Complement-Mediated Differential Immune Response of Human Macrophages to Sporothrix Species Through Interaction With Their Cell Wall Peptidorhamnomannans.

In this study, the human immune response mechanisms against Sporothrix brasiliensis and Sporothrix schenckii, two causative agents of human and animal sporotrichosis, were investigated. The interaction of S. brasiliensis and S. schenckii with human monocyte-derived macrophages (hMDMs) was shown to be dependent on the thermolabile serum complement protein C3, which facilitated the phagocytosis of Sporothrix yeast cells through opsonization. The peptidorhamnomannan (PRM) component of the cell walls of these two Sporothrix yeasts was found to be one of their surfaces exposed pathogen-associated molecular pattern (PAMP), leading to activation of the complement system and deposition of C3b on the Sporothrix yeast surfaces. PRM also showed direct interaction with CD11b, the specific component of the complement receptor-3 (CR3). Furthermore, the blockade of CR3 specifically impacted the interleukin (IL)-1ß secretion by hMDM in response to both S. brasiliensis and S. schenckii, suggesting that the host complement system plays an essential role in the inflammatory immune response against these Sporothrix species. Nevertheless, the structural differences in the PRMs of the two Sporothrix species, as revealed by NMR, were related to the differences observed in the host complement activation pathways. Together, this work reports a new PAMP of the cell surface of pathogenic fungi playing a role through the activation of complement system and via CR3 receptor mediating an inflammatory response to Sporothrix species.

Complement Factor H-Related 3 Enhanced Inflammation and Complement Activation in Human RPE Cells.

Complement Factor H-Related 3 (FHR-3) is a major regulator of the complement system, which is associated with different diseases, such as age-related macular degeneration (AMD). However, the non-canonical local, cellular functions of FHR-3 remained poorly understood. Here, we report that FHR-3 bound to oxidative stress epitopes and competed with FH for interaction. Furthermore, FHR-3 was internalized by viable RPE cells and modulated time-dependently complement component (C3, FB) and receptor (C3aR, CR3) expression of human RPE cells. Independently of any external blood-derived proteins, complement activation products were detected. Anaphylatoxin C3a was visualized in treated cells and showed a translocation from the cytoplasm to the cell membrane after FHR-3 exposure. Subsequently, FHR-3 induced a RPE cell dependent pro-inflammatory microenvironment. Inflammasome NLRP3 activation and pro-inflammatory cytokine secretion of IL-1ß, IL-18, IL-6 and TNF-α were induced after FHR-3-RPE interaction. Our previously published monoclonal anti-FHR-3 antibody, which was chimerized to reduce immunogenicity, RETC-2-ximab, ameliorated the effect of FHR-3 on ARPE-19 cells. Our studies suggest FHR-3 as an exogenous trigger molecule for the RPE cell "complosome" and as a putative target for a therapeutic approach for associated degenerative diseases.

Biologia Futura: stories about the functions of ß2-integrins in human phagocytes.

Integrins are essential membrane proteins that provide a tightly regulated link between the extracellular matrix and the intracellular cytoskeletal network. These cell surface proteins are composed of a non-covalently bound α chain and ß chain. The leukocyte-specific complement receptor 3 (CR3, αMß2, CD11b/CD18) and complement receptor 4 (CR4, αXß2, CD11c/CD18) belong to the family of ß2-integrins. These receptors bind multiple ligands like iC3b, ICAMs, fibrinogen or LPS, thus allowing them to partake in phagocytosis, cellular adhesion, extracellular matrix rearrangement and migration. CR3 and CR4 were generally expected to mediate identical functions due to their structural homology, overlapping ligand specificity and parallel expression on human phagocytes. Despite their similarities, the expression level and function of these receptors differ in a cell-type-specific manner, both under physiological and inflammatory conditions.We investigated comprehensively the individual role of CR3 and CR4 in various functions of human phagocytes, and we proved that there is a "division of labour" between these two receptors. In this review, I will summarize our current knowledge about this area.

