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.     

Antitumor immune responses induced by ionizing irradiation and further immune stimulation

The therapy of cancer emerged as multimodal treatment strategy. The major mode of action of locally applied radiotherapy (RT) is the induction of DNA damage that triggers a network of events that finally leads to tumor cell cycle arrest and cell death. Along with this, RT modifies the phenotype of the tumor cells and their microenvironment. Either may contribute to the induction of specific and systemic antitumor immune responses. The latter are boosted when additional immune therapy (IT) is applied at distinct time points during RT. We will focus on therapy-induced necrotic tumor cell death that is immunogenic due to the release of damage-associated molecular patterns. Immune-mediated distant bystander (abscopal) effects of RT when combined with dendritic cell-based IT and the role of fractionation of radiation in the induction of immunogenic tumor cell death will be discussed. Autologous whole-tumor-cell-based vaccines generated by high hydrostatic pressure technology will be introduced and the influence of cytokines and the immune modulator AnnexinA5 on the ex vivo generated or in situ therapy-induced vaccine efficacy will be outlined. RT should be regarded as immune adjuvant for metastatic disease and as a tool for the generation of an in situ vaccine when applied at distinct fractionation doses or especially in combination with IT to generate immune memory against the tumor. To identify the most beneficial combination and chronology of RT with IT is presumably one of the biggest challenges of innovative tumor research and therapies.     

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

ATP-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.     

Modulation of radiochemoimmunotherapy-induced B16 melanoma cell death by the pan-caspase inhibitor zVAD-fmk induces anti-tumor immunity in a HMGB1-, nucleotide- and T-cell-dependent manner

One prerequisite that radiotherapy (RT) and chemotherapy (CT) result in anti-tumor immune responses is triggering of immunogenic cell death forms such as necroptosis. The latter is inducible by inhibition of apoptosis with the pan-caspase inhibitor zVAD-fmk. The design of multimodal therapies that overcome melanoma''s resistance to apoptosis is a big challenge of oncoimmunology. As hints exist that immune stimulation by hyperthermia (HT) augments the efficacy of melanoma therapies and that tumors can be sensitized for RT with zVAD-fmk, we asked whether combinations of RT with dacarbazine (DTIC) and/or HT induce immunogenic melanoma cell death and how this is especially influenced by zVAD-fmk. Necroptosis was inducible in poorly immunogenic B16-F10 melanoma cells and zVAD-fmk generally increased melanoma cell necrosis concomitantly with the release of HMGB1. Supernatants (SNs) of melanoma cells whose cell death was modulated with zVAD-fmk induced an upregulation of the activation markers CD86 and MHCII on macrophages. The same was seen on dendritic cells (DCs), but only when zVAD-fmk was added to multimodal tumor treatments including DTIC. DCs of MyD88 KO mice and DCs incubated with SNs containing apyrase did not increase the expression of these activation markers on their surface. The in vivo experiments revealed that zVAD-fmk decreases the tumor growth significantly and results in a significantly reduced tumor infiltration of Tregs when added to multimodal treatment of the tumor with RT, DTIC and HT. Further, a significantly increased DC and CD8+ T-cell infiltration into the tumor and in the draining lymph nodes was induced, as well as an increased expression of IFNγ by CD8+ T cells. However, zVAD-fmk did not further reduce tumor growth in MyD88 KO mice, mice treated with apyrase or RAG KO mice. We conclude that HMGB1, nucleotides and CD8+ T cells mediate zVAD-fmk induced anti-melanoma immune reactions in multimodal therapy settings.The cancer immune editing concept raised by Schreiber and colleagues¹ and the findings that distinct chemotherapeutic agents induce immunogenic cancer cell death forms² opened our minds that standard tumor therapies alone and especially in combination with further immune therapies are capable of inducing anti-tumor immune responses.³ The phenotype of the tumor cells and the tumor microenvironment are altered during therapy and, thereby, the tumor might become visible for the immune system.⁴ A main prerequisite for induction of anti-tumor immunity is triggering of immunogenic tumor cell death forms.⁵Apoptosis is non- or even anti-inflammatory.⁶ In contrast, necrotic cells bear per se a high inflammatory and immunogenic potential. Damage-associated molecular patterns (DAMPs) are released because the plasma membrane of necrotic cells is disturbed.^{7, 8} Danger signals as the high mobility group protein B1 (HMGB1) and the nucleotide adenosine triphosphate (ATP) activate DCs, foster cross-presentation of antigens and consecutively the activation of T cells.⁹ DAMPs therefore link radio- and/or chemotherapy-induced local alterations of the tumor cells and subsequent systemic anti-tumor immune reactions.^{10, 11} HMGB1 is mostly passively released by therapy-induced necrotic tumor cells.¹² The activation of DCs by HMGB1 is induced by its binding to TLR2 or TLR4.^{13, 14} HMGB1 is further required for the migration of maturing DCs.¹⁵ The nucleotide ATP is often actively emitted and acts on purinergic receptors, especially on P2RX7.^{16, 17}Activation of DCs is crucial for the success of multimodal tumor treatments.¹⁸ Several preclinical and clinical studies have demonstrated that tumor cell death induced by radiochemotherapy in combination with intratumoral DC injection induces strong anti-tumor immune responses in several tumor entities.^{19, 20, 21} These responses can be enhanced by hyperthermia (HT). Mild HT is an additive therapy to radiotherapy (RT) and/or chemotherapy (CT) in which tumor tissue is locally heated to temperatures of 40–44 °C for a time period of 1 h. HT fosters protein aggregation and aggravates radiation- and chemotherapy-induced repair of DNA damage.²² In addition, locally applied HT is capable of inducing systemic anti-tumor responses.²³Melanoma is the most dangerous form of skin cancer and its response to CT and RT is poor.²⁴ To overcome melanoma''s resistance to apoptosis, the search for multimodal treatments that aim of inducing immunogenic cell death forms is a big challenge of innovative oncoimmunology,²⁵ as much as to understand the mechanisms of therapy-induced immunogenic melanoma cell death. Nowadays, evidence has come up that necrosis as immunogenic cell death form can also occur in a programmed manner.^{26, 27} Necroptosis is independent of caspases and mainly occurs when caspases are not activated or inhibited.²⁸ The pan-caspase inhibitor zVAD-fmk has been shown to inhibit apoptosis and concomitantly foster necroptosis.²⁹ Further, encouraging preclinical studies have been performed using caspase inhibitors to reduce apoptosis in neurological diseases³⁰ and to reduce angiogenesis in solid tumors.³¹ First hints exist that immune stimulation by HT is capable of augmenting the efficacy of CT and RT treatments in melanoma³² and that solid tumors can be rendered more sensitive to radiation by treatment with the pan-caspase inhibitor zVAD-fmk.³¹ Meaningful data regarding potential clinical efficacy of caspase inhibitors such as zVAD-fmk will only be yielded if the cell death pathways stimulated in model systems reflect that triggered in patients.³³ Therefore, we examined here for the first time whether combinations of the clinically relevant single dose of RT of 2 Gy with the only for metastatic melanoma FDA-approved CT agent dacarbazine (DTIC) or combinations with HT (41.5 °C for 1 h) induce immunogenic melanoma cell death and how zVAD-fmk is capable of improving the melanoma''s immunogenicity by modulating the therapy-induced melanoma cell death.     

