Biomedical subjects

A Aruffo

Publications and source records attributed to A Aruffo.

At least 73 records · Page 4Linked to original sources

PubMed1996

Identification of a novel inducible cell-surface ligand of CD5 on activated lymphocytes.

CD5 is a 67-kD glycoprotein that is expressed on most T lymphocytes and on a subset of mature B cells. Although its physiologic function is unknown, several lines of evidence suggest that CD5 may play a role in the regulation of T cell activation and in T cell-antigen presenting cell interactions. Using a CD5-immunoglobulin fusion protein (CD5Rg, for receptorglobulin) we have uncovered a new CD5 ligand (CD5L) expressed on the surface of activated splenocytes. Stimulation of murine splenocytes with anti-CD3 and anti-CD28 antibodies induce transient expression of CD5L on B lymphocytes that lasts for approximately 72 h. Binding of CD5Rg to activated splenocytes is trypsin resistant and independent of divalent cations. However, it is pronase sensitive and dependent on N-linked glycosylation of CD5, since treatment of CD5Rg with PNGaseF on N-glycanase completely abrogates its ability to bind activated splenocytes. It addition to splenocytes, CD5L is expressed on activated murine T cell clones. Immunoprecipitation, antibody, and recombinant protein blocking studies indicate that CD5L is distinct from CD72, which has been proposed to be a CD5 ligand. To determine whether CD5-CD5L interaction might play a role in vivo, we tested the effect of CD5Rg in a murine model of antibody-mediated membranous glomerulonephritis. Injection of CD5Rg was found to abrogate development of the disease. Taken together, our results help identify a novel ligand of CD5 and propose a role for CD5 in the regulation of immune responses.

Animals↗

PubMed1996

Identification of a costimulatory molecule rapidly induced by CD40L as CD44H.

The interaction between CD40 ligand and CD40 is critical for activation of T and B cells in vivo. We have recently demonstrated that this interaction rapidly induces a novel costimulatory activity distinct from B7 and independent of CD28. To study the molecular basis of the costimulatory activity, we have produced a novel monoclonal antibody, TM-1, that binds an 85-kilodalton costimulatory molecule rapidly induced by CD40L. Expression cloning reveals that TM-1 binds CD44H. CD44H expressed on Chinese hamster ovary cells has potent costimulatory activity for clonal expansion of T cells isolated from both wild-type mice and these with a targeted mutation of CD28. Thus, CD44H costimulates T cell proliferation by a CD28-independent mechanism. These results revealed that CD44H is a costimulatory molecule rapidly induced by CD40L.

Animals↗

PubMed1996

Differential expression of Fas (CD95) and Fas ligand on normal human phagocytes: implications for the regulation of apoptosis in neutrophils.

Human neutrophils, monocytes, and eosinophils are known to undergo apoptotic cell death. The Fas/Fas ligand pathway has been implicated as an important cellular pathway mediating apoptosis in diverse cell types. We conducted studies to examine the importance of the Fas/FasL system in normal human phagocytes. Although Fas expression was detected on neutrophils, monocytes, and eosinophils, constitutive expression of FasL was restricted to neutrophils. The three types of phagocytes demonstrated differential sensitivity to Fas-induced apoptosis. Only neutrophils were highly susceptible to rapid apoptosis in vitro after stimulation with activating anti-Fas IgM (mAb CH-11). Fas-mediated neutrophil apoptosis was suppressed by incubation with G-CSF, GM-CSF, IFN-gamma, TNF-alpha, or dexamethasone, as well as the selective tyrosine kinase inhibitors, herbimycin A and genistein. Spontaneous neutrophil death in vitro was partially suppressed by Fas-Ig fusion protein or antagonistic anti-Fas IgG1 (mAb ZB4). In coculture experiments, neutrophils released a soluble factor inducing death in Fas-susceptible Jurkat cells via a mechanism sensitive to the presence of Fas-Ig or anti-Fas IgG1. Immunoblot analysis using specific anti-human FasL IgG1 (mAb No. 33) identified a 37-kD protein in lysates of freshly isolated neutrophils and a 30-kD protein in the culture supernatant of neutrophils maintained in vitro. Our results suggest that mature neutrophils may be irrevocably committed to autocrine death by virtue of their constitutive coexpression of cell-surface Fas and FasL via a mechanism that is sensitive to proinflammatory cytokines, glucocorticoids, and inhibitors of tyrosine kinase activity. Furthermore, neutrophils can serve as a source of soluble FasL, which may function in a paracrine pathway to mediate cell death.

