New human leukocyte clusters of differentiation.
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Antigens, CD↗
Biomedical subjects
H Stockinger
Publications and source records attributed to H Stockinger.
Signal transduction via glycosyl phosphatidylinositol-anchored proteins in T cells is inhibited by lowering cellular cholesterol.
Glycosylphosphatidylinositol (GPI)-anchored proteins can deliver costimulatory signals to lymphocytes, but the exact pathway of signal transduction involved is not yet characterized. GPI-anchored proteins are fixed to the cell surface solely by a phospholipid moiety and are clustered in distinct membrane domains that are formed by an unique lipid composition requiring cholesterol. To elucidate the role of membrane lipids for signal transduction via GPI-anchored proteins, we studied the influence of reduced cellular cholesterol content on calcium signaling via GPI-anchored CD59 and CD48 in Jurkat T cells. Lowering cholesterol by different inhibitors of cellular cholesterol synthesis suppressed calcium response via GPI-anchored proteins by about 50%, whereas stimulation via CD3 was only minimally affected (<10%). The decrease in overall calcium response via GPI-anchored proteins was reflected by inhibition of calcium release from intracellular stores. Cell surface expression of GPI-anchored proteins was not changed quantitatively by lowering cellular cholesterol, and neither was the pattern of immunofluorescence in microscopic examination. In addition, the distribution of GPI-anchored proteins in detergent-insoluble complexes remained unaltered. These results suggest that cellular cholesterol is an important prerequisite for signal transduction via GPI-anchored proteins beyond formation of membrane domains.
CD59 Antigens↗
Analysis of the requirement for beta 2-microglobulin for expression and formation of human CD1 antigens.
Human CD1 form a group of nonpolymorphic leukocyte surface molecules with homology to major histocompatibility complex (MHC) proteins. Recent findings in human and in mouse demonstrate the capacity of CD1 molecules to present nonpeptide components like lipids or lipoglycans as well as peptides. We studied the involvement of beta 2-microglobulin (beta 2m) in expression of the classic human CD1 proteins CD1a, CD1b, and CD1c. The beta 2m-deficient human melanoma cell line FO-1 was transiently transfected with either CD1a, CD1b, or CD1c DNA alone, or in combination with beta 2m using the adenovirus-enhanced receptor-mediated transfer infection system. Only co-transfection of FO-1 cells with CD1+ beta 2m resulted in the detection of CD1 Ag by monoclonal antibodies (mAb). This indicated that CD1 mAb recognized determinants are dependent on beta 2m and raised the question whether beta 2m-free forms of CD1 can be expressed. Therefore, to visualize CD1 molecule expression independently of beta 2m, we expressed tagged recombinant forms. A full-length CD1b construct tagged at the very C terminus with a small peptide was transported to the plasma membrane only when beta 2m was co-transfected. beta 2m involvement in the transport of CD1 was confirmed by expression of soluble forms of CD1a, CD1b, and CD1c in three different cell types. Analogous to tagged full-length CD1b, secretion of the soluble CD1 constructs was strictly dependent on beta 2m. The soluble CD1 chimeras were secreted as complexes with endogenous beta 2m. Thus, similar to its role for MHC class I expression, beta 2m is essential for processing and surface transport of the classic human CD1 molecules CD1a, CD1b, and CD1c.
Antibodies, Monoclonal↗
Interaction of GPI-anchored cell surface proteins and complement receptor type 3.
Glycosylphosphatidylinositol (GPI)-anchored cell surface proteins lack transmembrane and cytoplasmic domains. For some of them, such as CD14, CD16b and CD87, it could be demonstrated recently that they are physically and functionally associated with complement receptor type 3 (CR3, CD11b/CD18). Also, there are indications that in certain cells these GPI proteins employ CR3 as an adapter for signal transduction across the plasma membrane.
Antigens, CD↗
Alpha L beta 2 integrin/LFA-1 binding to ICAM-1 induced by cytohesin-1, a cytoplasmic regulatory molecule.
The avidity of integrin adhesion receptors for extracellular ligands is subject to dynamic regulation by intracellular programs that have yet to be elucidated. We describe here a protein, cytohesin-1, which specifically interacts with the intracellular portion of the integrin beta 2 chain (CD18). The molecule shows homology to the yeast SEC7 gene product and bears a pleckstrin homology (PH) domain. Overexpression of either the full-length cytohesin-1 or the SEC7 domain induces beta 2 integrin-dependent binding of Jurkat cells to ICAM-1, whereas expression of the isolated cytohesin-1 PH domain inhibits T cell receptor-stimulated adhesion. Similar inhibition is not exhibited by PH domains taken from other proteins, showing that the interaction is specific and that individual PH domains are capable of discriminating between alternative targets.
