intestine, and pancreas. Up-regulation in the lung was not significant. Both the lung and liver had high constitutive levels of ICAM-1. CONCLUSIONS: These data show that (1) significant hepatic upregulation of ICAM-1 after hepatic ischemia/reperfusion and (2) significant ICAM-1 upregulation in other tissues (heart, kidney, and intestine) after hepatic ischemia/reperfusion. The ICAM-1 upregulation in distant organs is likely mediated by cytokines such as tumor necrosis factor (TNF). These data show that leukocyte endothelial cell interactions in distant organs may be mediated by hepatic ischemia/reperfusion. This is a possible explanation for how failure of one organ can lead to failure of others in multiple organ system failure.     

Monoclonal antibody GL1 and its possible involvement in the morphogenesis of the otic vesicle

In a previous study, a monoclonal antibody (MAB) named GL1 was identified that is expressed in a precise pattern during gastrulation and early neurulation stages in chick embryos. In this article we have further investigated the expression pattern of this MAB in the chick embryo. GL1 antigen is present in several organs that seem not to be related developmentally. Among them, GL1 is present during the early steps of the otic placode formation, in the pharyngeal endoderm, in some neural crest cells, in the somites, and in the ventricular surface of the nervous system. The distribution in the nervous system is well patterned with two broad lines of expression in the ventricular side of the metencephalic region, a unique and centered expression in the border between the metencephalon and the myelencephalon and again in two lines running along the myelencephalon and the rostral spinal cord. Additionally, GL1 can be induced by members of the FGF family, and we have used this system to elucidate its role in otic placode formation. The results obtained reveal that GL1 can be a useful marker for the study of developmental processes in the endoderm, the otic anlage, and the apical surface of the nervous system. Biochemical analysis of the antigen recognized by this MAB must be carried out to elucidate the molecular nature of the antigen.