CD47 and death signaling in the immune system

Receptor-mediated death signaling plays a critical role both in proper control of immune responses and in killing of target cells by T cells. In addition to the recognized death receptors which all belong to the tumor necrosis factor receptor family, recent studies suggest that also other cell surface antigens may be involved in apoptotic signaling in the immune system. New data on the Ig family member CD47 implicate a functional role of this molecule in growth regulation of lymphocytes and suggest that the antigen mediates cell death by activating a non-classical form of apoptosis. This mini review will focus on CD47 as a possible death receptor on lymphocytes and also summarize some of the current knowledge on death control in the immune system.     

Role of oxidative stress and caspase 3 in CD47-mediated neuronal cell death

CD47 or integrin-associated protein promotes cell death in blood and tumor cells. Recently, CD47 signaling has been identified in neurons as well. In this study, we investigated the role of CD47 in neuronal cell death. Exposure of primary mouse cortical neurons to the CD47 ligand thrombospondin-1 or the specific CD47-activating peptide 4N1K induced cell death. Activation of CD47 elevated levels of active caspase 3 and increased the generation of reactive oxygen species (ROS) in a time-dependent manner. Both ROS scavengers and caspase inhibitors attenuated cell death. But ROS scavenging did not reduce the activation of caspase 3, and combination treatments with a caspase inhibitor plus free radical scavenger did not yield additive protection. Taken together, these data suggest that parallel and redundant pathways of oxidative stress and caspase-mediated cell death are involved. We conclude that CD47 mediates neuronal cell death through caspase-dependent and caspase-independent pathways.     

Elimination of tumor by CD47/PD-L1 dual-targeting fusion protein that engages innate and adaptive immune responses

The host immune system generally serves as a barrier against tumor formation. Programmed death-ligand 1 (PD-L1) is a critical “don't find me” signal to the adaptive immune system, whereas CD47 transmits an anti-phagocytic signal, known as the “don't eat me” signal, to the innate immune system. These and similar immune checkpoints are often overexpressed on human tumors. Thus, dual targeting both innate and adaptive immune checkpoints would likely maximize anti-tumor therapeutic effect and elicit more durable responses. Herein, based on the variable region of atezolizumab and consensus variant 1 (CV1) monomer, we constructed a dual-targeting fusion protein targeting both CD47 and PD-L1 using “Knobs-into-holes” technology, denoted as IAB. It was effective in inducing phagocytosis of tumor cells, stimulating T-cell activation and mediating antibody-dependent cell-mediated cytotoxicity in vitro. No obvious sign of hematological toxicity was observed in mice administered IAB at a dose of 100 mg/kg, and IAB exhibited potent antitumor activity in an immune-competent mouse model of MC38. Additionally, the anti-tumor effect of IAB was impaired by anti-CD8 antibody or clodronate liposomes, which implied that both CD8+ T cells and macrophages were required for the anti-tumor efficacy of IAB and IAB plays an essential role in the engagement of innate and adaptive immune responses. Collectively, these results demonstrate the capacity of an elicited endogenous immune response against tumors and elucidate essential characteristics of synergistic innate and adaptive immune response, and indicate dual blockade of CD47 and PD-L1 by IAB may be a synergistic therapy that activates both innate and adaptive immune response against tumors.     

LsGRP1, a class II glycine-rich protein of Lilium,confers plant resistance via mediating innate immune activation and inducing fungal programmed cell death

