Almost half of the RTX domain is dispensable for complement receptor 3 binding and cell-invasive activity of the Bordetella adenylate cyclase toxin.

The whooping cough agent Bordetella pertussis secretes an adenylate cyclase toxin (CyaA) that through its large carboxy-proximal Repeat-in-ToXin (RTX) domain binds the complement receptor 3 (CR3). The RTX domain consists of five blocks (I-V) of characteristic glycine and aspartate-rich nonapeptides that fold into five Ca2+-loaded parallel ß-rolls. Previous work indicated that the CR3-binding structure comprises the interface of ß-rolls II and III. To test if further portions of the RTX domain contribute to CR3 binding, we generated a construct with the RTX block II/III interface (CyaA residues 1132-1294) linked directly to the C-terminal block V fragment bearing the folding scaffold (CyaA residues 1562-1681). Despite deletion of 267 internal residues of the RTX domain, the Ca2+-driven folding of the hybrid block III/V ß-roll still supported formation of the CR3-binding structure at the interface of ß-rolls II and III. Moreover, upon stabilization by N- and C-terminal flanking segments, the block III/V hybrid-comprising constructs competed with CyaA for CR3 binding and induced formation of CyaA toxin-neutralizing antibodies in mice. Finally, a truncated CyaAΔ1295-1561 toxin bound and penetrated erythrocytes and CR3-expressing cells, showing that the deleted portions of RTX blocks III, IV, and V (residues 1295-1561) were dispensable for CR3 binding and for toxin translocation across the target cell membrane. This suggests that almost a half of the RTX domain of CyaA is not involved in target cell interaction and rather serves the purpose of toxin secretion.

Structural Characterization of the Interaction between the αMI-Domain of the Integrin Mac-1 (αMß2) and the Cytokine Pleiotrophin.

Integrin Mac-1 (αMß2) is an adhesion receptor vital to many functions of myeloid leukocytes. It is also the most promiscuous member of the integrin family capable of recognizing a broad range of ligands. In particular, its ligand-binding αMI-domain is known to bind cationic proteins/peptides depleted in acidic residues. This contradicts the canonical ligand-binding mechanism of αI-domains, which requires an acidic amino acid in the ligand to coordinate the divalent cation within the metal ion-dependent adhesion site (MIDAS) of αI-domains. The lack of acidic amino acids in the αMI-domain-binding sequences suggests the existence of an as-yet uncharacterized interaction mechanism. In the present study, we analyzed interactions of the αMI-domain with a representative Mac-1 ligand, the cationic cytokine pleiotrophin (PTN). Through NMR chemical shift perturbation analysis, cross saturation, NOESY, and mutagenesis studies, we found the interaction between the αMI-domain and PTN is divalent cation-independent and mediated mostly by hydrophobic contacts between the N-terminal domain of PTN and residues in the α5-ß5 loop of αMI-domain. The observation that increased ionic strength weakens the interaction between the proteins indicates electrostatic forces may also play a significant role in the binding. On the basis of the results from these experiments, we formulated a model of the interaction between the αMI-domain and PTN.

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.

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.

Structural basis of the leukocyte integrin Mac-1 I-domain interactions with the platelet glycoprotein Ib.

Cell-surface receptor interactions between leukocyte integrin macrophage-1 antigen (Mac-1, also known as CR3, αMß2, CD11b/CD18) and platelet glycoprotein Ibα (GPIbα) are critical to vascular inflammation. To define the key residues at the binding interface, we used nuclear magnetic resonance (NMR) to assign the spectra of the mouse Mac-1 I-domain and mapped the residues contacting the mouse GPIbα N-terminal domain (GPIbαN) to the locality of the integrin metal ion-dependant adhesion site (MIDAS) surface. We next determined the crystal structures of the mouse GPIbαN and Mac-1 I-domain to 2 Å and 2.5 Å resolution, respectively. The mouse Mac-1 I-domain crystal structure reveals an active conformation that is stabilized by a crystal contact from the α7-helix with a glutamate side chain completing the octahedral coordination sphere of the MIDAS Mg2+ ion. The amino acid sequence of the α7-helix and disposition of the glutamic acid matches the C-terminal capping region α-helix of GPIbα effectively acting as a ligand mimetic. Using these crystal structures in combination with NMR measurements and docking analysis, we developed a model whereby an acidic residue from the GPIbα leucine-rich repeat (LRR) capping α-helix coordinates directly to the Mac-1 MIDAS Mg2+ ion. The Mac-1:GPIbαN complex involves additional interactions consolidated by an elongated pocket flanking the GPIbαN LRR capping α-helix. The GPIbαN α-helix has an HxxxE motif, which is equivalent by homology to RxxxD from the human GPIbαN. Subsequent mutagenesis of residues at this interface, coupled with surface plasmon resonance studies, confirmed the importance of GPIbαN residues H218, E222, and the Mac-1 MIDAS residue T209 to formation of the complex.

