Search PubMed⌕ Search

Biomedical subjects

K B Elkon

Publications and source records attributed to K B Elkon.

At least 37 records · Page 2Linked to original sources
PubMed1999

Structure and function of Fas/Fas ligand.

Fas is a member of the TNF receptor family, that contain 2-6 cysteine-rich domains (CRDs) in their extracellular regions, a single transmembrane domain and variably sized intracytoplasmic domains. Fas belongs to a subgroup of family members that have a "death domain" near the carboxy-terminal region of the molecule. This domain binds to adaptor molecules that transmit a death signal to the cell. Signal transduction is complex and involves caspases, ceramides and stress pathways. Fas ligand is biologically active as a homotrimer. Receptor binding has been localized to the C-terminus and a self-association motif to the N-terminus of the ligand extracellular domain. Expression of ligand in a functionally active form is highly regulated at the transcriptional level as well as by cleavage by metalloproteinases. Since Fas/Fas ligand delete activated cells in the peripheral immune system, defects in this pathway predispose to autoimmune disorders.

Animals↗
PubMed1998

Complement-dependent clearance of apoptotic cells by human macrophages.

Apoptotic cells are rapidly engulfed by phagocytes, but the receptors and ligands responsible for this phenomenon are incompletely characterized. Previously described receptors on blood- derived macrophages have been characterized in the absence of serum and show a relatively low uptake of apoptotic cells. Addition of serum to the phagocytosis assays increased the uptake of apoptotic cells by more than threefold. The serum factors responsible for enhanced uptake were identified as complement components that required activation of both the classical pathway and alternative pathway amplification loop. Exposure of phosphatidylserine on the apoptotic cell surface was partially responsible for complement activation and resulted in coating the apoptotic cell surface with C3bi. In the presence of serum, the macrophage receptors for C3bi, CR3 (CD11b/CD18) and CR4 (CD11c/CD18), were significantly more efficient in the uptake of apoptotic cells compared with previously described receptors implicated in clearance. Complement activation is likely to be required for efficient uptake of apoptotic cells within the systemic circulation, and early component deficiencies could predispose to systemic autoimmunity by enhanced exposure to and/or aberrant deposition of apoptotic cells.

Annexin A5↗
PubMed1998

Differential expression of Fas and Fas ligand in acute and chronic graft-versus-host disease: up-regulation of Fas and Fas ligand requires CD8+ T cell activation and IFN-gamma production.

The parent-into-F1 model of acute and chronic graft-vs-host disease (GVHD) was used as an example of in vivo cell-mediated or Ab-mediated responses, respectively, and the roles of Fas and Fas ligand (FasL) were investigated. Using both flow cytometry and PCR methodologies, we found that acute GVHD mice exhibited significant up-regulation of Fas and FasL, whereas Fas/FasL up-regulation in chronic GVHD mice was equal to or marginally greater than that in uninjected mice. Functional studies confirmed that Fas/FasL contributed to the anti-host CTL activity of splenocytes from acute GVHD mice, although a perforin-dependent pathway was also identified. Despite the presence of FasL on both donor CD4+ and CD8+ T cells in acute GVHD mice, depletion studies demonstrated that all the in vitro anti-host CTL activity resided in the CD8+ population. Furthermore, injection of CD8-depleted B6 spleen cells into F1 mice blocked Fas/FasL up-regulation and IFN-gamma production, resulting in chronic GVHD. Lastly, up-regulation of Fas/FasL in acute GVHD mice could be blocked by anti-IFN-gamma mAb in vivo. Thus, in this in vivo model of alloantigen immune responsiveness, Fas/FasL up-regulation is critically dependent on Ag-specific (donor) CD8+ T cell activation and IFN-gamma production. Donor CD4+ T cell activation in the absence of CD8+ T cell activation results in an autoantibody-mediated response, no significant Fas/FasL up-regulation, impaired elimination of autoreactive B cells, and persistent humoral autoimmunity.

Acute Disease↗
PubMed1998

Systemic exposure to irradiated apoptotic cells induces autoantibody production.

