J. R. Kalden

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

After shrinkage apoptotic cells expose internal membrane-derived epitopes on their plasma membranes

Apoptosis and phagocytosis of apoptotic cells are crucial processes. At best the phagocytic machinery detects and swallows all apoptotic cells in a way that progression to secondary necrosis is avoided. Otherwise, inflammation and autoimmune diseases may occur. Most apoptotic cells are phagocytosed instantaneously in a silent fashion; however, some dying cells escape their clearance. If the cells are not cleared early, they lose membranes due to extensive shedding of membrane surrounded vesicles (blebbing) and shrink. It is unclear how apoptotic cells compensate their massive loss of plasma membrane. Here, we demonstrate that endoplasmic reticulum- (ER) resident proteins (calnexin, the KDEL receptor and a dysfunctional immunoglobulin heavy chain) were exposed at the surfaces of shrunken late apoptotic cells. Additionally, these cells showed an increased binding of lectins, which recognize sugar structures predominantly found as moieties of incompletely processed proteins in ER and Golgi. In addition the ER resident lipophilic ER-Tracker™ Blue-White DPX, and internal GM1 were observed to translocate to the cell surfaces during late apoptosis. We conclude that during blebbing of apoptotic cells the surface membrane loss is substituted by immature membranes from internal stores. This mechanism explains the simultaneous appearance of preformed recognition structures for several adaptor proteins known to be involved in clearance of dead cells.

Viable, apoptotic and necrotic monocytes expose phosphatidylserine: cooperative binding of the ligand Annexin V to dying but not viable cells and implications for PS-dependent clearance

Viable, apoptotic and necrotic monocytes expose phosphatidylserine: cooperative binding of the ligand Annexin V to dying but not viable cells and implications for PS-dependent clearance

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

What triggers anti-dsDNA antibodies?

Analysis of somatic mutations revealed that induction of anti-dsDNA autoantibodies from SLE patients are antigen driven and thus T cell dependent. Since DNA per se has repeatedly been shown not to be immunogenic, various mechanisms leading to the production of anti-dsDNA-antibodies have been discussed including the role of oligonucleosomes. In the present study we demonstrate that the percentage of macrophage engulfing apoptotic cell material was significantly reduced in SLE as compared to control patients. These data suggest that, in contrast to a non-inflammatory clearance of apoptotic cell, phagocytosis of apoptotic cell material may be decreased in SLE patients, possibly leading to a presentation of autoantigens and thus possibly triggering an autoantibody response to nucleoproteins.

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.

Neue Therapieentwicklungen in der Rheumatoiden Arthritis

Zusammenfassung Trotz der in der Therapie der Rheumatoiden Arthritis (RA) erarbeiteten Fortschritte, die durch die TNFα-Blockade erzielt werden konnten, ist es notwendig, die Studienlage weiter zu optimieren (Etanercept/MTX Studie mit Erfassung radiologischer Daten; Veröffentlichung der D2E7 Studienergebnisse; Kombination von D2E7 mit MTX). Darüber hinaus werden neue Immunbiologika (PEG-TNFRI, PEG-TNFα-Antikörper, löslicher TNFRI; CTLA4Ig, CD40Ligand-Antikörper, Antikörper gegen IFN-γ, IL-6, IL-12, IL-15, IL-18, Complemente) oder Hemmstoffe der TNFα-Translation (Peptide, Anti-Sense-Konstrukte) oder TNFα-Syntheseinduktion (Inhibitoren des NFκB, der p38 MAP-Kinase, Phosphodiesterase IV, des TNFα converting enzymes) getestet. Prinzipiell davon unterscheiden sich Antagonisten der Complement-Convertase oder Collagenase sowie Ansätze zur Vakzinierung oder T-Zell-Anergisierung. Für Patienten mit MTX-Unverträglichkeit ist die Testung alternativer Basistherapeutika in Kombination mit den TNFα-Blockern wichtig, auch eine Kombination mit anderen Immunbiologika (anti-CD4-Antikörper, IL-4, IL-10, IL-1RA) ist sinnvoll. Der Einsatz von TNFα-Blockern bei Patienten mit einer Früh-RA ist derzeit in Studien unter Erprobung. Damit verbunden ist die Frage, inwieweit eine Auslassstudie, in der nach Remissionsinduktion mit TNFα-Blockern und MTX entweder die Therapie nur mit dem DMARD weitergeführt wird oder auf ein anderes Basistherapeutikum in Monotherapie oder Kombination umgestellt wird (z.B. Leflunomid/MTX), ein anhaltendes krankheitsinaktivierendes Ergebnis erzielen kann.Summary Despite the anti-TNFα based progress in the treatment of RA, it is necessary to further optimize study designs and reports (Etanercept/MTX combination with results of radiological progression; publication of D2E7 trials; combination of D2E7 with MTX). Moreover, innovative immunobiologicals (PEG-TNFRI, PEG-TNFα antibody fragments, soluble TNFRI, CTLA4-Ig, CD40 ligand antibody, antibodies against IFN-γ, IL-6, IL-12, IL-15, IL-18, complements), inhibitors of TNFα translation (peptides, anti-sense constructs) or TNFα synthesis (targeting NFκB, p38 MAP-kinase, phosphodiesterase IV, TNFα converting enzyme) are forthcoming. Principally different are inhibitors of complement convertases or collagenase as well as vaccination studies or trials trying to induce T cell anergy. Furthermore, for patients with MTX side effects, alternative DMARDs need to be tested along with TNFα blockers. Combination studies of TNFα constructs with other immunobiologicals (anti-CD4, IL-4, IL-10, IL-1RA) should be evaluated. To date, TNFα blockers have been evaluated in very early RA. Finally, a step-down trial will test whether – after induction of remission with a TNFα blocker plus MTX – replacement of the TNFα blocker with MTX alone or in combination with leflunomide will be able to keep disease activity suppressed for a longer duration.