Elimination of the fibrinogen integrin αMß2-binding motif improves renal pathology in mice with sickle cell anemia.

Sickle cell anemia (SCA) is caused by a point mutation in the ß-globin gene that leads to devastating downstream consequences including chronic hemolytic anemia, episodic vascular occlusion, and cumulative organ damage resulting in death. SCA patients show coagulation activation and inflammation even in the absence of vascular occlusion. The coagulation factor fibrinogen is not only central to hemostasis but also plays important roles in pathologic inflammatory processes, in part by engaging neutrophils/macrophages through the αMß2 integrin receptor. To determine whether fibrin(ogen)-mediated inflammation is a driver of SCA-associated pathologies, hematopoietic stem cells from Berkeley sickle mice were transplanted into homozygous Fibγ390-396A mice that express normal levels of a mutant form of fibrin(ogen) that does not engage αMß2 Fibγ390-396A mice with SCA displayed an impressive reduction of reactive oxygen species (ROS) in white blood cells (WBCs), decreased circulating inflammatory cytokines/chemokines, and significantly improved SCA-associated glomerular pathology highlighted by reduced glomerulosclerosis, inflammatory cell infiltration, ischemic lesions, mesangial thickening, mesangial hypercellularity, and glomerular enlargement. In addition, Fibγ390-396A mice with SCA had improved glomerular protective responses and podocyte/mesangial transcriptional signatures that resulted in reduced albuminuria. Interestingly, the fibrinogen γ390-396A mutation had a negligible effect on cardiac, lung, and liver functions and pathologies in the context of SCA over a year-long observation period. Taken together, our data support that fibrinogen significantly contributes to WBC-driven inflammation and ROS production, which is a key driver of SCA-associated glomerulopathy, and may represent a novel therapeutic target against irreversible kidney damage in SCA.

The pattern-recognition molecule mindin binds integrin Mac-1 to promote macrophage phagocytosis via Syk activation and NF-κB p65 translocation.

Mindin has a broad spectrum of roles in the innate immune system, including in macrophage migration, antigen phagocytosis and cytokine production. Mindin functions as a pattern-recognition molecule for microbial pathogens. However, the underlying mechanisms of mindin-mediated phagocytosis and its exact membrane receptors are not well established. Herein, we generated mindin-deficient mice using the CRISPR-Cas9 system and show that peritoneal macrophages from mindin-deficient mice were severely defective in their ability to phagocytize E  coli. Phagocytosis was enhanced when E  coli or fluorescent particles were pre-incubated with mindin, indicating that mindin binds directly to bacteria or non-pathogen particles and promotes phagocytosis. We defined that 131 I-labelled mindin binds with integrin Mac-1 (CD11b/CD18), the F-spondin (FS)-fragment of mindin binds with the αM -I domain of Mac-1 and that mindin serves as a novel ligand of Mac-1. Blockade of the αM -I domain of Mac-1 using either a neutralizing antibody or si-Mac-1 efficiently blocked mindin-induced phagocytosis. Furthermore, mindin activated the Syk and MAPK signalling pathways and promoted NF-κB entry into the nucleus. Our data indicate that mindin binds with the integrin Mac-1 to promote macrophage phagocytosis through Syk activation and NF-κB p65 translocation, suggesting that the mindin/Mac-1 axis plays a critical role during innate immune responses.

Hydroxyethyl Starch 130/0.4 Binds to Neutrophils Impairing Their Chemotaxis through a Mac-1 Dependent Interaction.