During apoptotic cell death, cell surface ligands initiate phagocytosis of the dying cell. Clearance of these apoptotic cells is thought to occur without an immune response. Since a number of autoantigens are located at the cell surface or within apoptotic blebs, we examined whether exposure of mice to syngeneic apoptotic cells by the intravenous route could induce autoantibody production. Normal mice injected with syngeneic apoptotic thymocytes developed antinuclear autoantibodies and anticardiolipin and anti-ssDNA antibodies. The autoantibody levels were generally lower than those observed in MRL/Faslpr mice and were transient. Surprisingly, six out of six immunized mice demonstrated immunoglobulin G deposition in the glomeruli several months after immunization. These findings indicate that systemic exposure to apoptotic cells can induce an immune response in normal mice, and may help to explain antigen selection and initiation of the immune response in diseases characterized by increased rates of apoptosis such as AIDS and, possibly, systemic lupus erythematosus.

Animals↗
PubMed1998

Induction of in vitro heart block is not restricted to affinity purified anti-52 kDa Ro/SSA antibody from mothers of children with neonatal lupus.

The ability of affinity purified anti-52 kDa Ro/SSA antibody from patients without obstetric history of neonatal lupus to cause heart block using an experimental model was investigated. IgG-enriched fractions from sera of 20 systemic lupus erythematosus (SLE) and one Sjögren's syndrome (SS) all positives for anti-Ro/SSA antibodies as detected by CIE, were perfused on isolated whole rabbit hearts. Only six (29%) samples induced A-V block, five of them presenting low anti-Ro/SSA titre. All of them recognized the 52 kDa isoform on ELISA whereas only one had a concomitant binding to the 60 kDa protein. Moreover, affinity purified antibodies from two sera previously known to induce A-V block were obtained by affinity chromatography using a column containing the full-length 52 kDa Ro/SSA fusion protein. Paired eluate and effluent devoid of anti-52 kDa activity from the same patient were individually perfused in whole hearts. The ability to cause cardiac blockade was restricted to the affinity anti-52 kDa eluates. In addition, anti-52 kDa eluates from three IgG fractions that primarily failed to induce blockade remained ineffective. The present study has added to our knowledge that affinity anti-52 kDa Ro/SSA antibodies from mothers with healthy infants are capable of causing in vitro cardiac conduction disturbances. A prospective follow up of these patients will better delineate the clinical usefulness of this experimental model.

Antibodies, Antinuclear↗
PubMed1997

Separate domains of the human fas ligand dictate self-association and receptor binding.

The Fas receptor rapidly induces apoptosis when activated by ligand binding or by cross-linking with anti-Fas antibody. The Fas ligand (FasL), a member of the tumor necrosis factor family of ligands, is a 40-kilodalton type II transmembrane protein which is cleaved to produce soluble ligand. Although the Fas-FasL interaction plays a critical role in peripheral T cell homeostasis and cytotoxic T lymphocyte-mediated target cell killing, the requirements for human FasL receptor binding and oligomerization have not been defined. Here we report two distinct domains of the ligand which are responsible for self-association and binding to the Fas receptor. A COOH-terminal sequence of the FasL was found to be required for binding and biological activity, as verified by deletion mutagenesis, use of the NOK-1 blocking antibody and the humanized gld FasL mutation. N-Linked glycosylation of the FasL was not required for biological activity. However, the FasL expression level was dependent upon the three N-linked glycosylation sites. Moreover, the ability of the FasL to self-associate was not dependent upon transmembrane or cytoplasmic sequences, but was localized to a 47-amino acid region in its extracellular domain. These results indicate that the FasL-Fas receptor complex depends upon independent motifs located within the extracellular domain of the FasL.

Animals↗
PubMed1997

Interleukin-10 promotes activation-induced cell death of SLE lymphocytes mediated by Fas ligand.

Immune function in SLE is paradoxically characterized by active T cell help for autoantibody production, along with impaired T cell proliferative and cytokine responses in vitro. To reconcile these observations, we investigated the possibility that the accelerated spontaneous cell death of SLE lymphocytes in vitro is caused by an activation-induced cell death process initiated in vivo. 27 SLE patients, three patients with systemic vasculitis, seven patients with arthritis, and 14 healthy subjects were studied. Patients with clinically active SLE or systemic vasculitis had accelerated spontaneous death of PBMC with features of apoptosis at day 5 of culture. A prominent role for IL-10 in the induction of apoptosis was observed, as neutralizing anti-IL-10 mAb markedly reduced cell death in the active SLE patients by 50%, from 22.3 +/- 5.2% to 11.2 +/- 2.8%, and the addition of IL-10 decreased viability in the active SLE group, but not in the control group, by 38%. In addition, apoptosis was shown to be actively induced through the Fas pathway. The potential clinical relevance of T cell apoptosis in active SLE is supported by the correlation of increased apoptosis and IL-10 levels in vitro with low lymphocyte counts in vivo. We conclude that the spontaneous cell death observed in vitro in lymphocytes from patients with SLE and other systemic autoimmune disorders results from in vivo T cell activation, is actively induced by IL-10 and Fas ligand, and reflects pathophysiologically important events in vivo. Activation-induced cell death in vivo provides a pathogenic link between the aberrant T helper cell activation and impaired T cell function that are characteristic features of the immune system of patients with SLE.

