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Heparanase is the only mammalian enzyme capable of cleaving heparan sulfate, a glycosaminoglycan of the extracellular matrix and cell surfaces. Most immune cells express heparanase that contributes to a range of functions including cell migration and cytokine expression. Heparanase also promotes natural killer (NK) cell migration; however, its role in other NK cell functions remains to be defined. In this study, heparanase‐deficient (Hpse–/–) mice were used to assess the role of heparanase in NK cell cytotoxicity, activation, and cytokine production. Upon challenge with the immunostimulant polyinosinic:polycytidylic acid (poly(I:C)), NK cells isolated from Hpse–/–mice displayed impaired cytotoxicity against EO771.LMB cells and reduced levels of activation markers CD69 and NKG2D. However, in vitro cytokine stimulation of wild‐type and Hpse–/–NK cells resulted in similar CD69 and NKG2D expression, suggesting the impaired NK cell activation in Hpse–/–mice results from elements within the in vivo niche. NK cells are activated in vivo by dendritic cells (DCs) in response to poly(I:C). Poly(I:C)‐stimulated Hpse–/–bone marrow DCs (BMDCs) expressed less IL‐12, and when cultured with Hpse–/–NK cells, less MCP‐1 mRNA and protein was detected. Although cell‐cell contact is important for DC‐mediated NK cell activation, co‐cultures of Hpse–/–BMDCs and NK cells showed similar levels of contact to wild‐type cells, suggesting heparanase contributes to NK cell activation independently of cell‐cell contact with DCs. These observations define a role for heparanase in NK cell cytotoxicity and activation and have important implications for how heparanase inhibitors currently in clinical trials for metastatic cancer may impact NK cell immunosurveillance. Heparanase contributes to natural killer cell cytotoxicity, activation, and cytokine production

Sepsis is a biphasic disease characterized by an acute inflammatory response, followed by a prolonged immunosuppressive phase. Therapies aimed at controlling inflammation help to reduce the time patients with sepsis spend in intensive care units, but they do not lead to a reduction in overall mortality. Recently, the focus has been on addressing the immunosuppressive phase, often caused by apoptosis of immune cells. However, molecular triggers of these events are not yet known. Using whole-genome CRISPR screening in mice, we identified a triggering receptor expressed on myeloid cells (TREM) family receptor, TREML4, as a key regulator of inflammation and immune cell death in sepsis. Genetic ablation of Treml4in mice demonstrated that TREML4 regulates calcium homeostasis, the inflammatory cytokine response, myeloperoxidase activation, the endoplasmic reticulum stress response and apoptotic cell death in innate immune cells, leading to an overall increase in survival rate, both during the acute and chronic phases of polymicrobial sepsis.

Many cell types are known to undergo a series of morphological changes during the progression of apoptosis, leading to their disassembly into smaller membrane-bound vesicles known as apoptotic bodies (ApoBDs). In particular, the formation of circular bulges called membrane blebs on the surface of apoptotic cells is a key morphological step required for a number of cell types to generate ApoBDs. Although apoptotic membrane blebbing is thought to be regulated by kinases including ROCK1, PAK2 and LIMK1, it is unclear whether these kinases exhibit overlapping roles in the disassembly of apoptotic cells. Utilising both pharmacological and CRISPR/Cas9 gene editing based approaches, we identified ROCK1 but not PAK2 or LIMK1 as a key non-redundant positive regulator of apoptotic membrane blebbing as well as ApoBD formation. Functionally, we have established an experimental system to either inhibit or enhance ApoBD formation and demonstrated the importance of apoptotic cell disassembly in the efficient uptake of apoptotic materials by various phagocytes. Unexpectedly, we also noted that ROCK1 could play a role in regulating the onset of secondary necrosis. Together, these data shed light on both the mechanism and function of cell disassembly during apoptosis.

A study was conducted to discuss the views of the Australian health and medical research (HMR) workforce on issues like employment and funding opportunities. Results confirmed that insecurity about employment and lack of funding are a major cause of concern among Australian health and medical researchers.