Amino Acid Sequence↗
Interaction of CD31 with a heterophilic counterreceptor involved in downregulation of human T cell responses.
CD31 is a 130-kD glycoprotein of the immunoglobulin (Ig) superfamily expressed on the surface of endothelial cells, platelets, and several leukocyte subsets. Previous reports indicated that CD31 can mediate intercellular adhesion via both homophilic and heterophilic interaction mechanisms. Using a soluble recombinant CD31-Ig fusion protein (CD31 receptor globulin [Rg]), we demonstrate here that human CD31- T lymphocytes and CD4+CD31- T cell clones express a heterophilic CD31 ligand that is upregulated 18 h after activation. Interaction of CD31Rg with CD31- T helper cell (Th) clones was divalent cation independent but could be blocked by heparin, thus indicating that the CD31 counterreceptor on T cells can be distinguished from the ligands identified on other cell types. Moreover, a single chain protein of 120 kD was precipitated by CD31Rg from the lysates of CD31- Th clones. CD31Rg completely downregulated the proliferative response and cytokine production (interleukin-4, interferon-gamma, and tumor necrosis factor-alpha) of CD31- Th clones when the cells were maximally stimulated via immobilized CD3 monoclonal antibody. These results suggest that interaction of CD31 with a heterophilic counterreceptor on T lymphocytes can interfere with a positive regulatory pathway of T cell activation, or directly signal T cells to downregulate immune function.
Antigens, Differentiation, Myelomonocytic↗
Noncovalent associations of T lymphocyte surface proteins.
A number of T cell surface transmembrane molecules such as CD2, CD4, CD8, lymphocyte functional antigen (LFA)-1 and CD45 are known to interact functionally with the T cell receptor (TCR) complex during T cell activation. Several previous communications have also reported physical associations between some of these molecules. On the other hand, there are indications that signaling through T cell surface molecules anchored via glycosylphosphatidylinositol (GPI), such as Thy-1, Ly-6 or CD59, is dependent on the TCR. Therefore, it was of interest to determine in a systematic way which T cell surface molecules are noncovalently associated with the TCR/CD3 complex and with the major intracellular signaling molecules, the protein tyrosine kinases of the Src family. To this aim, membrane proteins of human thymoma HPB-ALL cells were solubilized in a solution of the mild detergent Brij-58 and subjected to immunoprecipitation followed by in vitro kinase assays. Two types of large complexes containing protein tyrosine kinases were observed: the first one contained CD3 and the transmembrane proteins CD2, CD4, CD5, CD6, CD7, CD8, CD11a, CD38, CD43, CD45, CD71, CD98 and CD99 and the other contained mainly the GPI-anchored proteins CD48, CD55, CD59 and CDw108 as well as a fraction of CD4 and CD8. The GPI-anchored protein complexes were of larger size and lower buoyant density than the CD3 complexes. In agreement with these biochemical data, co-cross-linking of CD3 with most of the relevant transmembrane proteins on the surface of another T cell line, Jurkat, markedly enhanced tyrosine phosphorylation of several intracellular proteins. These data indicate the existence of at least two types of membrane microdomains of very different composition in the membranes of T cells which may play a role in signaling through different types of receptors and in functional cooperation between TCR/CD3 and various accessory molecules.
Antigens, Differentiation, T-Lymphocyte↗
CD4 monoclonal antibody VIT4 in human alloimmune response in vitro and in vivo.
In the present report the immunosuppressive effects of the murine anti-human CD4 monoclonal antibody (mAb) VIT4 on human alloimmune response in vitro were analyzed. Moreover, the antibody was tested for its activity to prolong allograft survival in seven patients with steroid-refractory allograft rejection. VIT4 inhibited the proliferative response to alloantigens in the mixed lymphocyte reaction (MLR) in a dose-dependent manner. At concentrations of 1 and 10 micrograms/ml VIT4 blocked MLR by 55 +/- 11% and 77 +/- 1%, respectively. Also alloantigen-specific proliferation of in vitro- generated memory T cells was dose-dependently reduced to 23 +/- 1% at a VIT4 concentration of 100 micrograms/ml. Furthermore, at the same dose level VIT4 blocked proliferation of antigen-specific short-term alloreactive CD4+ cell lines and significantly inhibited the in vitro generation of cytotoxic T lymphocytes (CTL). In a pilot study VIT4 (5 mg/d i.v.) was administered to 7 patients with steroid-refractory allograft rejection for 14 days. In 4 of 7 patients graft function transiently improved and graft survival in all patients was prolonged to a mean of 694 days (range 128-2163) from the beginning of the VIT4 treatment. In the light of our in vitro results and the preliminary clinical data, further clinical trials using higher antibody doses are greatly warranted to assess the efficacy of anti-CD4 mAb VIT4 in the treatment of allograft rejection.