Several studies showed that hydroxyethyl starch (HES), a synthetic colloid used in volume replacement therapies, interferes with leukocyte-endothelium interactions. Although still unclear, the mechanism seems to involve the inhibition of neutrophils' integrin. With the aim to provide direct evidence of the binding of HES to neutrophils and to investigate the influence of HES on neutrophil chemotaxis, we isolated and treated the cells with different concentrations of fluorescein-conjugated HES (HES-FITC), with or without different stimuli (N-Formylmethionine-leucyl-phenylalanine, fMLP, or IL-8). HES internalization was evaluated by trypan blue quenching and ammonium chloride treatment. Chemotaxis was evaluated by under-agarose assay after pretreatment of the cells with HES or a balanced saline solution. The integrin interacting with HES was identified by using specific blocking antibodies. Our results showed that HES-FITC binds to the plasma membrane of neutrophils without being internalized. Additionally, the cell-associated fluorescence increased after stimulation of neutrophils with fMLP (p < 0.01) but not IL-8. HES treatment impaired the chemotaxis only towards fMLP, event mainly ascribed to the inhibition of CD-11b (Mac-1 integrin) activity. Therefore, the observed effect mediated by HES should be taken into account during volume replacement therapies. Thus, HES treatment could be advantageous in clinical conditions where a low activation/recruitment of neutrophils may be beneficial, but may be harmful when unimpaired immune functions are mandatory.

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.

Cis interaction between sialylated FcγRIIA and the αI-domain of Mac-1 limits antibody-mediated neutrophil recruitment.

Vascular-deposited IgG immune complexes promote neutrophil recruitment, but how this process is regulated is still unclear. Here we show that the CD18 integrin Mac-1, in its bent state, interacts with the IgG receptor FcγRIIA in cis to reduce the affinity of FcγRIIA for IgG and inhibit FcγRIIA-mediated neutrophil recruitment under flow. The Mac-1 rs1143679 lupus-risk variant reverses Mac-1 inhibition of FcγRIIA, as does a Mac-1 ligand and a mutation in Mac-1's ligand binding αI-domain. Sialylated complex glycans on FcγRIIA interact with the αI-domain via divalent cations, and this interaction is required for FcγRIIA inhibition by Mac-1. Human neutrophils deficient in CD18 integrins exhibit augmented FcγRIIA-dependent recruitment to IgG-coated endothelium. In mice, CD18 integrins on neutrophils dampen IgG-mediated neutrophil accumulation in the kidney. In summary, cis interaction between sialylated FcγRIIA and the αI-domain of Mac-1 alters the threshold for IgG-mediated neutrophil recruitment. A disruption of this interaction may increase neutrophil influx in autoimmune diseases.

Rapid screening of anti-tumor metastasis drugs targeting integrin macrophage antigen-1 using immobilized cell capillary electrophoresis.

In this research a method called immobilized cell capillary electrophoresis (ICCE) was established under approximately physiological conditions for rapid screening of anti-tumor metastasis drugs targeting integrin macrophage antigen-1 (MAC-1). In this method, separation and purification of the target receptors on cell membranes was unnecessary, thus, maintaining their natural conformation and bioactivity. MAC-1-, CD11b-, or CD18-overexpressing HEK293 cells (human embryonic kidney) were cultured and immobilized on the inner wall of capillaries as stationary phase, and their interactions with lactosyl derivative Gu-4 (positive control)/dimethylsulfoxide (DMSO; negative control) were studied using ICCE. Using this method, 29 phenylethanoid glycosides from Cistanches Herba were screened, and the binding kinetic parameters (K, ka, kd, and k') of active compounds were calculated, and the specific subunits of MAC-1 were determined. Then, molecular docking studies were performed to discover the direct interaction sites between active compounds and MAC-1, and the order of Glide-calculated Emodel value obtained from the molecular docking study is consistent with that of the binding constants obtained using ICCE. Finally, pharmaceutical efficacy assays in vitro and in vivo were carried out to show that the anti-tumor metastasis activity of the active compound had better pharmaceutical efficacy and lower toxic side effects. The method was verified to be valid and practical for further use, and it is expected that it will be transferred to capillary array electrophoresis for use in high-throughput drug screening.

Functional studies of chronic lymphocytic leukemia B cells expressing ß2-integrin type complement receptors CR3 and CR4.

The expression and role of CR3 (CD11b/CD18) and CR4 (CD11c/CD18) in B cells are not yet explored in contrast to myeloid cells, where these ß2-integrin type receptors are known to participate in various cellular functions, including phagocytosis, adherence and migration. Here we aimed to reveal the expression and role of CR3 and CR4 in human B cells. In B cells of healthy donors CR3 and CR4 are scarcely expressed. However, two patients with chronic lymphocytic leukemia (CLL) characterized by a peculiar immune-phenotype containing both CD5-positive and CD5-negative B cell populations made possible to study these molecules in distinct B cell subsets. We found that CD11b and CD11c were expressed on both CD5-positive and CD5-negative B cells, albeit to different extents. Our data suggest that these receptors are involved in spreading, since this activity of CpG-activated B cells on fibrinogen could be partially blocked by monoclonal antibodies specific for CD11b or CD11c. CpG-stimulation lead to proliferation of both CD5-positive and CD5-negative B cells of the patients with a less pronounced effect on the CD5-positive cells. In contrast to normal B cells, CLL B cells of both patients reacted to CpG-stimulation with robust IL-10 production. The concomitant, suboptimal stimulus via the BCR and TLR9 exerted either a synergistic enhancing effect or resulted in inhibition of proliferation and IL-10 production of patients' B cells. Our data obtained studying B cells of leukemic patients point to the role of CR3 and probably CR4 in the interaction of tumor cells with the microenvironment and suggest the involvement of IL-10 producing B cells in the pathologic process.