Adult↗
PubMed1997

Requirement of cysteine-rich repeats of the Fas receptor for binding by the Fas ligand.

The Fas receptor is a member of a family of cell death receptors, including tumor necrosis factor receptor I (TNFR I), death receptor 3 and 4 (DR3 and DR4), and cytopathic avian receptor 1 (CAR1). The Fas receptor is composed of several discrete domains, including three cysteine-rich domains (CRDs), a transmembrane domain, and an intracellular domain responsible for transmitting an apoptotic signal. While the mechanism of Fas-mediated cell death has become elucidated, the requirements for Fas ligand binding to the receptor have not been fully defined. Using a series of chimeric Fc-receptor fusion proteins between the human Fas receptor and TNFR I, each cysteine-rich domain of Fas was found to be required for interaction with the Fas ligand. Interestingly, TNFR I CRD1 could partially substitute for the Fas CRD1. The importance of this domain was underscored by the analysis of a Fas extracellular mutation (C66R), which resulted in a complete loss of ligand binding. This mutation was cloned from a human patient suffering from Canale-Smith syndrome, which is characterized by autoimmunity resembling that observed in the lpr and lprcg mice. The localization of essential ligand binding domains in the Fas receptor correlated exactly with the ability of the Fas receptor fusion proteins to prevent cell death mediated by the Fas ligand.

Animals↗
PubMed1997

Fas ligand expression and function in systemic lupus erythematosus.

Mutations in the Fas receptor or its ligand (FasL) lead to lupus-like systemic autoimmune diseases in mice and in some humans. To determine whether a significant number of patients with systemic lupus erythematosus (SLE) have impaired FasL function, we compared T cell effector function by superantigen-activated CD4+ T cell lines or by anti-CD3- and IL-2-generated cytotoxic T cells. No differences were observed between SLE and normal control superantigen-derived CD4+ T cells in either the ability of these cells to up-regulate Fas expression or to induce apoptosis of the Fas-sensitive target B cells. When anti-CD3/IL-2-activated T cells were examined, SLE T cells had a modest reduction (-8%) in T cell cytotoxicity compared with normal controls, but the reduction was similar to the rheumatoid arthritis disease controls. A modest reduction in cytotoxicity was evident in both the Fas and perforin/granzyme pathways as determined by testing Fas-positive and -negative targets as well as by selective blockade of the perforin/granzyme pathway with concanamycin. These results indicate that no specific defects in FasL function are evident in the majority of SLE patients under the in vitro conditions tested. The proportional reduction in FasL and perforin/granzyme function in SLE and rheumatoid arthritis patients following anti-CD3/IL-2 stimulation most likely reflects subtle differences in activation in patient-derived vs normal control T cells.

Animals↗
PubMed1997

Tumor necrosis factor alpha plays a central role in immune-mediated clearance of adenoviral vectors.

Adenovirus (Ad) gene transfer vectors are rapidly cleared from infected hepatocytes in mice. To determine which effector mechanisms are responsible for elimination of the Ad vectors, we infected mice that were genetically compromised in immune effector pathways [perforin, Fas, or tumor necrosis factor alpha (TNF-alpha)] with the Ad vector, Ad5-chloramphenicol acetyl transferase (CAT). Mice were sacrificed at 7-60 days postinfection, and the levels of CAT expression in the liver determined by a quantitative enzymatic assay. When the livers of infected mice were harvested 28 days postinfection, the levels of CAT expression revealed that the effectors most important for the elimination of the Ad vector were TNF-alpha > Fas > perforin. TNF-alpha did not have a curative effect on infected hepatocytes, as the administration of TNF-alpha to infected severe combined immunodeficient mice or to infected cultures in vitro had no specific effect on virus persistence. However, TNF-alpha-deficient mice demonstrated a striking reduction in the leukocytic infiltration early on in the infection, suggesting that TNF-alpha deficiency resulted in impaired recruitment of inflammatory cells to the site of inflammation. In addition, the TNF-deficient mice had a significantly reduced humoral immune response to virus infection. These results demonstrate a dominant role of TNF-alpha in elimination of Ad gene transfer vectors. This result is particularly important because viral proteins that disable TNF-alpha function have been removed from most Ad vectors, rendering them highly susceptible to TNF-alpha-mediated elimination.