Phosphorylated phosphatidylinositol lipids, or phosphoinositides, critically regulate diverse cellular processes, including signalling transduction, cytoskeletal reorganisation, membrane dynamics and cellular trafficking. However, phosphoinositides have been inadequately investigated in the context of cell death, where they are mainly regarded as signalling secondary messengers. However, recent studies have begun to highlight the importance of phosphoinositides in facilitating cell death execution. Here, we cover the latest phosphoinositide research with a particular focus on phosphoinositides in the mechanisms of cell death. This progress article also raises key questions regarding the poorly defined role of phosphoinositides, particularly during membrane-associated events in cell death such as apoptosis and secondary necrosis. The review then further discusses important future directions for the phosphoinositide field, including therapeutically targeting phosphoinositides to modulate cell death.

Although there is much work to do before we see crocodile defensins in the clinic, we hope to one day harness the unique primal power of the crocodile's immune system to aid in the global fight against infectious disease. By using our knowledge of the crocodile's defensins, we could potentially engineer other proteins to take on CpoBD13's pH-sensing mechanism. By searching through the genome of the saltwater crocodile, we found that one particular defensin, named CpoBD13, was effective at killing the fungus Candida albicans - the leading cause of human fungal infections worldwide. [Extracted from the article]

ABSTRACTThe plant defensin NaD1 is a potent antifungal molecule that also targets tumor cells with a high efficiency. We examined the features of NaD1 that contribute to these two activities by producing a series of chimeras with NaD2, a defensin that has relatively poor activity against fungi and no activity against tumor cells. All plant defensins have a common tertiary structure known as a cysteine-stabilized α-β motif which consists of an α helix and a triple-stranded β-sheet stabilized by four disulfide bonds. The chimeras were produced by replacing loops 1 to 7, the sequences between each of the conserved cysteine residues on NaD1, with the corresponding loops from NaD2. The loop 5 swap replaced the sequence motif (SKILRR) that mediates tight binding with phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and is essential for the potent cytotoxic effect of NaD1 on tumor cells. Consistent with previous reports, there was a strong correlation between PI(4,5)P2binding and the tumor cell killing activity of all of the chimeras. However, this correlation did not extend to antifungal activity. Some of the loop swap chimeras were efficient antifungal molecules, even though they bound poorly to PI(4,5)P2, suggesting that additional mechanisms operate against fungal cells. Unexpectedly, the loop 1B swap chimera was 10 times more active than NaD1 against filamentous fungi. This led to the conclusion that defensin loops have evolved as modular components that combine to make antifungal molecules with variable mechanisms of action and that artificial combinations of loops can increase antifungal activity compared to that of the natural variants.

Annexin A5 and TO-PRO-3 (a nucleic acid–binding dye that stains early apoptotic and necrotic cells differentially) are used to distinguish six types of particles in a sample, including apoptotic bodies and cells at various stages of cell death.

The methylation of histones is a fundamental epigenetic process regulating gene expression programs in mammalian cells. Dysregulated patterns of histone methylation are directly implicated in malignant transformation. Here, we report the unexpected finding that the invasive extracellular matrix degrading endoglycosidase heparanase enters the nucleus of activated human T lymphocytes and regulates the transcription of a cohort of inducible immune response genes by controlling histone H3 methylation patterns. It was found that nuclear heparanase preferentially associates with euchromatin. Genome-wide ChIP-on-chip analyses showed that heparanase is recruited to both the promoter and transcribed regions of a distinct cohort of transcriptionally active genes. Knockdown and overexpression of the heparanase gene also showed that chromatin-bound heparanase is a prerequisite for the transcription of a subset of inducible immune response genes in activated T cells. Furthermore, the actions of heparanase seem to influence gene transcription by associating with the demethylase LSD1, preventing recruitment of the methylase MLL and thereby modifying histone H3 methylation patterns. These data indicate that heparanase belongs to an emerging class of proteins that play an important role in regulating transcription in addition to their well-recognized extra-nuclear functions.