Defence-related LsGRP1 is a leaf-specific plant class II glycine-rich protein (GRP) involved in salicylic acid-induced systemic resistance against grey mould caused by necrotrophic Botrytis elliptica in lily (Lilium) cultivar Stargazer. The C-terminal region of LsGRP1 (LsGRP1^C) can inhibit fungal growth in vitro via a mechanism of inducing fungal apoptosis programmed cell death (PCD). In this study, the role of LsGRP1 in induced defence mechanism was investigated using LsGRP1-silenced Stargazer lily and LsGRP1-transgenic Arabidopsis thaliana. LsGRP1 silencing in lily was found to slightly inhibit plant growth and greatly increase the susceptibility to B. elliptica by suppressing callose deposition and early reactive oxygen species (ROS) accumulation. In contrast, LsGRP1-transgenic Arabidopsis showed higher resistance to Botrytis cinerea and also to Pseudomonas syringae pv. tomato DC3000 as compared to the wild type, accompanied with the enhancement of callose deposition and ROS accumulation. Additionally, LsGRP1 silencing increased plant cell death caused by B. elliptica secretion and reduced pathogen-associated molecular pattern (PAMP)-triggered defence activation in Stargazer lily. Consistently, LsGRP1 expression boosted PAMP-triggered defence responses and effector recognition-induced hypersensitive response in Arabidopsis. Moreover, fungal apoptosis PCD triggered by LsGRP1 in an LsGRP1^C-dependent manner was demonstrated by leaf infiltration with LsGRP1^C-containing recombinant proteins in Stargazer lily. Based on these results, we presume that LsGRP1 plays roles in plant defence via functioning as a pathogen-inducible switch for plant innate immune activation and acting as a fungal apoptosis PCD inducer to combat pathogen attack.     

Mechanosensitive induction of apoptosis in fibroblasts is regulated by thrombospondin-1 and integrin associated protein (CD47)

Fibroblasts are cultured in three-dimensional collagen matrices to investigate the effect of mechanical tension on the regulation of apoptosis. Under the influence of mechanical loading, the cells show little apoptosis whereas releasing of tension leads to an increase up to tenfold during the first 24 h and remains constant for further 48 h. An autocrine loop of the integrin _V3/CD47 receptor complex and thrombospondin-1 is identified as the molecular coupling device between mechanical loading and apoptosis: The integrin _V3 is expressed under mechanical loading as well as unloading whereas the CD47 could only be identified after the release of tension. The secreted thrombospondin binds to the active receptor and induces apoptosis. The presented mechanosensitive regulation of apoptosis in fibroblast cultures could be an essential mechanism for the regression of the granulation tissue by apoptosis in the process of wound healing.     

The crystal structure of death receptor 6 (DR6): a potential receptor of the amyloid precursor protein (APP)

Death receptors belong to the tumor necrosis factor receptor (TNFR) super family and are intimately involved in the signal transduction during apoptosis, stress response and cellular survival. Here we present the crystal structure of recombinantly expressed death receptor six (DR6), one family member that was recently shown to bind to the amyloid precursor protein (APP) and hence to be probably involved in the development of Alzheimer's disease. The extracellular cysteine rich region of DR6, the typical ligand binding region of all TNFRs, was refined to 2.2 Å resolution and shows that its four constituting cysteine rich domains (CRDs) are arranged in a rod-like overall structure, which presents DR6-specific surface patches responsible for the exclusive recognition of its ligand(s). Based on the structural data, the general ligand binding modes of TNFRs and molecular modeling experiments we were able to elucidate structural features of the potential DR6-APP signaling complex.     

Calmodulin as a protein linker and a regulator of adaptor/scaffold proteins

Calmodulin (CaM) is a universal regulator for a huge number of proteins in all eukaryotic cells. Best known is its function as a calcium-dependent modulator of the activity of enzymes, such as protein kinases and phosphatases, as well as other signaling proteins including membrane receptors, channels and structural proteins. However, less well known is the fact that CaM can also function as a Ca^2 +-dependent adaptor protein, either by bridging between different domains of the same protein or by linking two identical or different target proteins together. These activities are possible due to the fact that CaM contains two independently-folded Ca^2 + binding lobes that are able to interact differentially and to some degree separately with targets proteins. In addition, CaM can interact with and regulates several proteins that function exclusively as adaptors. This review provides an overview over our present knowledge concerning the structural and functional aspects of the role of CaM as an adaptor protein and as a regulator of known adaptor/scaffold proteins.     

