Nanoscale imaging of CD47 informs how plasma membrane modifications shape apoptotic cell recognition.

CD47 recognized by its macrophage receptor SIRPα serves as a "don't eat-me" signal protecting viable cells from phagocytosis. How this is abrogated by apoptosis-induced changes in the plasma membrane, concomitantly with exposure of phosphatidylserine and calreticulin "eat-me" signals, is not well understood. Using STORM imaging and single-particle tracking, we interrogate how the distribution of these molecules on the cell surface correlates with plasma membrane alteration, SIRPα binding, and cell engulfment by macrophages.

Insights into the ligand binding specificity of SREC-II (scavenger receptor expressed by endothelial cells).

SREC-II (scavenger receptor expressed by endothelial cells II) is a membrane protein encoded by the SCARF2 gene, with high homology to class F scavenger receptor SR-F1, but no known scavenging function. We produced the extracellular domain of SREC-II in a recombinant form and investigated its capacity to interact with common scavenger receptor ligands, including acetylated low-density lipoprotein (AcLDL) and maleylated or acetylated BSA (MalBSA or AcBSA). Whereas no binding was observed for AcLDL, SREC-II ectodomain interacted strongly with MalBSA and bound with high affinity to AcBSA, a property shared with the SR-F1 ectodomain.

Molecular and Cellular Interactions of Scavenger Receptor SR-F1 With Complement C1q Provide Insights Into Its Role in the Clearance of Apoptotic Cells.

The scavenger receptor SR-F1 binds to and mediates the internalization of a wide range of ligands, and is involved in several immunological processes. We produced recombinant SR-F1 ectodomain and fragments deleted from the last 2 or 5 C-terminal epidermal growth factor-like modules and investigated their role in the binding of acetylated low density lipoprotein (AcLDL), complement C1q, and calreticulin (CRT). C1q measured affinity was in the 100 nM range and C1q interaction occurs its collagen-like region.

Cytosolic PCNA interacts with p47phox and controls NADPH oxidase NOX2 activation in neutrophils.

Neutrophils produce high levels of reactive oxygen species (ROS) by NADPH oxidase that are crucial for host defense but can lead to tissue injury when produced in excess. We previously described that proliferating cell nuclear antigen (PCNA), a nuclear scaffolding protein pivotal in DNA synthesis, controls neutrophil survival through its cytosolic association with procaspases. We herein showed that PCNA associated with p47phox, a key subunit of NADPH oxidase, and that this association regulated ROS production.

Proteomic analysis of neutrophils in ANCA-associated vasculitis reveals a dysregulation in proteinase 3-associated proteins such as annexin-A1 involved in apoptotic cell clearance.

Granulomatosis with polyangiitis (GPA) is an autoimmune vasculitis associated with anti-neutrophil-cytoplasmic antibodies (ANCA) against proteinase 3 leading to kidney damage. Neutrophils from those patients have increased expression of membrane proteinase 3 during apoptosis. Here we examined whether neutrophils from patients with GPA have dysregulated protein expressions associated with apoptosis.

Proteinase 3 Interferes With C1q-Mediated Clearance of Apoptotic Cells.

Proteinase 3 (PR3) is the autoantigen in granulomatosis with polyangiitis, an autoimmune necrotizing vasculitis associated with anti-neutrophil cytoplasmic antibodies (ANCAs). Moreover, PR3 is a serine protease whose membrane expression can potentiate inflammatory diseases such as ANCA-associated vasculitis and rheumatoid arthritis. During apoptosis, PR3 is co-externalized with phosphatidylserine (PS) and is known to modulate the clearance of apoptotic cells through a calreticulin (CRT)-dependent mechanism.

Calreticulin Release at an Early Stage of Death Modulates the Clearance by Macrophages of Apoptotic Cells.

Calreticulin (CRT) is a well-known "eat-me" signal harbored by dying cells participating in their recognition by phagocytes. CRT is also recognized to deeply impact the immune response to altered self-cells. In this study, we focus on the role of the newly exposed CRT following cell death induction.

Relative contribution of c1q and apoptotic cell-surface calreticulin to macrophage phagocytosis.