Adenoviridae↗
PubMed1997

Intercellular adhesion molecule-1 deficiency protects MRL/MpJ-Fas(lpr) mice from early lethality.

MRL/MpJ-Fas(lpr) (Fas(lpr)) mice develop a rapidly fatal form of systemic autoimmune disease characterized by glomerulonephritis and vasculitis similar to severe cases of systemic lupus erythematosus in humans. To evaluate the requirement for intercellular adhesion molecule-1 (ICAM-1) in the pathogenesis of tissue injury in this model, we created ICAM-1-deficient MRL/MpJ-Fas(lpr) (ICAM-1/Fas(lpr)) mice. ICAM-1 deficiency resulted in a striking improvement in the survival of Fas(lpr) mice (median +/- SEM survival of Fas(lpr) = 26 +/- 1.7 vs ICAM-1/Fas(lpr) = 47 +/- 2.4 wk, p < 0.0001) and the increased survival was associated with delayed elevations of blood urea nitrogen levels in the ICAM-1/Fas(lpr) mice. Histologic examination of the ICAM-1/Fas(lpr) mice revealed an overall reduction in glomerular disease and a significant reduction in vasculitis in the kidney, lung, skin, and salivary glands when compared with Fas(lpr). These findings indicate that ICAM-1 plays a major role in development of glomerular and vascular injury in Fas(lpr) mice.

Animals↗
PubMed1997

Anti-P antibodies and neuropsychiatric lupus erythematosus.

A high proportion of patients with SLE develop neuropsychiatric lupus during the course of their disease. The expression of disease varies significantly in terms of clinical manifestations, onset, and severity, so that this form of lupus remains a major diagnostic and therapeutic challenge. Because affected tissue cannot be sampled and animal models are not readily available, scientific investigation is considerably hampered. Approximately 15% of lupus patients have anti-P antibodies. These autoantibodies are highly specific for SLE. Most series show a highly significant association between anti-P and lupus psychosis, and this result is now confirmed in a series of 336 SLE patients. The mechanism explaining this association is uncertain and may simply reflect an immune response to damaged tissue. However, the possibility that the antibodies are pathogenic by directly binding to cell-surface receptors on neuronal cells or that they penetrate cells and inhibit protein synthesis within the cell requires further investigation. Whether T cells are involved in the pathogenesis of CNS disease is an important question that has received little attention. As in the CNS of patients with multiple sclerosis and patients with other autoimmune diseases, excess production of cytokines may contribute significantly to organ inflammation. We report associations between anti-P autoantibodies and certain MHC class II alleles, particularly HLA-DQB1*0602. These findings support a role for T cells in anti-P autoantibody production and encourage further studies of the role of autoantigen-specific T and B cells in injury to the central nervous system.

Autoantibodies↗
PubMed1997

Immunohistochemical analysis of murine CD95/Fas/Apo-1 receptor and its ultrastructural distribution in the thymus.

CD95/Fas/Apo-1 is a cell surface receptor that, upon contact with its ligand, induces cells to die by apoptosis. In view of the importance of Fas receptor (FasR) in immunologic tolerance, an immunohistochemical analysis of FasR expression was performed in the lymphoid and certain parenchymal tissues of normal and mutant MRL/lpr mice using a rabbit polyclonal anti-Fas receptor antibody. FasR was expressed by immunoperoxidase (IP) in the cortex and at the corticomedullary junction of the thymus of normal mice. By immunoelectron microscopy FasR was detected on the cell membrane of normal thymocytes. In MRL/lpr mice, FasR protein expression could not be clearly detected. FasR protein expression was not detected in the heart, liver or ovary by IP, presumably reflecting the low number of receptors in these tissues.

Animals↗
PubMed1997

Perforin, a cytotoxic molecule which mediates cell necrosis, is not required for the early control of mycobacterial infection in mice.