Histidine-rich glycoprotein (HRG), also known as histidine-proline-rich glyco-protein, is an abundant and well-characterized protein of vertebrate plasma. HRG has a multidomain structure that allows the molecule to interact with many ligands, including heparin, phospholipids, plasminogen, fibrinogen, immunoglobulin G, C1q, heme, and Zn2+. The ability of HRG to interact with various ligands simultaneously has suggested that HRG can function as an adaptor molecule and regulate numerous important biologic processes, such as immune complex/necrotic cell/pathogen clearance, cell adhesion, angiogenesis, coagulation, and fibrinolysis. The present review covers the proposed multifunctional roles of HRG with a focus on recent findings that have led to its emergence as a key regulator of immunity and vascular biology. Also included is a discussion of the striking functional similarities between HRG and other important multifunctional proteins found in plasma, such as C-reactive protein, C1q, β2 glycoprotein I, and thrombospondin-1.

HRG enhances the phagocytosis of necrotic cells via a heparan sulfate‐dependent pathway that is inhibitable by heparin. Dying cells, such as apoptotic and necrotic cells, are cleared rapidly from the site of cell death to prevent the exposure of intracellular antigenic and immunostimulatory molecules that may cause tissue injury or facilitate the development of autoimmune diseases. For the immune system to recognize and remove dying cells efficiently, professional phagocytes use a variety of mechanisms that distinguish healthy cells from dying cells. HRG, a relatively abundant heparin/HS‐binding protein in human plasma, has been shown recently to tether IgG specifically to necrotic cells and aid the phagocytic uptake of necrotic cells via a FcγRI‐dependent pathway. In this study, we provide direct evidence that HRG can function cooperatively with cell surface HS on the monocytic cell line THP‐1 to promote necrotic cell removal. In addition, we found that the presence of heparin can markedly inhibit HRG‐enhanced necrotic cell clearance by THP‐1 cells, possibly by blocking the ability of HRG to interact with necrotic cells as well as THP‐1 cells. Thus, these data suggest that HRG can aid the phagocytosis of necrotic cells via a HS‐dependent pathway, and this process can be regulated by the presence of certain HRG ligands, such as heparin.

Under normal physiologic conditions, necrotic cells resulting from tissue injury are rapidly removed from the circulation and tissues by phagocytes, thus preventing the exposure of intracellular antigenic and immunostimulatory molecules that can aid the development of autoimmune disease. Histidine-rich glycoprotein (HRG), a relatively abundant plasma glycoprotein, has a multidomain structure that can interact with many ligands including components of the fibrinolytic and immune systems. Recently, it has been reported that HRG can bind strongly to cytoplasmic ligand(s) exposed in necrotic cells to enhance clearance by phagocytes. Here we describe the molecular mechanisms underpinning this process. A complex consisting of both HRG and immunoglobulin G (IgG) was found as necessary to aid necrotic cell uptake by monocytes, predominantly via an FcγRI-dependent mechanism. The findings in this study also show that HRG can potentially interact with anionic phospholipids exposed in necrotic cells. Furthermore, the enhanced phagocytosis of necrotic cells induced by HRG-IgG complexes triggers phagocytes to release proinflammatory cytokines such as interleukin-8 and tumor necrosis factor. Thus, HRG has the unique property of complexing with IgG and facilitating a proinflammatory innate immune response to promote the clearance of necrotic cells.