Distinct recognition of complement iC3b by integrins αXß2 and αMß2.

Recognition by the leukocyte integrins αXß2 and αMß2 of complement iC3b-opsonized targets is essential for effector functions including phagocytosis. The integrin-binding sites on iC3b remain incompletely characterized. Here, we describe negative-stain electron microscopy and biochemical studies of αXß2 and αMß2 in complex with iC3b. Despite high homology, the two integrins bind iC3b at multiple distinct sites. αXß2 uses the αX αI domain to bind iC3b on its C3c moiety at one of two sites: a major site at the interface between macroglobulin (MG) 3 and MG4 domains, and a less frequently used site near the C345C domain. In contrast, αMß2 uses its αI domain to bind iC3b at the thioester domain and simultaneously interacts through a region near the αM ß-propeller and ß2 ßI domain with a region of the C3c moiety near the C345C domain. Remarkably, there is no overlap between the primary binding site of αXß2 and the binding site of αMß2 on iC3b. Distinctive binding sites on iC3b by integrins αXß2 and αMß2 may be biologically beneficial for leukocytes to more efficiently capture opsonized pathogens and to avoid subversion by pathogen factors.

Structural Basis for Simvastatin Competitive Antagonism of Complement Receptor 3.

The complement system is an important part of the innate immune response to infection but may also cause severe complications during inflammation. Small molecule antagonists to complement receptor 3 (CR3) have been widely sought, but a structural basis for their mode of action is not available. We report here on the structure of the human CR3 ligand-binding I domain in complex with simvastatin. Simvastatin targets the metal ion-dependent adhesion site of the open, ligand-binding conformation of the CR3 I domain by direct contact with the chelated Mg(2+) ion. Simvastatin antagonizes I domain binding to the complement fragments iC3b and C3d but not to intercellular adhesion molecule-1. By virtue of the I domain's wide distribution in binding kinetics to ligands, it was possible to identify ligand binding kinetics as discriminator for simvastatin antagonism. In static cellular experiments, 15-25 µm simvastatin reduced adhesion by K562 cells expressing recombinant CR3 and by primary human monocytes, with an endogenous expression of this receptor. Application of force to adhering monocytes potentiated the effects of simvastatin where only a 50-100 nm concentration of the drug reduced the adhesion by 20-40% compared with untreated cells. The ability of simvastatin to target CR3 in its ligand binding-activated conformation is a novel mechanism to explain the known anti-inflammatory effects of this compound, in particular because this CR3 conformation is found in pro-inflammatory environments. Our report points to new designs of CR3 antagonists and opens new perspectives and identifies druggable receptors from characterization of the ligand binding kinetics in the presence of antagonists.

The cationic peptide LL-37 binds Mac-1 (CD11b/CD18) with a low dissociation rate and promotes phagocytosis.

As a broad-spectrum anti-microbial peptide, LL-37 plays an important role in the innate immune system. A series of previous reports implicates LL-37 as an activator of various cell surface receptor-mediated functions, including chemotaxis in integrin CD11b/CD18 (Mac-1)-expressing cells. However, evidence is scarce concerning the direct binding of LL-37 to these receptors and investigations on the associated binding kinetics is lacking. Mac-1, a member of the ß2 integrin family, is mainly expressed in myeloid leukocytes. Its critical functions include phagocytosis of complement-opsonized pathogens. Here, we report on interactions of LL-37 and its fragment FK-13 with the ligand-binding domain of Mac-1, the α-chain I domain. LL-37 bound the I-domain with an affinity comparable to the complement fragment C3d, one of the strongest known ligands for Mac-1. In cell adhesion assays both LL-37 and FK-13 supported binding by Mac-1 expressing cells, however, with LL-37-coupled surfaces supporting stronger cell adhesion than FK-13. Likewise, in phagocytosis assays with primary human monocytes both LL-37 and FK-13 enhanced uptake of particles coupled with these ligands but with a tendency towards a stronger uptake by LL-37.