Mac-1 Receptor Clustering Initiates Production of Pro-Inflammatory, Antibacterial Extracellular Vesicles From Neutrophils.

Depending on the prevailing environmental conditions, neutrophilic granulocytes release extracellular vesicles (EV) which have either anti-inflammatory effects on other neutrophils or pro-inflammatory and antibacterial effects. In the present study we investigated the molecular mechanisms underlying the biogenesis of functionally heterogenic EVs. We show that selective stimulation of Mac-1 integrin (complement receptor 3) by specific ligands initiates the generation of EVs which are able to impair bacterial growth and to induce the secretion of the pro-inflammatory cytokine IL-8 (aEV). However, direct Mac-1 stimulation results in aEV release only if neutrophils were activated on ligand coated surfaces whereas soluble ligands are ineffective. Using total internal reflection fluorescence (TIRF) microcopy, an increased clustering of Mac-1 molecules could be visualized in neutrophils added to C3bi coated surfaces; moreover antibody induced cluster formation triggers aEV release as well. Mac-1 induced production of aEV apparently necessitates a strong calcium signal as it fully depends on the presence of extracellular calcium. However, initiation of a strong calcium signal by an ionophore only results the generation of EV devoid of any antibacterial or pro-inflammatory effect. Our results thus demonstrate that stimulation and clustering of Mac-1 is necessary and sufficient for initiation of aEV biogenesis. In contrast, an intracellular calcium signal is necessary but by itself not sufficient for the production of antibacterial and pro-inflammatory EVs.

Complement Receptor 3 Forms a Compact High-Affinity Complex with iC3b.

Complement receptor 3 (CR3, also known as Mac-1, integrin αMß2, or CD11b/CD18) is expressed on a subset of myeloid and certain activated lymphoid cells. CR3 is essential for the phagocytosis of complement-opsonized particles such as pathogens and apoptotic or necrotic cells opsonized with the complement fragment iC3b and, to a lesser extent, C3dg. Although the interaction between the iC3b thioester domain and the ligand binding CR3 αM I-domain is structurally and functionally well characterized, the nature of additional CR3-iC3b interactions required for phagocytosis of complement-opsonized objects remains obscure. In this study, we analyzed the interaction between iC3b and the 150-kDa headpiece fragment of the CR3 ectodomain. Surface plasmon resonance experiments demonstrated a 30 nM affinity of the CR3 headpiece for iC3b compared with 515 nM for the iC3b thioester domain, whereas experiments monitoring binding of iC3b to CR3-expressing cells suggested an affinity of 50 nM for the CR3-iC3b interaction. Small angle x-ray scattering analysis revealed that iC3b adopts an extended but preferred conformation in solution. Upon interaction with CR3, iC3b rearranges to form a compact receptor-ligand complex. Overall, the data suggest that the iC3b-CR3 interaction is of high affinity and relies on minor contacts formed between CR3 and regions outside the iC3b thioester domain. Our results rationalize the more efficient phagocytosis elicited by iC3b than by C3dg and pave the way for the development of specific therapeutics for the treatment of inflammatory and neurodegenerative diseases that do not interfere with the recognition of noncomplement CR3 ligands.

Insights on the Functional Role of Beta-Glucans in Fungal Immunity Using Receptor-Deficient Mouse Models.

Understanding the host anti-fungal immunity induced by beta-glucan has been one of the most challenging conundrums in the field of biomedical research. During the last couple of decades, insights on the role of beta-glucan in fungal disease progression, susceptibility, and resistance have been greatly augmented through the utility of various beta-glucan cognate receptor-deficient mouse models. Analysis of dectin-1 knockout mice has clarified the downstream signaling pathways and adaptive effector responses triggered by beta-glucan in anti-fungal immunity. On the other hand, assessment of CR3-deficient mice has elucidated the compelling action of beta-glucans in neutrophil-mediated fungal clearance, and the investigation of EphA2-deficient mice has highlighted its novel involvement in host sensing and defense to oral mucosal fungal infection. Based on these accounts, this review focuses on the recent discoveries made by these gene-targeted mice in beta-glucan research with particular emphasis on the multifaceted aspects of fungal immunity.