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.

Vaskuläre Veränderungen in der Pathogenese der systemischen Sklerose

Systemic sclerosis (SSc, scleroderma) is a connective tissue disease of unknown etiology. Perivascular inflammatory infiltrates and endothelial apoptosis with an impaired angiogenesis are observed in early stages of the disease, whereas later stages are characterized by an excessive accumulation of extracellular matrix proteins in the skin and various internal organs. Consistent with the ongoing endothelial cell damage, various markers of endothelial cells such as endothelin-1, sICAM-1, s-VCAM-1 and thrombomodulin are found in high levels in the serum of SSc patients. Surprisingly, the vascular endothelial growth factor (VEGF), a potent angiogenic molecule, is overexpressed in the skin of patients with SSc despite insufficient angiogenesis. Interestingly, patients suffering from diffuse SSc and patients without finger tip ulcers show higher VEGF levels compared to age- and sex-matched controls. These results indicate that a controlled overexpression of VEGF might help to protect against the manifestation of ischemic conditions. On the other hand, data from animal models indicate that a long-term, uncontrolled overexpression of VEGF might have paradox effects on the formation of new vessels leading to capillary changes similar to those observed in SSc. In addition to the impaired angiogenesis, defective vasculogenesis might contribute to the vascular symptoms of SSc.ZusammenfassungDie systemische Sklerose (SSc, Sklerodermie) ist eine Erkrankung des Bindegewebes, die durch eine Akkumulation von Proteinen der Extrazellularmatrix in der Haut und in inneren Organen gekennzeichnet ist. Weitere histopathologische Merkmale der SSc sind perivaskuläre Entzündungsinfiltrate und vaskuläre Veränderungen mit einer Reduktion der Gefäßdichte, welche beide bereits in frühen Krankheitsstadien nachweisbar sind. Die Ursachen der vaskulären Veränderungen sind vielschichtig. So konnte als eine der ersten pathologischen Veränderung bei SSc-Patienten eine erhöhte Apoptoserate der Endothelzellen nachgewiesen werden. Als Zeichen der Endothelzellschädigung finden sich verschiedene Endothelzell-Marker wie Endothelin-1, sICAM-1 und s-VCAM-1 sowie Thrombomodulin im Serum von SSc-Patienten erhöht. Daneben existieren Hinweise auf Defekte der Vaskulogenese mit einer erniedrigten Anzahl von zirkulierenden Endothelzell-Vorläuferzellen. Trotz einer Überexpression des potenten angiogenen Faktors Vascular Endothelial Growth Factor (VEGF) in der Haut von Patienten mit SSc ist auch die Angiogenese gestört. Interessanterweise zeigten Patienten ohne Fingerkuppenulzera höhere VEGF-Werte als die jeweiligen Vergleichspopulationen. Diese Ergebnisse deuten daraufhin, dass eine zeitlich begrenzte Überexpression von VEGF präventiv auf die Manifestation ischämischer Symptome wirken könnte. Ergebnisse von Tiermodellen deuten andererseits darauf hin, dass eine zeitlich unkontrollierte Expression von VEGF über mehrere Krankheitsstadien hinweg eher deletäre Effekte auf eine suffiziente Gefäßneubildung ausübt.SummarySystemic sclerosis (SSc, scleroderma) is a connective tissue disease of unknown etiology. Perivascular inflammatory infiltrates and endothelial apoptosis with an impaired angiogenesis are observed in early stages of the disease, whereas later stages are characterized by an excessive accumulation of extracellular matrix proteins in the skin and various internal organs. Consistent with the ongoing endothelial cell damage, various markers of endothelial cells such as endothelin-1, sICAM-1, s-VCAM-1 and thrombomodulin are found in high levels in the serum of SSc patients. Surprisingly, the vascular endothelial growth factor (VEGF), a potent angiogenic molecule, is overexpressed in the skin of patients with SSc despite insufficient angiogenesis. Interestingly, patients suffering from diffuse SSc and patients without finger tip ulcers show higher VEGF levels compared to age- and sex-matched controls. These results indicate that a controlled overexpression of VEGF might help to protect against the manifestation of ischemic conditions. On the other hand, data from animal models indicate that a long-term, uncontrolled overexpression of VEGF might have paradox effects on the formation of new vessels leading to capillary changes similar to those observed in SSc. In addition to the impaired angiogenesis, defective vasculogenesis might contribute to the vascular symptoms of SSc.