Loss of GM1 surface expression precedes annexin V-phycoerythrin binding of neutrophils undergoing spontaneous apoptosis during in vitro aging.

BACKGROUND: Apoptosis of neutrophil granulocytes is an important determinant of the resolution of inflammation. Apoptotic neutrophils undergo specific alterations in their receptor profiles. These alterations are likely to contribute to the characteristic functional silencing of the dying cells. METHODS: By flow cytometry and fluorescence microscopy, we analyzed the ganglioside GM1, a lipid raft marker, with respect to its surface expression on neutrophil and eosinophil granulocytes. Apoptosis was monitored by morphological changes and by the binding of annexin V-phycoerythrin (AxV-PE). RESULTS: GM1, which was stained by the cholera toxin subunit B, was found only on neutrophil granulocytes; eosinophil granulocytes did not bind cholera toxin subunit B. GM1 was lost from the surfaces of neutrophils before AxV-PE binding (early apoptosis). Surprisingly, GM1 reappeared during the late stages of apoptosis, although without functional consequences. GM1 was found on the cell surface and in intracellular membranes, whereas CD16 was found only at the cell surface. CONCLUSIONS: Loss of surface GM1 is a new marker for the detection of the aging of neutrophils. Its loss precedes the binding of AxV-PE of neutrophils.     

Photopheresis with UV-A light and 8-methoxypsoralen leads to cell death and to release of blebs with anti-inflammatory phenotype in activated and non-activated lymphocytes

Background: Extracorporeal photopheresis is a therapy for treatment of autoimmune diseases, cutaneous T-cell lymphoma, organ graft rejection as well as graft-versus-host diseases. The exact mechanism how the combination of 8-methoxypsoralen plus UV-A irradiation (PUVA) acts is still unclear. We investigated the cell death of activated and non-activated lymphocytes after PUVA treatment as well as the rate of released blebs and their antigen composition. Results: In presence of 8-MOP, UV-A light highly significantly increased the cell death of activated lymphocytes. The same was observed to a lesser extent in non-activated cells. Blebs derived from activated lymphocytes after PUVA treatment showed the highest surface exposition of phosphatidylserine. These blebs also displayed a high exposure of the antigens CD5 and CD8 as well as a low exposure of CD28 and CD86. Conclusion: PUVA treatment exerts anti-inflammatory effects by inducing apoptosis and apoptotic cell-derived blebs with immune suppressive surface composition.