Structural assessment of SARS-CoV2 accessory protein ORF7a predicts LFA-1 and Mac-1 binding potential.

ORF7a is an accessory protein common to SARS-CoV1 and the recently discovered SARS-CoV2, which is causing the COVID-19 pandemic. The ORF7a protein has a structural homology with ICAM-1 which binds to the T lymphocyte integrin receptor LFA-1. As COVID-19 has a strong immune component as part of the disease, we sought to determine whether SARS-CoV2 would have a similar structural interaction with LFA-1. Using molecular docking simulations, we found that SARS-CoV2 ORF7a has the key structural determinants required to bind LFA-1 but also the related leukocyte integrin Mac-1, which is also known to be expressed by macrophages. Our study shows that SARS-CoV2 ORF7a protein has a conserved Ig immunoglobulin-like fold containing an integrin binding site that provides a mechanistic hypothesis for SARS-CoV2's interaction with the human immune system. This suggests that experimental investigation of ORF7a-mediated effects on immune cells such as T lymphocytes and macrophages (leukocytes) could help understand the disease further and develop effective treatments.

Differential Interactions of Serum and Bronchoalveolar Lavage Fluid Complement Proteins with Conidia of Airborne Fungal Pathogen Aspergillus fumigatus.

Even though both cellular and humoral immunities contribute to host defense, the role played by humoral immunity against the airborne opportunistic fungal pathogen Aspergillus fumigatus has been underexplored. In this study, we aimed at deciphering the role of the complement system, the major humoral immune component, against A. fumigatus Mass spectrometry analysis of the proteins extracted from A. fumigatus conidial (asexual spores and infective propagules) surfaces opsonized with human serum indicated that C3 is the major complement protein involved. Flow cytometry and immunolabeling assays further confirmed C3b (activated C3) deposition on the conidial surfaces. Assays using cell wall components of conidia indicated that the hydrophobin RodAp, ß-(1,3)-glucan (BG) and galactomannan (GM) could efficiently activate C3. Using complement component-depleted sera, we showed that while RodAp activates C3 by the alternative pathway, BG and GM partially follow the classical and lectin pathways, respectively. Opsonization facilitated conidial aggregation and phagocytosis, and complement receptor (CR3 and CR4) blockage on phagocytes significantly inhibited phagocytosis, indicating that the complement system exerts a protective role against conidia by opsonizing them and facilitating their phagocytosis mainly through complement receptors. Conidial opsonization with human bronchoalveolar lavage fluid (BALF) confirmed C3 to be the major complement protein interacting with conidia. Nevertheless, complement C2 and mannose-binding lectin (MBL), the classical and lectin pathway components, respectively, were not identified, indicating that BALF activates the alternative pathway on the conidial surface. Moreover, the cytokine profiles were different upon stimulation of phagocytes with serum- and BALF-opsonized conidia, highlighting the importance of studying interaction of conidia with complement proteins in their biological niche.

Physical Constraints and Forces Involved in Phagocytosis.

Phagocytosis is a specialized process that enables cellular ingestion and clearance of microbes, dead cells and tissue debris that are too large for other endocytic routes. As such, it is an essential component of tissue homeostasis and the innate immune response, and also provides a link to the adaptive immune response. However, ingestion of large particulate materials represents a monumental task for phagocytic cells. It requires profound reorganization of the cell morphology around the target in a controlled manner, which is limited by biophysical constraints. Experimental and theoretical studies have identified critical aspects associated with the interconnected biophysical properties of the receptors, the membrane, and the actin cytoskeleton that can determine the success of large particle internalization. In this review, we will discuss the major physical constraints involved in the formation of a phagosome. Focusing on two of the most-studied types of phagocytic receptors, the Fcγ receptors and the complement receptor 3 (αMß2 integrin), we will describe the complex molecular mechanisms employed by phagocytes to overcome these physical constraints.