C1q has been shown to recognize apoptotic cells, to enhance their uptake and to modulate cytokine release by phagocytes and thus promote immune tolerance. Surface-exposed calreticulin (CRT), known as a C1q receptor, is also considered to be an early eat-me signal that enhances the phagocytosis of apoptotic cells and is capable of eliciting an immunogenic response. However, the molecular mechanisms that trigger these functions are not clear.

Human and pneumococcal cell surface glyceraldehyde-3-phosphate dehydrogenase (GAPDH) proteins are both ligands of human C1q protein.

C1q, a key component of the classical complement pathway, is a major player in the response to microbial infection and has been shown to detect noxious altered-self substances such as apoptotic cells. In this work, using complementary experimental approaches, we identified the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a C1q partner when exposed at the surface of human pathogenic bacteria Streptococcus pneumoniae and human apoptotic cells. The membrane-associated GAPDH on HeLa cells bound the globular regions of C1q as demonstrated by pulldown and cell surface co-localization experiments.

Proteinase 3, the autoantigen in granulomatosis with polyangiitis, associates with calreticulin on apoptotic neutrophils, impairs macrophage phagocytosis, and promotes inflammation.

Proteinase 3 (PR3) is the target of anti-neutrophil cytoplasm Abs in granulomatosis with polyangiitis, a form of systemic vasculitis. Upon neutrophil apoptosis, PR3 is coexternalized with phosphatidylserine and impaired macrophage phagocytosis. Calreticulin (CRT), a protein involved in apoptotic cell recognition, was found to be a new PR3 partner coexpressed with PR3 on the neutrophil plasma membrane during apoptosis, but not after degranulation.

Investigations on the C1q-calreticulin-phosphatidylserine interactions yield new insights into apoptotic cell recognition.

Both C1q and calreticulin (CRT) are involved in the recognition of apoptotic cells. CRT was initially characterized as a receptor for the C1q collagen-like fragment (CLF), whereas C1q was shown to bind apoptotic cells through its globular region (GR). Using purified CRT and recombinant CRT domains, we now provide unambiguous experimental evidence that, in addition to its CLF, the C1q GR also binds CRT and that both types of interactions are mediated by the CRT globular domain.

How phagocytes track down and respond to apoptotic cells.

The uptake of apoptotic cells by macrophages and dendritic cells or nonprofessional phagocytes is crucial for development and tissue homeostasis. This is of special importance because deficiencies in the recognition or removal of apoptotic cells may result in autoimmune diseases. The efficient elimination of an apoptotic cell involves contact between the altered cell and the phagocyte, specific recognition, and phagocytosis of the target.

The lectin-like activity of human C1q and its implication in DNA and apoptotic cell recognition.

C1q, the binding subunit of the C1 complex of complement, is an archetypal pattern recognition molecule known for its striking ability to recognize a wide variety of targets, ranging from pathogenic non self to altered self. DNA is one of the C1q ligands, but the precise region of C1q and the DNA motifs that support interaction have not been characterized yet. Here, we report for the first time that the peripheral globular region of the C1q molecule displays a lectin-like activity, which contributes to DNA binding through interaction with its deoxy-d-ribose moiety and may participate in apoptotic cell recognition.

C1q binds phosphatidylserine and likely acts as a multiligand-bridging molecule in apoptotic cell recognition.

Efficient apoptotic cell clearance is critical for maintenance of tissue homeostasis, and to control the immune responses mediated by phagocytes. Little is known about the molecules that contribute "eat me" signals on the apoptotic cell surface. C1q, the recognition unit of the C1 complex of complement, also senses altered structures from self and is a major actor of immune tolerance.

Interaction of C1q and mannan-binding lectin with viruses.

As soluble recognition molecules of innate immunity, C1q and MBL are able to bind directly to various viruses, including retroviruses and influenza viruses. Interaction of C1q with retroviruses and certain infected cells was shown to involve the globular region of C1q and viral envelope glycoproteins, such as p15E of MuLV, gp41 and gp120 of HIV-1, gp21 of HTLV-1. C1q binding was found to trigger antibody-independent activation of the classical pathway of complement, but did not lead to virus destruction and had even an adverse effect on infection in humans, because of subversion of the complement system by the virus.