Apoptosis↗

PubMed1996

The amino-terminal immunoglobulin-like domain of activated leukocyte cell adhesion molecule binds specifically to the membrane-proximal scavenger receptor cysteine-rich domain of CD6 with a 1:1 stoichiometry.

Activated leukocyte cell adhesion molecule (ALCAM) was recently identified as a ligand for CD6, a signaling receptor expressed on T cells, a subset of B cells, and some cells in the brain. Receptor-ligand binding assays, antibody blocking experiments, and examination of the tissue distribution of these two cell surface proteins suggest that CD6-ALCAM interactions play an important role in mediating the binding of thymocytes to thymic epithelial cells and of T cells to activated leukocytes. Presently, the details of CD6-ALCAM interactions and of signaling through CD6 are unknown. A series of truncated human ALCAM and CD6 immunoglobulin fusion proteins were produced and tested in different binding assays to analyze ALCAM-CD6 interactions in more detail. In this study, we report that the amino-terminal Ig-like domain of human ALCAM specifically binds to the third membrane-proximal scavenger receptor cysteine-rich (SRCR) domain of human CD6. Using thrombin-cleaved Ig fusion proteins containing single or multiple ALCAM or CD6 domains, we were able to determine that the stoichiometry of the interaction between the amino-terminal ALCAM domains and the membrane-proximal CD6 SRCR domain is 1:1. These results provide the first example of an Ig-like domain mediating an interaction with an SRCR domain.

Activated-Leukocyte Cell Adhesion Molecule↗

PubMed1996

Long-term acceptance of skin and cardiac allografts after blocking CD40 and CD28 pathways.

The receptor-ligand pairs CD28-B7 and CD40-gp39 are essential for the initiation and amplification of T-cell-dependent immune responses. CD28-B7 interactions provide 'second signals' necessary for optimal T-cell activation and IL-2 production, whereas CD40-gp39 signals co-stimulate B-cell, macrophage, endothelial cell and T-cell activation. Nonetheless, blockade of either of these pathways alone is not sufficient to permit engraftment of highly immunogenic allografts. Here we report that simultaneous but not independent blockade of the CD28 and CD40 pathways effectively aborts T-cell clonal expansion in vitro and in vivo, promotes long-term survival of fully allogeneic skin grafts, and inhibits the development of chronic vascular rejection of primarily vascularized cardiac allografts. The requirement for simultaneous blockade of these pathways for effective inhibition of alloimmunity indicates that, although they are interrelated, the CD28 and CD40 pathways are critical independent regulators of T-cell-dependent immune responses.

Abatacept↗

PubMed1996

CD40-CD40 ligand interactions in experimental allergic encephalomyelitis and multiple sclerosis.

We investigated the role of CD40-CD40 ligand (CD40L) interactions in multiple sclerosis (MS) and experimental allergic encephalomyelitis (EAE). Activated helper T cells expressing CD40L (gp39) surface protein were found in MS patient brain sections, but not in brain tissue sections of normal controls or patients with other neurological disease. CD40L-positive cells were co-localized with CD40-bearing cells in active lesions (perivascular infiltrates). Most of these CD40-bearing cells proved to be of the monocytic lineage (macrophages or microglial cells), and relatively few were B cells. To functionally evaluate CD40-CD40L interactions, EAE was elicited in mice by means of proteolipid-peptide immunization. Treatment with anti-CD40L monoclonal antibody completely prevented the development of disease. Furthermore, administration of anti-CD40L monoclonal antibody, even after disease onset, shortly before maximum disability score was reached led to dramatic disease reduction. The presence of helper T cells expressing CD40L in brain tissue of MS patients and EAE animals, together with the functional evidence provided by successful experimental prevention and therapy in an animal model, indicates that blockade of CD40-CD40L-mediated cellular interactions may be a method for interference in active MS.

Animals↗

PubMed1996

CD40-gp39 interactions play a critical role during allograft rejection. Suppression of allograft rejection by blockade of the CD40-gp39 pathway.