The differential role of CR3 (CD11b/CD18) and CR4 (CD11c/CD18) in the adherence, migration and podosome formation of human macrophages and dendritic cells under inflammatory conditions.

CR3 and CR4, the leukocyte specific ß2-integrins, involved in cellular adherence, migration and phagocytosis, are often assumed to have similar functions. Previously however, we proved that under physiological conditions CR4 is dominant in the adhesion to fibrinogen of human monocyte-derived macrophages (MDMs) and dendritic cells (MDDCs). Here, using inflammatory conditions, we provide further evidence that the expression and function of CR3 and CR4 are not identical in these cell types. We found that LPS treatment changes their expression differently on MDMs and MDDCs, suggesting a cell type specific regulation. Using mAb24, specific for the high affinity conformation of CD18, we proved that the activation and recycling of ß2-integrins is significantly enhanced upon LPS treatment. Adherence to fibrinogen was assessed by two fundamentally different approaches: a classical adhesion assay and a computer-controlled micropipette, capable of measuring adhesion strength. While both receptors participated in adhesion, we demonstrated that CR4 exerts a dominant role in the strong attachment of MDDCs. Studying the formation of podosomes we found that MDMs retain podosome formation after LPS activation, whereas MDDCs lose this ability, resulting in a significantly reduced adhesion force and an altered cellular distribution of CR3 and CR4. Our results suggest that inflammatory conditions reshape differentially the expression and role of CR3 and CR4 in macrophages and dendritic cells.

Mechanistic Understanding of Cell Recognition and Immune Reaction via CR1/CR3 by HAP- and SiO2-NPs.

Nanodrug carrier will eventually enter the blood when intravenously injected or in other ways. Meanwhile, a series of toxic effects were caused to the body with the formation of nanoparticle protein corona. In our studies, we try to reveal the recognition mechanism of nanoparticle protein corona by monocyte and the damage effect on immune cells by activated complement of hydroxyapatite nanoparticles (HAP-NPs) and silicon dioxide nanoparticles (SiO2-NPs). So expressions of TLR4/CR1/CR were analyzed by flow cytometry (FCM) in order to illuminate the recognition mechanism of nanoparticle protein corona by monocyte. And the expression of ROS, cytokines, adhesion molecules, and arachidonic acid was measured when THP-1 and HUVECs were stimulated by NP-activated complement. The results showed that HAP-NPs can be recognized by the opsonin receptor (iC3b/CR3) model, while plasma protein, opsonin receptor, and Toll-like receptors are all likely launch cell recognition of SiO2-NPs. And it was considerate that NP-activated complement can damage THP-1 and HUVECs, including oxidative stress, inflammation, and increased vascular permeability. So the surface of nanodrug carrier can be modified to avoid being clear and reduce the efficacy according to the three receptors (TLR4/CR1/CR3).

ß2 Integrins-Multi-Functional Leukocyte Receptors in Health and Disease.

ß2 integrins are heterodimeric surface receptors composed of a variable α (CD11a-CD11d) and a constant ß (CD18) subunit and are specifically expressed by leukocytes. The α subunit defines the individual functional properties of the corresponding ß2 integrin, but all ß2 integrins show functional overlap. They mediate adhesion to other cells and to components of the extracellular matrix (ECM), orchestrate uptake of extracellular material like complement-opsonized pathogens, control cytoskeletal organization, and modulate cell signaling. This review aims to delineate the tremendous role of ß2 integrins for immune functions as exemplified by the phenotype of LAD-I (leukocyte adhesion deficiency 1) patients that suffer from strong recurrent infections. These immune defects have been largely attributed to impaired migratory and phagocytic properties of polymorphonuclear granulocytes. The molecular base for this inherited disease is a functional impairment of ß2 integrins due to mutations within the CD18 gene. LAD-I patients are also predisposed for autoimmune diseases. In agreement, polymorphisms within the CD11b gene have been associated with autoimmunity. Consequently, ß2 integrins have received growing interest as targets in the treatment of autoimmune diseases. Moreover, ß2 integrin activity on leukocytes has been implicated in tumor development.