The heparan sulfate‐cleaving enzyme heparanase (HPSE) plays an important role in remodeling of the basement membrane and extracellular matrix during inflammation. Inducible HPSE enzymatic activity has been reported in leukocytes; however, little is known of the molecular mechanisms that regulate HPSEgene expression during inflammatory disease. In this study, HPSEexpression and regulation in the T cell‐mediated disease model, experimental autoimmune encephalomyelitis (EAE), were investigated. Expression analysis showed that HPSEmRNA is induced in rat CD4+ antigen‐specific T lymphocytes upon activation and correlates with the encephalitogenicity of the cells. Examination of the kinetics and cell type‐specific expression of HPSE throughout the progression of active EAE in rats, indicated that HPSE was highly expressed in CD4+ T cells infiltrating the central nervous system (CNS) during clinical disease. Little or no HPSE expression was observed in CD8+ T cells, macrophages, or astrocytes during disease progression. To investigate the mechanism of inducible HPSEgene regulation in T cells, studies were extended into human primary T cells. HPSEmRNA, protein, and enzymatic activity were induced upon activation. Functional analysis of the human HPSEpromoter identified an EGR1 binding motif that contained high inducible activity and was transactivated by EGR1. Furthermore, the treatment of primary T lymphocytes with an EGR1 siRNA inhibited inducible HPSEmRNA expression. These data provide evidence to suggest that inducible HPSE expression in primary T lymphocytes is regulated at the transcriptional level by EGR1 and is important in facilitating CD4+ T cell infiltration into the CNS to promote EAE.

Eosinophilic inflammation is a common feature of numerous eosinophil‐associated gastrointestinal (EGID) diseases. Central to eosinophil migration into the gastrointestinal tract are the integrin‐mediated interactions with adhesion molecules. Although the mechanisms regulating eosinophil homing into the small intestine have begun to be elucidated, the adhesion pathways responsible for eosinophil trafficking into the large intestine are unknown. We investigated the role of adhesion pathways in eosinophil recruitment into the large intestine during homeostasis and disease. First, using a hapten‐induced colonic injury model, we demonstrate that in contrast to the small intestine, eosinophil recruitment into the colon is regulated by a β7‐integrin addressin cell adhesion molecule‐1‐independent pathway. Characterization of integrin expression on colonic eosinophils by flow cytometry analysis revealed that colonic CC chemokine receptor 3+eosinophils express the intercellular adhesion molecule‐1 (ICAM‐1) counter‐receptor integrins αL, αM, and β2. Using ICAM‐1‐deficient mice and anti‐ICAM‐1 neutralizing antibodies, we show that hapten‐induced colonic eosinophilic inflammation is critically dependent on ICAM‐1. These studies demonstrate that β2‐integrin/ICAM‐1‐dependent pathways are integral to eosinophil recruitment into the colon during GI inflammation associated with colonic injury.

Currently most attempts at cancer immunotherapy involve the generation of CD8+ cytotoxic T lymphocytes (CTLs) against tumor-associated antigens. Many tumors, however, have been immunoselected to evade recognition by CTLs and thus alternative approaches to cancer immunotherapy are urgently needed. Here we demonstrate that CD4+ T cells that recognize a secreted tumor-specific antigen and exhibit a cytokine secretion profile characteristic of Th2 cells, are capable of clearing established lung and visceral metastases of a CTL-resistant melanoma. Clearance of lung metastases by the Th2 cells was found to be totally dependent on the eosinophil chemokine, eotaxin, and partially dependent on the transcription activator signal transducer and activator of transcription 6 (STAT6), with degranulating eosinophils within the tumors inducing tumor regression. In contrast, tumor-specific CD4+ Th1 cells, that recruited macrophages into the tumors, had no effect on tumor growth. This work provides the basis for a new approach to adoptive T cell immunotherapy of cancer.

CD20 and the β subunit of the high affinity receptor for IgE (FcεRIβ) are related four-transmembrane molecules that are expressed on the surface of hematopoietic cells and play crucial roles in signal transduction. Herein, we report the identification and characterization of a human gene, TETM4, that encodes a novel four-transmembrane protein related to CD20 and FcεRIβ. The predicted TETM4 protein is 200 amino acids and contains four putative transmembrane regions, N- and C-terminal cytoplasmic domains, and three inter-transmembrane loop regions. TETM4 shows 31.0 and 23.2% overall identity with CD20 and FcεRIβ respectively, with the highest identity in the transmembrane regions, whereas the N- and C-termini and inter-transmembrane loops are more divergent. Northern blot and RT-PCR analysis suggest that TETM4 mRNA has a highly restricted tissue distribution, being expressed selectively in the testis. Using fluorescence in situ hybridization and radiation hybrid analysis, the TETM4 gene has been localized to chromosome 11q12. The genes for CD20 and FcεRIβ have also been mapped to the same region of chromosome 11 (11q12-13.1), suggesting that these genes have evolved by duplication to form a family of four-transmembrane genes. TETM4 is the first nonhematopoietic member of the CD20/FcεRIβ family, and like its hematopoietic-specific relatives, it may be involved in signal transduction as a component of a multimeric receptor complex.