Studies in vivo have documented the importance of CD40-gp39 interactions in the development of T-dependent antibody responses to foreign and auto-antigens. In this report, we demonstrate that allograft rejection is also associated with strong induction of CD40 and gp39 transcripts. When treatment was initiated at the time of transplant, MR1, a mAb specific for gp39, induced markedly prolonged survival of fully disparate murine cardiac allografts in both naive and sensitized hosts. However, when therapy was delayed until postoperative day 5, anti-gp39 failed to prolong graft survival. Allografts from recipients treated with MR1 from the time of transplantation showed decreased expression of transcripts for the macrophage effector molecule, inducible nitric oxide synthase, but essentially unaltered expression of B7 molecules and T cell cytokine transcripts (interleukin [IL]-2, interferon-gamma, IL-10, and IL-4) relative to control allografts. In addition, alloantibody responses in the MR1-treated mice were profoundly inhibited. However, our studies using B cell-deficient mice indicated that the ability of MR1 to prolong allograft survival was not dependent on B cells. These data suggest that blockade of CD40-gp39 interactions may inhibit allograft rejection primarily by interfering with T cell help for effector functions, rather than by interference with T cell activation.

Animals↗

PubMed1996

The murine homolog of human Ep-CAM, a homotypic adhesion molecule, is expressed by thymocytes and thymic epithelial cells.

In this report, we demonstrate that gp40, a molecule previously shown to be expressed by thymic epithelial cell lines in vitro and by thymic epithelial cells in vivo, is the murine homolog of human Ep-CAM, a calcium-independent homotypic adhesion molecule. gp40 is also expressed at low levels by thymocytes and peripheral T cells. In the adult thymus, gp40 expression was inversely related to the state of thymocyte maturation, with the highest levels associated with CD4-CD8- and CD4+CD8+ thymocyte populations. Ultrastructural immunohistochemistry revealed gp40 localization to areas of thymocyte/epithelial contact and demonstrated that gp40 is also expressed by thymic dendritic cells. During fetal development, thymocytes at days 14-16 of gestation expressed high levels of gp40. At later stages, the observed decline in the frequency of gp40+ cells and levels of expression correlated with the emergence of alpha beta+ thymocytes by day 18 of gestation. In short-term cultures, stimulation of unfractionated adult thymocytes with concanavalin A increased gp40 expression, particularly among CD3hi and CD3int thymocyte populations. This demonstration that Ep-CAM, initially considered to be expressed primarily by epithelial cells, is also expressed by thymocytes, T cells and antigen-presenting cells, raises the possibility that Ep-CAM may contribute to adhesive interactions between thymocytes and epithelial cells or dendritic cells, either in the context of thymocyte development or peripheral T cell trafficking and function.

Animals↗

PubMed1996

CD40 is functionally expressed on human keratinocytes.

The CD40/gp39 pathway is known to be an important feature of B/T cell collaboration leading to T cell-dependent activation, proliferation or differentiation of B cells. Additionally, CD40 is involved in the regulation of B cell survival and apoptosis. Recently, CD40 has been shown to be expressed functionally on non-hematopoietic cells, i.e. endothelial cells. Here, we demonstrate that human keratinocytes (KC) cultured in vitro express CD40 constitutively. The surface expression of CD40 is markedly up-regulated following stimulation with interferon (IFN)-gamma, but not with tumor necrosis factor-alpha or interleukin (IL)-1 beta. This process is regulated at the CD40 mRNA level as demonstrated by Northern blot analysis. Furthermore, ligation of CD40 via soluble gp39, the CD40 ligand, enhances intercellular adhesion molecule (ICAM)-1 and Bcl-x up-regulation on IFN-gamma-stimulated KC, but not lymphocyte function-associated antigen (LFA)-3, B7-2, HLA-DR, or Fas expression. The release of IL-8 is also induced following CD40 ligation on KC. In psoriasis, a T cell-mediated inflammatory skin disease, KC have a markedly enhanced expression of CD40. This expression co-localizes with the expression of ICAM-1, Bcl-x, and an influx of CD3+ T cells. These findings suggest a functional role of CD40 on KC in inflammatory skin disorders such as psoriasis and could make a therapeutic intervention by disrupting the CD40/gp39 pathway an approach to consider in these inflammatory skin diseases.

