Wasilis Kolowos

Impaired uptake of apoptotic cells into tingible body macrophages in germinal centers of patients with systemic lupus erythematosus

OBJECTIVE To investigate the fate of apoptotic cells in the germinal centers (GCs) of patients with systemic lupus erythematosus (SLE). METHODS Lymph node biopsy specimens obtained from 7 SLE patients with benign follicular hyperplasia, 5 non-SLE patients with benign follicular hyperplasia (non-SLE), 5 patients with malignant follicular lymphoma, and 3 patients with dermatopathic lymphadenitis were stained with monoclonal antibodies against macrophages (CD68) and follicular dendritic cells (CR2/CD21). TUNEL staining and transmission electron microscopy were performed to detect apoptotic cells. Confocal microscopy was used to evaluate the in vivo capacity of tingible body macrophages to remove apoptotic cell material. RESULTS In a subgroup of patients with SLE, apoptotic cells accumulated in the GCs of the lymph nodes. The number of tingible body macrophages, which usually contained engulfed apoptotic nuclei, was significantly reduced in these patients. In contrast to what was observed in all controls, TUNEL-positive apoptotic material from SLE patients was observed to be directly associated with the surfaces of follicular dendritic cells (FDCs). CONCLUSION Our findings suggest that in a sub-group of SLE patients, apoptotic cells are not properly cleared by tingible body macrophages of the GCs. Consequently, nuclear autoantigens bind to FDCs and may thus provide survival signals for autoreactive B cells. This action may override an important control mechanism for B cell development, resulting in the loss of tolerance for nuclear antigens.

Complement binding is an early feature of necrotic and a rather late event during apoptotic cell death

The phagocytosis of dying cells is an integral feature of apoptosis and necrosis. There are many receptors involved in recognition of dying cells, however, the molecular mechanisms of the scavenging process remain elusive. The activation by necrotic cells of complement is well established, however, the importance of complement in the scavenging process of apoptotic cells was just recently described. Here we report that the complement components C3 and C4 immediately bound to necrotic cells. The binding of complement was much higher for lymphocytes compared to granulocytes. In case of apoptotic cell death complement binding was a rather late event, which in lymphocytes was preceded by secondary necrosis. Taken together complement binding is an immediate early feature of necrosis and a rather late event during apoptotic cell death. We conclude that complement may serve as an opsonin for fragments of apoptotic cells that have escaped regular scavenging mechanisms. Cell Death and Differentiation (2001) 8, 327–334

Treatment with annexin V increases immunogenicity of apoptotic human T-cells in Balb/c mice

Exposure of phosphatidylserine on the outer leaflet of the cytoplasmic membrane is an early event during apoptotic cell death and serves as a recognition signal for phagocytes. Usually the clearance of apoptotic cells does not initiate inflammation or immune response. We investigated the immune response in Balb/c mice towards apoptotic human T-cells. Animals injected with apoptotic cells showed significantly reduced humoral immune responses, especially Th1-dependent IgG2a titres, compared to controls immunised with viable cells. However, treatment of apoptotic cells with annexin V (AxV) significantly increased the humoral immune response. AxV binds with high affinity to anionic phospholipids and as a result interferes with the phosphatidylserine recognition by phagocytes. Our results indicate that AxV treatment may be used to increase the efficiency of apoptotic cell-based vaccines, e.g. some tumour vaccines. Cell Death and Differentiation (2000) 7, 911–915

UV irradiation inhibits ABC transporters via generation of ADP-ribose by concerted action of poly(ADP-ribose) polymerase-1 and glycohydrolase.

AbstractATP-binding cassette (ABC) transporters are involved in the transport of multiple substrates across cellular membranes, including metabolites, proteins, and drugs. Employing a functional fluorochrome export assay, we found that UVB irradiation strongly inhibits the activity of ABC transporters. Specific inhibitors of poly(ADP-ribose) polymerase-1 (PARP-1) restored the function of ABC transporters in UVB-irradiated cells, and PARP-1-deficient cells did not undergo UVB-induced membrane transport inhibition. These data suggest that PARP-1 activation is necessary for ABC transporter functional downregulation. The hydrolysis of poly(ADP-ribose) by poly(ADP-ribose) glycohydrolase (PARG) was also required, since specific PARG inhibitors, which limit the production of ADP-ribose molecules, restored the function of ABC transporters. Furthermore, ADP-ribose molecules potently inhibited the activity of the ABC transporter P-glycoprotein. Hence, poly(ADP-ribose) metabolism appears to play a novel role in the regulation of ABC transporters.