The high affinity receptor for IgE, FcεRI, binds IgE through the second Ig-like domain of the α subunit. The role of the first Ig-like domain is not well understood, but it is required for optimal binding of IgE to FcεRI, either through a minor contact interaction or in a supporting structural capacity. The results reported here demonstrate that domain one of FcεRI plays a major structural role supporting the presentation of the ligand-binding site, by interactions generated within the interdomain interface. Analysis of a series of chimeric receptors and point mutants indicated that specific residues within the A′ strand of domain one are crucial to the maintenance of the interdomain interface, and IgE binding. Mutation of the Arg15and Phe17residues caused loss in ligand binding, and utilizing a homology model of FcεRI-α based on the solved structure of FcγRIIa, it appears likely that this decrease is brought about by collapse of the interface and consequently the IgE-binding site. In addition discrepancies in results of previous studies using chimeric IgE receptors comprising FcεRIα with either FcγRIIa or FcγRIIIA can be explained by the presence or absence of Arg15and its influence on the IgE-binding site. The data presented here suggest that the second domain of FcεRI-α is the only domain involved in direct contact with the IgE ligand and that domain one has a structural function of great importance in maintaining the integrity of the interdomain interface and, through it, the ligand-binding site.

Fcγ receptors bind IgG to initiate cellular responses against pathogens and soluble antigens. We have determined the three–dimensional structure of the extracellular portion of human FcγRIIa to 2.0 Å resolution providing a structural basis for the unique functions of the leukocyte FcR family. The receptor is composed of two immunoglobulin domains and arranged to expose the ligand–binding site at one end of domain 2. Using alanine mutants we find that the binding sites for IgG1 and 2 are similar but the relative importance of specific regions on the receptor varies. In crystals, FcγRIIa molecules associate to resemble VLVHdimers, suggesting that two FcγRIIa molecules could cooperate to bind IgG in an asymmetric manner.

The high affinity receptor for IgE (FcεRI) plays an integral role in triggering IgE-mediated hypersensitivity reactions. The IgE-interactive site of human FcεRI has previously been broadly mapped to several large regions in the second extracellular domain (D2) of the α-subunit (FcεRIα). In this study, the IgE binding site of human FcεRIα has been further localized to subregions of D2, and key residues putatively involved in the interaction with IgE have been identified. Chimeric receptors generated between FcεRIα and the functionally distinct but structurally homologous low affinity receptor for IgG (FcγRIIa) have been used to localize two IgE binding regions of FcεRIα to amino acid segments Tyr129–His134and Lys154–Glu161. Both regions were capable of independently binding IgE upon placement into FcγRIIa. Molecular modeling of the three-dimensional structure of FcεRIα-D2 has suggested that these binding regions correspond to the “exposed” C′-E and F-G loop regions at the membrane distal portion of the domain. A systematic site-directed mutagenesis strategy, whereby each residue in the Tyr129–His134and Lys154–Glu161regions of FcεRIα was replaced with alanine, has identified key residues putatively involved in the interaction with IgE. Substitution of Tyr131, Glu132, Val155, and Asp159decreased the binding of IgE, whereas substitution of Trp130, Trp156, Tyr160, and Glu161increased binding. In addition, mutagenesis of residues Trp113, Val115, and Tyr116in the B-C loop region, which lies adjacent to the C′-E and F-G loops, has suggested Trp113also contributes to IgE binding, since the substitution of this residue with alanine dramatically reduces binding. This information should prove valuable in the design of strategies to intervene in the FcεRIα-IgE interaction for the possible treatment of IgE-mediated allergic disease.