Adult↗

PubMed1996

Structure-based modeling of the ligand binding domain of the human cell surface receptor CD23 and comparison of two independently derived molecular models.

CD23, a type II membrane receptor protein, recognizes four different ligands via its extracellular C-type lectin domain: immunoglobulin E (IgE), CD21, and the beta 2-integrins CD11b and CD11c. CD23 specifically interacts in a calcium-dependent manner, "lectin-like" with carbohydrate moieties expressed on CD21 and CD11b/c, but also "lectin-unlike" with protein epitopes on IgE. As a first step in analyzing the multiple binding specificities associated with CD23 in more detail, we report a detailed molecular model of the lectin-like domain of human CD23 (hCD23). The model was built based on information provided by X-ray structures of mannose binding protein (MBP) and E-selectin, both of which are members of the calcium-dependent (C-type) lectin superfamily. Sequence-structure comparisons suggest that hCD23 is structurally more similar to MBP than to E-selectin. The hCD23 model is compared to an independently derived model. Although the CD23-carbohydrate and CD23-protein interactions are both calcium dependent, analysis of the model suggests the presence of distinct binding sites for these ligands.

Amino Acid Sequence↗

PubMed1996

Classification of mutations in the human CD40 ligand, gp39, that are associated with X-linked hyper IgM syndrome.

The interaction between the T cell activation antigen gp39 and CD40, its receptor CD40 on B cells, plays a critical role in the regulation of humoral immune responses. Using a detailed three-dimensional model of the gp39 extracellular region, we have analyzed 20 mutations in gp39 that were, with one exception, isolated from patients with X-linked hyper IgM (XHIM) syndrome. On the basis of this analysis, the mutations were classified according to their predicted locations and effects. Twelve mutations are thought to compromise the gp39 structure by affecting interactions in hydrophobic core regions or at monomer interfaces, whereas seven others map closely to gp39 residues important for interaction with CD40. The latter mutations may thus, directly or indirectly, interfere with CD40 binding. One naturally occurring mutant whose carrier displays normal immune responses maps to a solvent-exposed position in a loop region of the molecule.

CD40 Antigens↗

PubMed1996

CD40 ligand triggers interleukin-6 mediated B cell differentiation.

Interleukin-6 (IL-6) is the major cytokine to date mediating antigen (Ag)- or mitogen-driven B cell differentiation. Recently, CD40 ligand (CD40L), with the co-stimulatory cytokines IL-4 and IL-10, has been shown to trigger immunoglobulin (Ig) secretion and class switching. In the present report, we have examined the role of IL-6 in mediating B cell differentiation and Ig secretion triggered with CD40L and/or these cytokines. Culture of splenic B cells with CD40L triggered (1) significant (5.4-fold) increases in IL-6 secretion; (2) differentiation, evidenced by sequential loss of B cell (CD20, CD21) and acquisition of plasma cell (CD38, PCA-1) surface antigens (Ags); and (3) Ig secretion. Interleukin-4 increased both IL-6 and IgG secretion stimulated by CD40L. Interleukin-10+ CD40L triggered 100-fold increments in IgG, IgA and IgM secretion, but IL-10 suppressed IL-6 secretion triggered with CD40L +/- IL-4. Exogenous IL-6 can further increase IgG secretion induced by CD40L + IL-10; moreover, the anti-IL-6 monoclonal antibody partially blocked IgG secretion triggered by CD40L +/- IL-4 or IL-10. Finally, IL-10 suppressed differentiation of B cells induced by CD40L. These studies suggest that CD40L augments Ig secretion in at least two mechanisms: by triggering IL-6 secretion and related differentiation, and by priming B cells for responsiveness to IL-10.

B-Lymphocytes↗

PubMed1996

Glycosylation of CD44 is implicated in CD44-mediated cell adhesion to hyaluronan.