Molecular mechanism of leukocidin GH-integrin CD11b/CD18 recognition and species specificity.

Host-pathogen interactions are central to understanding microbial pathogenesis. The staphylococcal pore-forming cytotoxins hijack important immune molecules but little is known about the underlying molecular mechanisms of cytotoxin-receptor interaction and host specificity. Here we report the structures of a staphylococcal pore-forming cytotoxin, leukocidin GH (LukGH), in complex with its receptor (the α-I domain of complement receptor 3, CD11b-I), both for the human and murine homologs. We observe 2 binding interfaces, on the LukG and the LukH protomers, and show that human CD11b-I induces LukGH oligomerization in solution. LukGH binds murine CD11b-I weakly and is inactive toward murine neutrophils. Using a LukGH variant engineered to bind mouse CD11b-I, we demonstrate that cytolytic activity does not only require binding but also receptor-dependent oligomerization. Our studies provide an unprecedented insight into bicomponent leukocidin-host receptor interaction, enabling the development of antitoxin approaches and improved animal models to explore these approaches.

Coupling of ß2 integrins to actin by a mechanosensitive molecular clutch drives complement receptor-mediated phagocytosis.

αMß2 integrin (complement receptor 3) is a major receptor for phagocytosis in macrophages. In other contexts, integrins' activities and functions are mechanically linked to actin dynamics through focal adhesions. We asked whether mechanical coupling of αMß2 integrin to the actin cytoskeleton mediates phagocytosis. We found that particle internalization was driven by formation of Arp2/3 and formin-dependent actin protrusions that wrapped around the particle. Focal complex-like adhesions formed in the phagocytic cup that contained ß2 integrins, focal adhesion proteins and tyrosine kinases. Perturbation of talin and Syk demonstrated that a talin-dependent link between integrin and actin and Syk-mediated recruitment of vinculin enable force transmission to target particles and promote phagocytosis. Altering target mechanical properties demonstrated more efficient phagocytosis of stiffer targets. Thus, macrophages use tyrosine kinase signalling to build a mechanosensitive, talin- and vinculin-mediated, focal adhesion-like molecular clutch, which couples integrins to cytoskeletal forces to drive particle engulfment.

Human Neutrophils Will Crawl Upstream on ICAM-1 If Mac-1 Is Blocked.

The recruitment of neutrophils to sites of inflammatory insult is a hallmark of the innate immune response. Neutrophil recruitment is regulated by a multistep process that includes cell rolling, activation, adhesion, and transmigration through the endothelium commonly referred to as the leukocyte adhesion cascade. After selectin-mediated braking, neutrophils migrate along the activated vascular endothelium on which ligands, including intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), are expressed. Previous studies have shown that two cells that commonly home from blood vessel to tissue-T cells and hematopoietic stem and progenitor cells-use the integrin lymphocyte functional antigen-1 (LFA-1) to migrate against the direction of shear flow once adherent on ICAM-1 surfaces. Like T cells and hematopoietic stem and progenitor cells, neutrophils express LFA-1, but they also express macrophage-1 antigen (Mac-1), which binds to ICAM-1. Previous reports have shown that neutrophils will not migrate against the direction of flow on ICAM-1, but we hypothesized this was due to the influence of Mac-1. Here, we report that both the HL-60 neutrophil-like cell line and primary human neutrophils can migrate against the direction of fluid flow on ICAM-1 surfaces via LFA-1 if Mac-1 is blocked; otherwise, they migrate downstream. We demonstrate this both on ICAM-1 surfaces and on activated endothelium. In sum, both LFA-1 and Mac-1 binding ICAM-1 play a critical role in determining the direction of neutrophil migration along the endothelium, and their interaction may play an important role in controlling neutrophil trafficking during inflammation.