Rat natural killer cell Met-ase-1 (RNK-Met-1) is a 30 000 Mr serine protease (granzyme) found in the cytolytic granules of CD3- large granular lymphocytes (LGL) with natural killer (NK) activity. To characterize the genomic sequences responsible for the CD3- LGL-restricted expression of this gene, we screened a rat genomic library with RNK-Met-1 cDNA, and obtained bacteriophage clones that contained the RNK-Met-1 gene. The RNK-Met-1 gene comprises 5 exons and spans approximately 5.2 kilobases (kb), exhibiting a similar structural organization to a class of CTL-serine proteases with protease catalytic residues encoded near the borders of exons 2, 3, and 5. The 5'-flanking region of the RNK-Met-1 gene contains a number of putative promoter and enhancer regulatory elements and shares several regions of homology with the 5'-flanking region of the mouse perforin gene. We have prepared nested deletions from approximately 3.3 kb of the 5'-flanking region of the RNK-Met-1 gene, and inserted these upstream of the chloramphenicol acetyltransferase (CAT) reporter gene. These 5'-flanking RNK-Met-1-CAT constructs were transiently transfected into rat LGL leukemia, T-lymphoma, and basophilic leukemia cell lines.

Abstract: Met-ase-1 is a 30000M r serine protease (granzyme) that was first isolated in the cytolytic granules of rat CD3− large granular lymphocytes. We screened a mouse genomic library with ratMet-ase-1 cDNA, and obtained bacteriophage clones that contained the mouseMet-ase-1 gene. The mouseMet-ase-1 gene comprises five exons spanning approximately 5.2 kilobases (kb) and exhibits a similar structural organization to its rat homologue and a family of neutrophil elastase-like serine proteases. MouseMet-ase-1 mRNA was only detected in total cellular and poly A mRNA of mouse CD3− GM1+ large granular lymphocytes derived from splenocytes stimulated with IL-2 and the mouse NK1.1+ cell line 4–16. Spleen T-cell populations generated by Concanavalin A stimulation and a number of mouse pre-NK and T cell lines did not express mouseMet-ase-1 mRNA. The 5′ flanking region of the mouseMet-ase-1 gene also shares considerable regions of identity with the 5′ flanking region of the ratMet-ase-1 gene. A 3.3 kb segment of 5′ sequence flanking the mouseMet-ase-1 gene was inserted upstream of the chloramphenicol acetyltransferase reporter gene and this construct transiently transfected into a variety of mouse and rat large granular lymphocyte leukemia and T-cell lines. The transcriptional activity of the mouseMet-ase-1 5′ flanking region was significant in the RNK-16 large granular lymphocyte leukemia, strongest in the 4–16 mouse NK1.1+ cell line, and weak in several mouse pre-NK cell lines. Reverse transcriptase polymerase chain reaction of mouse large granular lymphocyte mRNA was used to derive the full-length coding sequence for mouseMet-ase-1. The predicted hexapropeptide of mouseMet-ase-1 (Asn−6 to Gln−1), was deleted by polymerase chain reaction mutagenesis to enable expression of active mouseMet-ase-1 in mammalian COS-7 cells. Northern blot analysis and protease assays of transfected COS cell lysates against a panel of thiobenzyl ester substrates formally demonstrated that the mouseMet-ase-1 gene encodes a serine proteinase that hydrolyzes substrates containing a long narrow hydrophobic amino acids like methionine, norleucine, and leucine in the P1.