Host defense against mycobacterial infection requires the participation of monocytes and T cells. Both CD4+ and CD8+ T cells have been shown to be important in resistance to mycobacterial infection in vivo. The main contribution of CD4+ T cells to the protective antituberculosis response involves the production of Th1-type cytokines, including interleukin-2 (IL-2) and gamma interferon (IFN-gamma). CD8+ T cells have been considered to be responsible primarily for cytotoxicity mediated by toxic molecules, including perforin. CD8+ T cells may also elaborate Th1-type cytokines, such as IFN-gamma, in response to the infection. To elucidate the contribution of perforin-mediated target cell death to the control of mycobacterial infection in vivo, mice with a disruption in the perforin gene (P-/-) were infected with Mycobacterium bovis BCG or M. tuberculosis Erdman for 5 and 13 weeks, respectively. At 1, 3, 5, and 13 weeks postinfection, the number of viable mycobacteria in the lungs, spleens, and livers of mice were determined by CFU assay. The infected tissues were examined histologically, and cytokine mRNA levels in the spleens of these mice were determined. Similar studies were carried out in Fas receptor-defective (CBA/lpr(cg)) mice to evaluate the contribution of this alternative cytotoxic pathway to the control of mycobacterial infection. The absence of either perforin gene function or Fas receptor gene function did not modify the course of experimental mycobacterial infection in these mice. In addition, both P-/- and Fas receptor-defective mice appeared to have a compensatory activation of cytokine genes, even in the absence of the experimental infection. P-/- mice had a mean 3.4- to 5-fold increase in mRNA levels for IL-10, IL-12p35, IL-6, and IFN-gamma. Similarly, Fas receptor-defective mice had a mean 3- to 3.6-fold increase in mRNA levels for IFN-gamma, IL-12p35, and IL-10. Our results indicate that both perforin-mediated cytotoxicity and Fas-mediated cytotoxicity do not appear to be necessary for the early control of mycobacterial infection in vivo.

Animals↗
PubMed1996

A major role for the Fas pathway in acute graft-versus-host disease.

Recent studies have suggested a role for the Fas pathway in the wasting syndrome associated with lpr-->wild-type bone marrow transplants. To directly examine whether Fas ligand has a major role in the development of acute graft-vs-host disease (GVHD), Fas ligand-deficient (gld) mice were used as donors and C3H/HeJ x C57BL/6F1 as recipients in the parent-into-F1 model of acute GVHD. Transplantation of C3H/gld spleen cells induced significantly less host lymphoid depletion and was associated with less antihost cytotoxic activity in vitro when compared with wild-type C3H donor cells. The reduced depletion of host lymphocytes was explained by both impaired antihost T cell cytolytic activity and by reduced expansion of gld donor T cells in F1 recipients. These findings not only indicate that the Fas ligand is an important effector molecule in acute GVHD, but also provide in vivo evidence supporting a role for Fas/Fas ligand interactions in T cell expansion and maturation.

Acute Disease↗
PubMed1996

Fas gene mutations in the Canale-Smith syndrome, an inherited lymphoproliferative disorder associated with autoimmunity.

BACKGROUND: The Canale-Smith syndrome is a childhood disorder characterized by lymphadenopathy and autoimmunity. The similarity between this syndrome and that in mice with the lymphoproliferation (lpr) phenotype or the generalized-lymphoproliferative-disease (gld) phenotype led us to investigate whether it too is caused by mutations of the Fas gene (lpr mice) or the Fas ligand (gld mice), which regulate apoptosis in lymphocytes. METHODS: We studied four patients with the syndrome and their families. T-lymphocyte phenotypes were analyzed, and the susceptibility of activated T cells to Fas-mediated apoptosis in vitro was determined. Mutations of Fas were sought by nucleotide-sequence analysis. RESULTS: Patients with the Canale-Smith syndrome had increased numbers of circulating double-negative T cells (>20 percent) and profoundly impaired apoptosis of activated T cells incubated with an anti-Fas antibody. Three novel Fas mutations were identified, all of which were heterozygous and predicted to impair signal transduction by Fas. Autoimmune manifestations of the disease, such as hemolytic anemia and thrombocytopenia, persisted into adolescence. Two patients followed into adulthood had intermittent lymphadenopathy, which diminished over time. Neoplasms developed in both, and one died of hepatocellular carcinoma at the age of 43. CONCLUSIONS: Patients with the Canale-Smith syndrome have mutations in Fas, which implicates this gene in the accumulation of lymphocytes and the autoimmunity characteristic of the syndrome.

Adult↗