CD44-mediated cell adhesion to hyaluronate is controlled by mechanisms which are poorly understood. In the present work we examine the role of N-linked glycosylation and Ser-Gly motifs in regulating CD44-hyaluronate interaction. Our results show that treatment of a panel of human cell lines which constitutively express CD44 with the inhibitor of N-linked glycosylation tunicamycin results in the loss of attachment of these cells to hyaluronate-coated substrate. In contrast, treatment of the same cells with deoxymannojirimycin, which inhibits the conversion of high mannose oligosaccharides to complex N-linked carbohydrates, results in either no change or an increase in CD44-mediated adhesion to hyaluronate, suggesting that complex N-linked oligosaccharides may not be required for and may even inhibit CD44-HA interaction. Using human melanoma cells stably transfected with CD44 N-linked glycosylation site-specific mutants, we show that integrity of five potential N-linked glycosylation sites within the hyaluronate recognition domain of CD44 is critical for hyaluronate binding. Mutation of any one of these potential N-linked glycosylation sites abrogates CD44-mediated melanoma cell attachment to hyaluronate-coated surfaces, suggesting that all five sites are necessary to maintain the HA-recognition domain in the appropriate conformation. We also demonstrate that mutation of serine residues which constitute the four Ser-Gly motifs in the membrane proximal domain, and provide potential sites for glycosaminoglycan side chain attachment, impairs hyaluronate binding. Taken together, these observations indicate that changes in glycosylation of CD44 can have profound effects on its interaction with hyaluronic acid and suggest that glycosylation may provide an important regulatory mechanism of CD44 function.

1-Deoxynojirimycin↗

PubMed1996

The major surface glycoprotein of Trypanosoma cruzi amastigotes are ligands of the human serum mannose-binding protein.

Trypanosoma cruzi, an obligate intracellular protozoan parasite, chronically infects mammals and causes Chagas' disease in humans. T. cruzi evasion of the mammalian immune response and establishment of chronic infection are poorly understood. During T. cruzi infection, amastigotes and trypomastigotes disseminate in the mammalian host and invade multiple cell types. Parasite surface carbohydrates and mammalian lectins have been implicated in the invasion of mammalian cells. A recent study has demonstrated that the human mannose-binding protein and the macrophage mannose receptor, two mammalian C-type lectins, bind to T. cruzi (S. J. Kahn, M. Wleklinski, A. Aruffo, A. Farr, D. Coder, and M. Kahn, J. Exp. Med. 182:1243-1258,1995). In this report we identify the major surface glycoproteins, including the SA85-1 glycoproteins, as T. cruzi ligands of the mannose-binding protein. Further characterization of the interaction between the mannose-binding protein and T. cruzi demonstrates that (i) the SA85-1 glycoproteins are expressed by amastigotes and trypomastigotes but only amastigotes express the mannose-binding protein ligand, (ii) treatment of amastigotes with alpha-mannosidase inhibits the binding of mannose-binding protein, and (iii) amastigote binding of mannose-binding protein is stable despite the spontaneous shedding of some glycoproteins from its surface. Together, the data indicate that developmentally regulated glycosylation of surface glycoproteins controls the expression of ligands that affect the interactions between T. cruzi and mannose-binding protein. It has been established that the binding of mannose-binding protein to microorganisms facilitates their uptake into phagocytic cells. Preferential opsonization of amastigotes with mannose-binding proteins may account for their clearance from the circulation and may contribute to the parasite's ability to invade different cell types.

Animals↗

PubMed1996

Helicobacter pylori and Porphyromonas gingivalis lipopolysaccharides are poorly transferred to recombinant soluble CD14.

Helicobacter pylori and Porphyromonas gingivalis are gram-negative bacteria associated with chronic inflammatory diseases. These bacteria possess lipopolysaccharides (LPSs) that are able to activate human monocytes to produce tumor necrosis factor alpha but fail to activate human endothelial cells to express E-selectin. With Escherichia coli LPS, tumor necrosis factor alpha activation requires membrane-bound CD14 and E-selectin expression requires soluble CD14 (sCD14). Therefore, the ability of H. pylori and P. gingivalis LPSs to transfer to and bind sCD14 was examined by using immobilized recombinant sCD14 and human serum or recombinant LPS-binding protein (LBP). H. pylori and P. gingivalis LPSs were transferred to sCD14 when serum or LBP was present. However, the transfer of these LPSs to CD14 in serum was significantly slower than the transfer of E. coli LPS. Quantitation of the transfer rates by Michaelis-Menten kinetics yielded K(m) values of 6 and 0.1 nM for H. pylori and E. coli LPSs, respectively. The amount of P. gingivalis LPS required to obtain half-maximum binding to CD14 was approximately 10-fold greater than the amount of E. coli LPS required. The slower transfer rates displayed by these LPSs can be explained by the poor binding to LBP observed in direct binding assays. These results are consistent with the proportionately lower ability of these LPSs to activate monocytes compared with E. coli LPS. However, the ability of H. pylori and P. gingivalis LPSs to bind LBP and transfer to sCD14 demonstrates that the lack of endothelial cell CD14-dependent cell activation by these LPSs occurs distal to sCD14 binding.