ß-1,3-Glucan/CR3/SYK pathway-dependent LC3B-II accumulation enhanced the fungicidal activity in human neutrophils.

Since molecular genotyping has been established for the Candida species, studies have found that a single Candida strain (endemic strain) can persist over a long period of time and results in the spread of nosocomial invasive candidiasis without general characteristics of horizontal transmissions. Our previous study also found the existence of endemic strains in a cancer center in Tianjin, China. In the current study, we performed further investigation on endemic and non-endemic Candida albicans strains, with the aim of explaining the higher morbidity of endemic strains. In an in vivo experiment, mice infected with endemic strains showed significantly shorter survival time and higher kidney fungal burdens compared to mice infected with non-endemic strains. In an in vitro experiment, the killing percentage of neutrophils to endemic strains was significantly lower than that to non-endemic strains, which is positively linked to the ratio of LC3B-II/I in neutrophils. An immunofluorescence assay showed more ß-1,3-glucan exposure on the cell walls of non-endemic strains compared to endemic strains. After blocking the ß-glucan receptor (CR3) or inhibiting downstream kinase (SYK) in neutrophils, the killing percent to C. albicans (regardless of endemic and non-endemic strains) and the ratio of LC3B-II/I of neutrophils were significantly decreased. These data suggested that the killing capability of neutrophils to C. albicans was monitored by ß-1,3-glucan via CR3/SYK pathway-dependent LC3B-II accumulation and provided an explanation for the variable killing capability of neutrophils to different strains of C. albicans, which would be beneficial in improving infection control and therapeutic strategies for invasive candidiasis.

Neutrophils disturb pulmonary microcirculation in sepsis-induced acute lung injury.

The lung is highly vulnerable during sepsis, yet its functional deterioration accompanied by disturbances in the pulmonary microcirculation is poorly understood. This study aimed to investigate how the pulmonary microcirculation is distorted in sepsis-induced acute lung injury (ALI) and reveal the underlying cellular pathophysiologic mechanism.Using a custom-made intravital lung microscopic imaging system in a murine model of sepsis-induced ALI, we achieved direct real-time visualisation of the pulmonary microcirculation and circulating cells in vivo We derived the functional capillary ratio (FCR) as a quantitative parameter for assessing the fraction of functional microvasculature in the pulmonary microcirculation and dead space.We identified that the FCR rapidly decreases in the early stage of sepsis-induced ALI. The intravital imaging revealed that this decrease resulted from the generation of dead space, which was induced by prolonged neutrophil entrapment within the capillaries. We further showed that the neutrophils had an extended sequestration time and an arrest-like dynamic behaviour, both of which triggered neutrophil aggregates inside the capillaries and arterioles. Finally, we found that Mac-1 (CD11b/CD18) was upregulated in the sequestered neutrophils and that a Mac-1 inhibitor restored the FCR and improved hypoxaemia.Using the intravital lung imaging system, we observed that Mac-1-upregulated neutrophil aggregates led to the generation of dead space in the pulmonary microcirculation that was recovered by a Mac-1 inhibitor in sepsis-induced ALI.

CR3 Engaged by PGL-I Triggers Syk-Calcineurin-NFATc to Rewire the Innate Immune Response in Leprosy.

Mycobacterium leprae, the causative agent of leprosy, is unique amongst human pathogens in its capacity to produce the virulence factor phenolic glycolipid (PGL)-I. In addition to mediating bacterial tropism for neurons, PGL-I interacts with Complement Receptor (CR)3 on macrophages (MPs) to promote infection. We demonstrate here that PGL-I binding to CR3 also enhances bacterial invasion of both polymorphonuclear neutrophils (PMNs) and dendritic cells (DCs). Moreover, in all cell types CR3 engagement by PGL-I activates the Syk tyrosine kinase, inducing calcineurin-dependent nuclear translocation of the transcription factor NFATc. This selectively augments the production of IL-2 by DCs, IL-10 by PMNs and IL-1ß by MPs. In intranasally-infected mice PGL-I binding to CR3 heightens mycobacterial phagocytosis by lung PMNs and MPs, and stimulates NFATc-controlled production of Syk-dependent cytokines. Our study thus identifies the CR3-Syk-NFATc axis as a novel signaling pathway activated by PGL-I in innate immune cells, rewiring host cytokine responses to M. leprae.