Expression of the gene encoding the cytolytic granule protein perforin is restricted to cytotoxic lymphocytes. To undertake a functional analysis of the immediate 5′‐promoter region of the mouse perforin gene, we transiently transfected mouse perforin promoter‐chloramphenicol acetyltransferase (CAT) reporter gene constructs into cytotoxic T, T lymphoid, B‐lymphoid, and nonlymphoid cell lines. The transcriptional activity of the perforin promoter was restricted to cytotoxic lymphocytes. The perforin promoter was controlled by several positive (in perforin‐positive cells) and negative (in perforin‐negative cells) cis‐acting regions, spread over at least 1.1 kilobases. The most specific expression of the CAT reporter gene in the interleukin‐2‐dependent cytotoxic T cell line CTLL‐R8 was obtained with the mouse perforin promoter encompassing positions ‐1104 to +1 in relation to the RNA cap site. This construct expressed 65‐ to 70‐fold higher CAT activity than the promoterless CAT construct in perforin‐expressing cells but only 1‐ to 5‐fold higher CAT activity than the promoterless construct in nonlymphoid cells. On the basis of these data, we used this most specifically active mouse perforin promoter, ‐1104 to +1, to express in CTLL‐R8, a chimeric human receptor comprising the extracellular domains of human FcγRI and the transmembrane and intracellular domains of TCR . Selection in G418‐containing medium produced CTLL‐R8 transfectant clones that (1) expressed high levels of human FcγRI mRNA; (2) expressed cell surface FcγRI as demonstrated by immunoprecipitation and their ability to bind the Fc portion of human and mouse monoclonal antibodies (mAbs) in an isotype‐specific manner, and (3) bound RBC expressing mucin‐1 (Muc‐1) peptide in the presence of a chimeric mouse‐human anti‐Muc‐1 mAb. Activation of CTLL‐R8 transfectants upon engagement of the human FcγRI was evidenced by their ability to lyse tumor target cells in an mAb isotype‐dependent manner. The successful expression of a functional chimeric gene in CTLL‐R8 suggests that the mouse perforin promoter represents a novel reagent for expressing exogenous genes in cytotoxic T lymphocytes. J. Leukoc. Biol.55: 514–522; 1994.

FcγRI (CD64) is functionally unique as it is the only FcγR able to bind monomeric IgG with high affinity. FcγRI is also structurally distinct, containing an extracellular Ig-interactive region of three Ig-like domains in contrast to the two domains of the low affinity receptors FcγRII and FcγRIII. Previous studies have demonstrated that the third domain of FcγRI plays a crucial role in high affinity IgG binding of the receptor, with the first and second domains together forming a low affinity IgG binding motif. In this study the individual functional contributions of the first and second domains of FcγRI to IgG binding have been investigated. Chimeric FcγR were generated by exchanging extracellular domains between mouse FcγRI and the structurally related yet distinct low affinity receptor for IgG, mouse FcγRII. The replacement of both domains 1 and 2 of FcγRI with domains 1 and 2 of FcγRII results in a dramatic change in IgG binding characteristics, as this receptor loses the capacity to bind monomeric IgG with high affinity and also demonstrates a broader specificity (binding not only IgG2a but also IgG1 and 2b. IgG3 was not tested). However, the substitution of FcγRII domain 2 of this chimeric receptor with domain 2 of FcγRI (generating a chimeric receptor with domain 1 of FcγRII linked to domains 2 and 3 of FcγRI) was found to reconstitute the specific high affinity monomeric IgG2a binding of wild-type FcγRI, albeit with a slightly reduced affinity (1.8-fold lower than wild-type FcγRI). These findings suggest that it is the specific interaction between domains 2 and 3 of FcγRI, with domain 1 playing a supporting role in maintaining the conformational stability of the receptor, that is the major structural requirement to confer the unique Ig binding characteristics of FcγRI.

The low-affinity receptor for IgG, FcγRll, and the high-affinity receptor for IgE, FcεRI, are functionally distinct but structurally homologous receptors. These characteristics have been exploited using a chimeric receptor strategy to examine segments of human FcγRII for IgG-binding function. A series of chimeric receptors was generated by exchanging coding regions of the extracellular ligand-binding regions between FcγRll and the FcεRI α chain using splice overlap extension by the polymerase chain reaction. The expression of these chimeric receptors in COS-7 cells and analysis of their IgG/IgE binding capacities have enabled the Ig-binding region of FcγRll to be localized to a subregion of the second extracellular domain. The localization of the Ig-binding region of FcγRII has provided the opportunity of performing site-directed mutagenesis to determine the key amino acids involved in the interaction of the receptor with IgG. These findings demonstrate that the chimeric receptor approach is a powerful technique for the dissection of structure/function relationships of structurally related yet functionally different molecules.Copyright 1994, 1999 Academic Press