Acute-Phase Proteins↗

PubMed1996

Immune regulation by CD40 and its ligand GP39.

Over the past three years, CD40 and its ligand (gp39, CD40L, TBAM) have been shown to be essential for humoral immune responses to thymus-dependent antigens. However, as the tissue distribution widens for those cells that express CD40 and gp39, we can now show that this ligand-receptor pair also plays an important role in the selection of self-reactive T cells in the thymus (central tolerance) and the regulation of tolerance in mature T cells (peripheral tolerance). Advances in our understanding of the molecular basis for CD40 biology is based in two areas of research. First, a major breakthrough in our understanding of how CD40 transduces biological events centers on the identification of a novel protein that binds to the cytoplasmic tail of CD40 and may act as a signal transducing molecule. Secondly, advances in molecular modeling and mutagenesis of this ligand-receptor pair have helped to identify the critical receptor/ligand contacts in the gp39/CD40 complex. Advances in each of these areas are discussed.

Animals↗

PubMed1995

Selective activation of c-Jun kinase mitogen-activated protein kinase by CD40 on human B cells.

The B cell surface antigen receptor, surface IgM (sIgM), is involved in B cell activation and proliferation. CD40 is involved in regulating IgE production and B cell survival. Cross-linking of B cell sIgM activates the Ras/Raf/p42erk2 pathway. In contrast, ligation of CD40 by antibody or soluble gp39 (CD40 ligand) leads to activation of the c-Jun kinase (JNK)/stress-activated protein kinase pathway. JNK/stress-activated protein kinase activation correlated with the stimulation of MEK kinase activity. CD40 does not activate the p42erk2 pathway, and sIgM fails to regulate the JNK/stress-activated protein kinase pathway in B cells. Thus, two important cell surface receptors involved in controlling specific B cell response differentially regulate sequential protein kinase pathways involving different members of the mitogen-activated protein kinase family. Anti-CD40 also rescued B cell apoptosis induced by anti-IgM. CD40 ligation did not affect the sIgM stimulation of p42erk2 activity. Conversely, sIgM ligation did not influence CD40 stimulation of JNK/stress-activated protein kinase. These results suggest that independent, parallel protein kinase response pathways are involved in the integration of sIgM and CD40 control of B cell phenotype and function.

Antibodies↗

PubMed1995

Cleavable CD40Ig fusion proteins and the binding to sgp39.

Recombinant immunoglobulin (Ig) fusion proteins of cell surface and intracellular proteins have wide applications. For example, fusion proteins have been used in the isolation, identification and study of ligands and the effects of binding or blocking a receptor-ligand pair, either in vivo or in vitro. For some applications, removal of the immunoglobulin Fc region is advantageous. We have developed two vectors for the expression of Ig fusion proteins that contain recognition sequences for protease cleavage using thrombin. In one vector, the sequence encoding the thrombin cleavage site is located at the junction of the DNA fragment encoding the protein or protein fragment to be studied and the hinge and constant regions of the immunoglobulin, allowing the generation of a monomeric form of the protein of interest. In the second vector, the sequence encoding the thrombin cleavage site is located between the sequences encoding the hinge and constant regions of the immunoglobulin, allowing for the generation of covalent dimers of the recombinant protein without the constant Fc domains. We have used these vectors to produce the constructs encoding two forms of the extracellular domain of CD40, CD40ThrIg and CD40HinThrIg, allowing production of a monomeric and dimeric form of recombinant CD40. Cleavage is efficient and complete. Following cleavage, there was no detectable binding of the monomeric form of CD40 to a soluble form of gp39, the ligand of CD40, while the dimeric form was able to bind. These vectors have been constructed to allow facile substitution with other sequences to generate cleavable forms of other proteins of interest.

Base Sequence↗