Animals↗
1995 directory for the human leukocyte clusters of differentiation.
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Antibodies, Monoclonal↗
Acquisition of host cell-surface-derived molecules by HIV-1.
OBJECTIVE: To determine the acquisition of host cell-membrane-derived molecules by HIV-1 during the budding process, and to investigate whether the uptake of these molecules is cell-type-specific and selective. DESIGN: Virions, propagated by four different cell types were analysed for the presence of adhesion molecules, glycosylphosphatidylinositol (GPI)-anchored proteins and various cell-surface markers. The pattern was compared with the phenotype of the HIV-1-infected cell. METHODS: For phenotypic analysis of virions a two-step assay was used. In the first step, virus was captured with monoclonal antibodies (in some cases polyclonal sera) against different cell-membrane proteins. In a second step, the presence of virus was measured by determining the concentration of the virus-specific p24 core antigen. The expression of surface molecules on uninfected and HIV-1IIIB-infected cells was analysed by FACS. RESULTS: Depending on the cell type used for virus propagation, different cell-membrane molecules were found on the virus surface reflecting the corresponding cell type. The uptake of these molecules was selective to a certain degree. No CD4 and CD87 molecules were detectable on HIV-1, although both molecules were present on uninfected and HIV-1-infected cells. CR3 and CDw108 could not be seen on uninfected cells, but wre detectable on infected cells and virions. CONCLUSIONS: During the budding process HIV-1 acquires a variety of cell-type-specific cell-surface molecules. Certain cell-membrane molecules become upregulated during HIV-1-infection and are then found on virions, whereas other molecules remain on the cell surface and do not become incorporated.
Cell Line↗
Identification of the TS2/18-recognized epitope on the CD2 molecule as a target for suppression of T cell cytokine synthesis.
CD2 mAb can inhibit T cell activation, but the mechanisms involved are still unclear. In this study, we identify the mAb TS2/18, previously reported to bind to an epitope on the distal domain of the CD2 molecule at amino acids 87-99 (1), as a particularly potent inhibitor of T cell cytokine synthesis. Although TS2/18 is comitogenic with the CD2R mAb VIT13, this mAb combination does not induce the secretion of substantial amounts of cytokines. When added to T cells stimulated with the CD2 mAb pair OKT11-VIT13, TS2/18 efficiently blocks the induction of cytokine synthesis induced by that CD2 mAb pair, although it does not interfere with the binding of OKT11. In addition, TS2/18 inhibits the increase in protein tyrosine phosphorylation and the accumulation of phosphatidic acid induced by either OKT11-VIT13 or a cross-linked CD3 mAb. Finally, TS2/18 disrupts CD2 clusters induced by the CD2 mAb pair OKT11-VIT13. We conclude that TS2/18 blocks T cell cytokine synthesis by interfering with early signal transduction, possibly by impairing the formation of signal-transducing molecule complexes on the T cell surface. Together, these data identify the CD2 epitope recognized by the mAb TS2/18 as a candidate epitope for T cell-specific immunosuppressive ligands.
Antibodies, Monoclonal↗
Urokinase plasminogen activator receptor, beta 2-integrins, and Src-kinases within a single receptor complex of human monocytes.
The glycosylphosphatidylinositol (GPI)-anchored membrane protein urokinase plasminogen activator-receptor (uPA-R; CD87) is one of the key molecules involved in migration of leukocytes and tumor cells. uPA bound to uPA-R provides the cell proteolytic potential used for degradation of extracellular matrix. uPA-R is also involved in induction of cell adhesion and chemotaxis. Here, we provide a molecular explanation for these uPA-R-related cellular events. By size fractionation of monocyte lysate and affinity isolation on its natural ligand uPA, we demonstrate uPA-R as a component of a receptor complex of relatively large size. Reprecipitation and immunoblotting techniques allowed us to detect the protein tyrosine kinases (PTKs) p60fyn, p53/56lyn, p58/64hck, and p59fgr as components of this "uPA-R complex". Activation of monocytes even with enzymatically inactivated uPA resulted in induction of tyrosine phosphorylation, suggesting modulation of uPA-R-associated PTKs upon ligand binding. In spite of their presence in large complexes, we did not find the GPI-linked proteins CD14, CD58, and CD59 in the uPA-R complex, which indicates the presence of different receptor domains containing GPI-linked proteins in monocytes. However, we identified the leukocyte integrins LFA-1 and CR3 as components of the uPA-R complex as indicated by coisolation of these molecules, as well as by cocapping and comodulation of uPA-R and leukocyte integrins on the monocyte surface. The assemblage of uPA-R, PTKs and membrane spanning beta 2-integrins in one receptor complex indicates functional cooperation. In regard to the involvement of these molecules in pericellular proteolysis, signal transduction, as well as adhesion and chemotactic movement, we suggest uPA-R complex as a potential cellular device for cell migration.