Cell death in the injured brain: Roles of metallothioneins

In traumatic brain injury (TBI), the primary, irreversible damage associated with the moment of impact consists of cells dying from necrosis. This contributes to fuelling a chronic central nervous system (CNS) inflammation with increased formation of proinflammatory cytokines, enzymes and reactive oxygen species (ROS). ROS promote oxidative stress, which leads to neurodegeneration and ultimately results in programmed cell death (secondary injury). Since this delayed, secondary tissue loss occurs days to months following the primary injury it provides a therapeutic window where potential neuroprotective treatment could alleviate ongoing neurodegeneration, cell death and neurological impairment following TBI. Various neuroprotective drug candidates have been described, tested and proven effective in pre-clinical studies, including glutamate receptor antagonists, calcium-channel blockers, and caspase inhibitors. However, most of the scientific efforts have failed in translating the experimental results into clinical trials. Despite intensive research, effective neuroprotective therapies are lacking in the clinic, and TBI continues to be a major cause of morbidity and mortality.This paper provides an overview of the TBI pathophysiology leading to cell death and neurological impairment. We also discuss endogenously expressed neuroprotectants and drug candidates, which at this stage may still hold the potential for treating brain injured patients.     

Proteomic analysis of B-cell malignancies

The identification of proteins aberrantly expressed in malignant B-cells can potentially be used to develop new diagnostic, prognostic or therapeutic targets. Proteomic studies of B-cell malignancies have made significant progress, but further studies are needed to increase our coverage of the B-cell malignant proteome. To achieve this goal we stress the advantages of using sub-cellular fractionation, protein separation, quantitation and affinity purification techniques to identify hitherto unidentified signalling and regulatory proteins. For example, proteomic analysis of B-cell plasma membranes isolated from patients with mantle cell lymphoma (MCL) identified the voltage-gated proton channel (HVCN1,[1]). This protein has now been characterised as a key modulator of B-cell receptor (BCR) signalling and abrogation of HVCN1 function could have a role in the treatment of B-cell malignancies dependent on maintained BCR signalling [2]. Similarly, proteomic studies on cell lysates from prognostic subtypes of CLL, distinguished by the absence (UM-CLL) or presence (M-CLL) of somatic hypermutation of the immunoglobulin heavy chain locus identified nucleophosmin 1 (NMP1) as a potential prognostic marker [3,4]. Thus, targeted proteomic analysis on selected organelles or sub-cellular compartments can identify novel proteins with unexpected localisation or function in malignant B-cells that could be developed for clinical purposes.     

Potential use of tight junction modulators to reversibly open membranous barriers and improve drug delivery

The epithelial and endothelial barriers of the human body are major obstacles for drug delivery to the systemic circulation and to organs with unique environment and homeostasis, like the central nervous system. Several transport routes exist in these barriers, which potentially can be exploited for enhancing drug permeability. Beside the transcellular pathways via transporters, adsorptive and receptor-mediated transcytosis, the paracellular flux for cells and molecules is very limited. While lipophilic molecules can diffuse across the cellular plasma membranes, the junctional complexes restrict or completely block the free passage of hydrophilic molecules through the paracellular clefts. Absorption or permeability enhancers developed in the last 40 years for modifying intercellular junctions and paracellular permeability have unspecific mode of action and the effective and toxic doses are very close. Recent advances in barrier research led to the discovery of an increasing number of integral membrane, adaptor, regulator and signalling proteins in tight and adherens junctions. New tight junction modulators are under development, which can directly target tight or adherens junction proteins, the signalling pathways regulating junctional function, or tight junction associated lipid raft microdomains. Modulators acting directly on tight junctions include peptides derived from zonula occludens toxin, or Clostridium perfringens enterotoxin, peptides selected by phage display that bind to integral membrane tight junction proteins, and lipid modulators. They can reversibly increase paracellular transport and drug delivery with less toxicity than previous absorption enhancers, and have a potential to be used as pharmaceutical excipients to improve drug delivery across epithelial barriers and the blood-brain barrier.