Microparticles Shed from Different Antigen‐Presenting Cells Display an Individual Pattern of Surface Molecules and a Distinct Potential of Allogeneic T‐Cell Activation

Various cells such as platelets, lymphocytes, endothelial cells, red blood cells and monocytes do release surface‐derived microparticles (mps). We analysed mp isolated from supernatants of cultured antigen‐presenting human cells (APCs) and human cell lines. Particle sizing by dynamic light scattering revealed a characteristic size of the particles ranging from 80 nm to 300 nm in viable cells and from 400 nm to 1200 nm in irradiated cells. Employing flow‐cytometry, we observed partly an altered surface protein composition of the mp compared to their cellular source. Mp originating from dendritic cells (DCs) differed in their surface composition from those released from monocytes and monocyte‐derived macrophages. In functional assays, these mp stimulated alloreactive T‐cells. The treatment of the cells with either UV‐B or lipopolysaccharide strongly influenced the quantity, the immunostimulatory features and the surface composition of the mp. Mp from apoptotic macrophages were able to reduce the stimulatory capacity of vital macrophages but not of DC. Apoptotic mps from DC, on the other hand, were always stimulatory. This is the first report regarding the study of mp released from DC and compared with those released from other APC.

Increased spontaneous in vitro apoptosis in double negative T cells of humans with a fas/apo-1 mutation.

We describe a 17 year old patient suffering from Canale-Smith syndrome (CSS) including chronic lymphadenopathy, splenomegaly, hypergammaglobulinemia and recurrent Coombs positive hemolytic crises. The parents are not consanguine, all other family members including two brothers are healthy. Peripheral blood mononuclear cells of the patient showed an increased rate of CD3 positive, CD4/CD8 double negative T-lymphocytes. In vitro assays showed these cells to have an increased rate of spontaneous apoptosis. Though expression of Fas/Apo-1 (CD95) and Fas-ligand (FasL) was detected on RNA- and protein level we found Fas/Apo-1 mediated apoptosis being significantly reduced. Sequencing of the fas/apo-1 gene proved the patient RT and his father to carry a point mutation at position 804 located in exon 9 (death domain) leading to an amino acid substitution. For developing of CSS, a fas/apo-1 mutation seems to be necessary but not sufficient. An additional independent mechanism must be involved in the pathogenesis of human lpr-phenotype.

Apoptosis of the Teratocarcinoma Cell Line Tera-1 Leads to the Cleavage of HERV-K10gag Proteins by Caspases and/or Granzyme B

Redistribution, post‐translational modifications and coclustering with viral antigens contribute to the immunogenicity of apoptotic cell‐derived autoantigens. Almost all known targets of the humoral autoimmune response in systemic lupus erythematosus (SLE) are cleaved by caspases or granzyme B during apoptosis. Antibodies against retroviral proteins can frequently be detected in the sera of SLE patients without overt retroviral infections. These antibodies may represent cross‐reactive antibodies or may have been induced by proteins encoded by endogenous retroviral sequences. We used Tera‐1 cells that abundantly express a group‐specific antigen of human endogenous retroviruses, HERV‐K10gag polyprotein, to investigate its processing during apoptosis. Tera‐1 cells induced to undergo apoptosis showed an altered HERV‐K10gag processing compared with viable cells. In addition, granzyme B was able to cleave HERV‐K10gag isolated from viable Tera‐1 cells.

Ätiopathogenese des systemischen Lupus erythematodes (SLE)

Die Atiopathogenese des SLE, bislang nur partiell verstanden, ist ein multifaktorieller Prozess. Die Erkrankung entsteht vermutlich aus der Interaktion von Suszeptibilitatsgenen mit Umweltfaktoren wie Viren, anderen infektiosen Agenzien, Medikamenten, Chemikalien, UV-Exposition u. A. Suszeptibilitatsgene sind als Gene definiert, die das relative Risiko einer bestimmten Erkrankungsmanifestation erhohen, ohne dass die meisten Individuen, die Trager dieses Gens sind, erkranken. Basierend auf Konkordanzanalysen bei Zwillingsstudien wird vermutet, dass mindestens 3 oder 4 solcher Gene fur die Manifestation des SLE notwendig sind. Beweisend fur eine genetische Predisposition sind u.a. die um das 3-bis lOfache erhohte Erkrankungswahrscheinlichkeit bei monozygoten im Vergleich zu dizygoten Zwillingen, das 8-bis 9fache relative Erkrankungsrisiko bei Verwandten ersten und zweiten Grads und Kopplungsanalysen, die die Erkrankung mit bestimmten Haplotypen und dem MHC-Locus assoziieren.