Antigens, CD↗
Secretory group II phospholipase A2 in human atherosclerotic plaques.
Atherosclerotic plaques exhibit a series of features that are similar to those of chronic inflammation. Based on the fact that during inflammation several cell types synthesize and secrete a group II phospholipase A2 (PLA2), an immunohistochemical study was undertaken to explore whether this enzyme can be identified in human atherosclerotic lesions. Tissue specimens obtained from 13 patients who had undergone arteriectomy and three specimens with advanced atherosclerotic plaques obtained at autopsy were analyzed and compared to arteries free of atherosclerosis. The results showed that in all areas with atherosclerotic lesions, a staining with monoclonal antibodies raised against group II PLA2 was evident. In normal arteries without thickened intima, this immunostaining was completely negative. With the use of specific monoclonal antibodies against macrophages (anti-KP-1) and smooth muscle cells (anti-alpha-actin), PLA2-positive cells were identified as foam cells mainly derived from macrophages. In addition to these cells, other regions of the thickened intima gave a partially positive reaction with anti-PLA2 antibodies, but could not be stained with either anti-KP-1 or anti-alpha-actin. Some of these regions were localized on edges of calcification and cell necrosis. Other PLA2-positive regions seem to be associated with extracellular matrix structures. In summary, the findings of this study may be regarded as further evidence to support the link between atherosclerosis and chronic inflammatory processes. In view of the fact that the in vitro modification of lipoproteins by PLA2-treatment induces lipid deposition in macrophages, the results of this study suggest that group II PLA2 may actively be involved in the formation of foam cells in vivo.
Aged↗
Selective recognition of the activated form of transcription factor NF-kappa B by a monoclonal antibody.
Transcription factor NF-kappa B is a central regulator of inflammatory, immune and acute phase reactions. It rapidly initiates the transcription of a wide variety of target genes in response to various pathogenic stimuli. Because NF-kappa B is predominantly controlled at a posttranscriptional level through association with the inhibitory I kappa B subunits, its activation cannot be monitored directly at a cellular level by means of detecting new mRNA or protein synthesis. In this study, we describe a monoclonal antibody, designated alpha-p65MAb, that recognizes an epitope which includes the nuclear location signal (NLS) of p65, the DNA binding subunit mainly responsible for the strong gene-inductory potential of NF-kappa B. alpha-p65MAb recognized human and rodent p65 only when I kappa B alpha was not bound to p65. Thus, the IgG3 selectively stained the activated, nuclear form of NF-kappa B in cultured cells. Unlike I kappa B, the MAb and its Fab fragments did not inhibit the DNA binding activity of NF-kappa B in mobility shift assays. We show that alpha-p65MAb is suitable to study the activation state of NF-kappa B in cryosections of tissues.
Amino Acid Sequence↗
Co-ligation of CD31 and Fc gamma RII induces cytokine production in human monocytes.
The CD31 (PECAM-1) cell surface glycoprotein is considered to be involved in intercellular recognition and adhesion. Cytokines play a major role in cellular interactions, and therefore it was of interest to study whether engagement of CD31 affects synthesis and release of proadhesive cytokines. Here we demonstrate that immobilized CD31 mAb 1B5 induces the release of TNF-alpha, IL-1 beta, and IL-8 from human PBMCs. CD11b mAb VIM12 and HLA-D mAb VID1, both of which are of the same Ig subclass as mAb 1B5 (IgG1), as well as nonbinding isotype control mAb VIAP, were ineffective. That the effect was caused by the mAb, but not endotoxin contamination, was shown by negative Limulus amebocyte lysate tests and coculture with polymyxin B, which did not abolish TNF-alpha release. Cytokine production through intact mAb 1B5 was completely blocked by soluble F(ab) fragments of anti-IgG Fc gamma RII mAb IV.3, suggesting a significant contribution of that FcR. Cross-linking of neither CD31 nor Fc gamma RII molecules with the respective F(ab) fragments induced TNF-alpha release, but nonbinding control IgG1 Ab was able to restore the response of PBMC to 1B5 F(ab) fragments, when both Ab preparations were coated concomitantly. Therefore, only coligation of CD31 and Fc gamma RII appears to transduce activation signals leading to cytokine production. Our findings thus indicate a novel functional aspect of CD31 molecules that might play an important role in the propagation of an ongoing immune response as well as in the regulation of cell-cell interactions during inflammatory reactions.