The low affinity receptor for IgG, FcγRII (CD32), has a wide distribution on hematopoietic cells where it is responsible for a diverse range of cellular responses crucial for immune regulation and resistance to infection. FcγRII is a member of the immunoglobulin superfamily, containing an extracellular region of two Ig-like domains. The IgG binding site of human FcγRII has been localized to an 8-amino acid segment of the second extracellular domain, Asn154-Ser161. In this study, evidence is presented to suggest that domain 1 and two additional regions of domain 2 also contribute to the binding of IgG by FcγRII. Chimeric receptors generated by exchanging the extracellular domains and segments of domain 2 between FcγRII and the structurally related Fc∊RI α chain were used to demonstrate that substitution of domain 1 in its entirety or the domain 2 regions encompassing residues Ser109-Val116and Ser130-Thr135resulted in a loss of the ability of these receptors to bind hIgG1 in dimeric form. Site-directed mutagenesis performed on individual residues within and flanking the Ser109-Val116and Ser130-Thr135domain 2 segments indicated that substitution of Lys113, Pro114, Leu115, Val116, Phe129, and His131profoundly decreased the binding of hIgG1, whereas substitution of Asp133and Pro134increased binding. These findings suggest that not only is domain 1 contributing to the affinity of IgG binding by FcγRII but, importantly, that the domain 2 regions Ser109-Val116and Phe129-Thr135also play key roles in the binding of hIgG1. The location of these binding regions on a molecular model of the entire extracellular region of FcγRII indicates that they comprise loops that are juxtaposed in domain 2 at the interface with domain 1, with the putative crucial binding residues forming a hydrophobic pocket surrounded by a wall of predominantly aromatic and basic residues.

The innate immune system in mammals include pattern recognition receptors (PRRs), which initiate immune responses to microbial infection via several mechanisms. These PRRs include cell surface Toll-like receptors (TLRs) and cytosolic Nod-like receptors (NLRs) that recognizes extracellular and intracellular danger signals respectively. NLRs are poised to respond specifically to pathogens that access the host cell cytosol. The molecular mechanisms by which NLRs are activated to form inflammasomes and exert downstream inflammatory responses remain poorly understood. Additionally, very little is known about the regulation of cytosolic pathogen sensory NLR family members, except for NLRP3. Recently a deubiquitinase known as STAMBP has been implicated as a regulator of NLRP7 inflammasome assembly. We have investigated the role of STAMBP in regulation of other inflammasome components and its broader role in inflammation using genetic removal of STAMBP protein from cells using CRISPR/Cas9 gene editing and challenging these gene edited cells with an inflammatory stimuli. Our study demonstrated that STAMBP has a critical role in inflammation both in the context of NLR pathway, through NLRP stabilization and TLR pathway, through JNK signaling and downstream cytokine production. The findings indicate that STAMBP has a wider role in inflammation than previously thought to be the case.

Billions of cells undergo apoptosis daily and often fragment into small, membrane-bound extracellular vesicles termed apoptotic bodies (ApoBDs). We demonstrate that apoptotic monocytes undergo a highly coordinated disassembly process and form long, beaded protrusions (coined as beaded apoptopodia), which fragment to release ApoBDs. Here, we find that the protein plexin B2 (PlexB2), a transmembrane receptor that regulates axonal guidance in neurons, is enriched in the ApoBDs of THP1 monocytes and is a caspase 3/7 substrate. To determine whether PlexB2 is involved in the disassembly of apoptotic monocytes, we generate PlexB2-deficient THP1 monocytes and demonstrate that lack of PlexB2 impairs the formation of beaded apoptopodia and ApoBDs. Consequently, the loss of PlexB2 in apoptotic THP1 monocytes impairs their uptake by both professional and non-professional phagocytes. Altogether, these data identify PlexB2 as a positive regulator of apoptotic monocyte disassembly and demonstrate the importance of this process in apoptotic cell clearance.