Different Calcium and Src Family Kinase Signaling in Mac-1 Dependent Phagocytosis and Extracellular Vesicle Generation.

Encountering opsonized particles by neutrophils results in phagocytosis of the particle and generation of extracellular vesicles with antibacterial property (aEV). The aim of the present study is to compare the involvement of different receptors and receptor-proximal signaling pathways in these two parallel processes. Investigating human neutrophils from peripheral blood, we show that complement receptors are decisive for both processes whereas immunoglobulin binding Fc receptors (FcR) only participate moderately in phagocytosis and pattern recognition receptors induce mild EV production but only minimal phagocytosis. Studying bone marrow derived neutrophils of genetically modified animals we verify that the involved complement receptor is CR3, also known as the ß2 integrin Mac-1. We show that genetic deletion of the adaptor molecules FcRγ chain or DAP12 does not influence either process, suggesting potential redundant function. Combined absence of the Src family kinases Hck, Fgr, and Lyn drastically impairs phagocytosis but does not influence aEV production. In contrast, deletion of PLCγ2 has no influence on phagocytosis, but reduces aEV formation. In accord with the essential role of PLCγ2, aEV biogenesis both from murine and from human neutrophils is dependent on presence of extracellular calcium. Absence of external calcium prevented the generation of antibacterial EVs, whereas the spontaneous EV formation was not influenced. We thus show that phagocytosis and biogenesis of antibacterial EVs are independent processes and proceed on different signaling pathways although the same receptor plays the critical role in both. Our data reveal the possibility in neutrophilic granulocytes to modulate aEV production without disturbing the phagocytic process.

Structural Immunology of Complement Receptors 3 and 4.

Complement receptors (CR) 3 and 4 belong to the family of beta-2 (CD18) integrins. CR3 and CR4 are often co-expressed in the myeloid subsets of leukocytes, but they are also found in NK cells and activated T and B lymphocytes. The heterodimeric ectodomain undergoes considerable conformational change in order to switch the receptor from a structurally bent, ligand-binding in-active state into an extended, ligand-binding active state. CR3 binds the C3d fragment of C3 in a way permitting CR2 also to bind concomitantly. This enables a hand-over of complement-opsonized antigens from the cell surface of CR3-expressing macrophages to the CR2-expressing B lymphocytes, in consequence acting as an antigen presentation mechanism. As a more enigmatic part of their functions, both CR3 and CR4 bind several structurally unrelated proteins, engineered peptides, and glycosaminoglycans. No consensus motif in the proteinaceous ligands has been established. Yet, the experimental evidence clearly suggest that the ligands are primarily, if not entirely, recognized by a single site within the receptors, namely the metal-ion dependent adhesion site (MIDAS). Comparison of some recent identified ligands points to CR3 as inclined to bind positively charged species, while CR4, by contrast, binds strongly negative-charged species, in both cases with the critical involvement of deprotonated, acidic groups as ligands for the Mg2+ ion in the MIDAS. These properties place CR3 and CR4 firmly within the realm of modern molecular medicine in several ways. The expression of CR3 and CR4 in NK cells was recently demonstrated to enable complement-dependent cell cytotoxicity toward antibody-coated cancer cells as part of biological therapy, constituting a significant part of the efficacy of such treatment. With the flexible principles of ligand recognition, it is also possible to propose a response of CR3 and CR4 to existing medicines thereby opening a possibility of drug repurposing to influence the function of these receptors. Here, from advances in the structural and cellular immunology of CR3 and CR4, we review insights on their biochemistry and functions in the immune system.