Antibodies, Monoclonal↗
Signaling and induction of enhanced cytoadhesiveness via the hematopoietic progenitor cell surface molecule CD34.
The transmembrane glycoprotein CD34 shows a highly restricted expression on a crucial subset of hematopoietic cells. We show here that engagement of particular determinants of CD34 can lead to signal transduction and to enhanced adhesiveness of CD34+ hematopoietic cells. Monoclonal antibodies (MoAbs) directed against O-sialoglycoprotease-sensitive epitopes of CD34 (QBEND10, ICH3, BI.3C5, MY10) but not MoAbs against O-sialoglycoprotease-resistant epitopes (9F2, 8G12) induce actin polymerization in KG-1a and KG-1 cells and strongly enhanced cytoadhesiveness. The capacity to induce adhesion requires cellular energy, divalent cations, and intact cytoskeleton but not de novo protein synthesis. The observed cytoadhesion seems at least in part to be caused by a concomitant activation of the beta 2 integrin cytoadhesion pathway. It can be significantly inhibited with lymphocyte function-associated antigen-1 and intercelluar adhesion molecule-1 antibodies. Protein kinase inhibition analyses suggest that the pathways initiated by engagement of the CD34 molecule with certain CD34 MoAbs involves protein tyrosine kinases but that protein kinase C is not critically involved.
Antibodies, Monoclonal↗
CD1 molecule expression on human monocytes induced by granulocyte-macrophage colony-stimulating factor.
In this paper we demonstrate that granulocyte-macrophage CSF (GM-CSF) specifically induces the expression of CD1 molecules, CD1a, CD1b and CD1c, upon human monocytes. CD1 molecules appeared upon monocytes on day 1 of stimulation with rGM-CSF, and expression was up-regulated until day 3. Monocytes cultured in the presence of LPS, FMLP, PMA, recombinant granulocyte-CSF, rIFN-gamma, rTNF-alpha, rIL-1 alpha, rIL-1 beta, and rIL-6 remained negative. The induction of CD1 molecules by rGM-CSF was restricted to monocytes, since no such effect was observed upon peripheral blood granulocytes, PBL, and the myeloid cell lines Monomac1, Monomac6, MV4/11, HL60, U937, THP1, KG1, and KG1A. CD1a mRNA was detectable in rGM-CSF-induced monocytes but not in those freshly isolated. SDS-PAGE and immunoblotting analyses of CD1a mAb VIT6 immunoprecipitate from lysate of rGM-CSF-activated monocytes revealed an appropriate CD1a polypeptide band of 49 kDa associated with beta 2-microglobulin. Expression of CD1 molecules on monocytes complements the distribution of these structures on accessory cells, and their specific induction by GM-CSF strengthens the suggestion that CD1 is a family of crucial structures required for interaction between accessory cells and T cells.
Antibodies, Monoclonal↗
Monoclonal antibodies to the carbohydrate structure Lewis(x) stimulate the adhesive activity of leukocyte integrin CD11b/CD18 (CR3, Mac-1, alpha m beta 2) on human granulocytes.
Several carbohydrate structures on human granulocytes have been discussed as potential ligands for C-type lectins (selectins) on endothelial cells. Among them are the lacto-series type II chain antigens sialyl-Lewis(x) (SLe(x), Lewis(x) (Le(x)), and VIM2. We demonstrated in this study that monoclonal antibodies (mAbs) to Le(x) and to SLe(x), but not other anticarbohydrate mAbs (VIM2, CDw17, CD24), can stimulate granulocytes to form homotypic aggregates. This effect was particularly noticeable with three distinct anti-Le(x) mAbs (3C6, 4D1, 6C7). Much less impressive effects were also seen with nine other anti-Le(x) mAbs and with the anti-SLe(x) mAb CSLEX1. Aggregation was shown to be an active process. It is temperature and energy dependent, requires divalent cations, and is selective in terms of mAb specificity. Anti-Le(x)-induced homoaggregate formation could be inhibited with CD11b mAb JML-H11 and CD54 (ICAM1) mAb LB-2 and thus seems to be associated with activation of the beta 2-integrin cytoadhesion pathway.
Antibodies, Monoclonal↗