Differential effects of volatile anesthetics on leukocyte integrin macrophage-1 antigen.

Macrophage-1 antigen (Mac-1, αMß2) is a leukocyte adhesion molecule that plays a significant role in leukocyte crawling and phagocytosis, and is homologous to its sister protein leukocyte function-associated antigen-1 (LFA-1, αLß2). The authors have previously demonstrated that volatile anesthetics isoflurane and sevoflurane bound to and inhibited LFA-1. Here, the hypothesis tested was that isoflurane and sevoflurane would inhibit Mac-1. A binding assay of Mac-1 to its ligand inter-cellular adhesion molecule-1 (ICAM-1) using V-bottom plates was established. The effect of isoflurane and sevoflurane on Mac-1 was examined using this ICAM-1 binding assay and by probing exposure of activation-sensitive epitopes. The docking program Glide was used to predict anesthetic binding site(s) on Mac-1. The functional role of this predicted binding site was then assessed by introducing point mutations in this region. Lastly, the effect of anesthetic on activating mutants was evaluated. The results indicated that isoflurane inhibited binding of Mac-1 to ICAM-1, but sevoflurane did not. Isoflurane also attenuated the exposure of the activation-sensitive epitopes. The docking simulation predicted the isoflurane binding site to be at the cavity underneath the α7 helix of the ligand binding domain (the αM I domain). Point mutants at this predicted binding site contained both activating and deactivating mutants, suggesting its functional significance. The binding of activating mutants αM-Y267A ß2-WT and αM-L312A ß2-WT to ICAM-1 was not affected by isoflurane, but binding of another activating mutant αM-WT ß2-L132A was inhibited supporting the binding of isoflurane to this cavity. The conclusion reached from these findings was that isoflurane inhibited Mac-1 binding to ICAM-1 by binding to the cavity underneath the α7 helix of the αM I domain, but sevoflurane did not. Thus, because these common clinical volatile anesthetics demonstrated different effects on Mac-1, this implied their effects on the immune system might differ.

[Production and stabilization of an integrin-binding moiety of complement component 3].

The third component of complement, C3, plays a central role in human innate immunity. The subsequent proteolysis product of native C3, iC3b, is the primary ligand of complement receptors (CRs) CR3 and CR4. CR3 and CR4 are ß2-family integrins, and their binding to iC3b contributes to phagocytosis. How iC3b binds to its receptors and transmits signals into the cells is not clear. To perform structural and functional studies on the interaction between iC3b and its receptors CR3/CR4, we isolated the integrin-binding fragment of iC3b, MG3-4. Low temperature is required for its soluble expression in Escherichia coli. Purified MG3-4 existed as a dimer in solution and was easy to aggregate. We tried different agents and found glycerol could efficiently stabilize the MG3-4 fragment to avoid aggregation. Using surface plasmon resonance (SPR) analysis, we confirmed MG3-4 could bind I domain, the iC3b-binding domain of CR3. Here, we report the successful production of a soluble, stable, and biologically active integrin-binding moiety of human iC3b for further studies.

Reduced Fluorescence versus Forward Scatter Time-of-Flight and Increased Peak versus Integral Fluorescence Ratios Indicate Receptor Clustering in Flow Cytometry.

Clustering of surface receptors is often required to initiate signal transduction, receptor internalization, and cellular activation. To study the kinetics of clustering, we developed an economic high-throughput method using flow cytometry. The quantification of receptor clustering by flow cytometry is based on the following two observations: first, the fluorescence signal length (FL time-of-flight [ToF]) decreases relative to the forward scatter signal length (FSc-ToF), and second, the peak FL (FL-peak) increases relative to the integral FL (FL-integral) upon clustering of FL-labeled surface receptors. Receptor macroclustering can therefore be quantified using the ratios FL-ToF/FSc-ToF (method ToF) or FL-peak/FL-integral (method Peak). We have used these methods to analyze clustering of two immune receptors known to undergo different conformational and oligomeric states: the BCR and the complement receptor 3 (CR3), on murine splenocytes, purified B cells, and human neutrophils. Engagement of both the BCR and CR3, on immortalized as well as primary murine B cells and human neutrophil, respectively, resulted in decreased FL-ToF/FSc-ToF and increased FL-peak/FL-integral ratios. Manipulation of the actin-myosin cytoskeleton altered BCR clustering which could be measured using the established parameters. To confirm clustering of CR3 on neutrophils, we applied imaging flow cytometry. Because receptor engagement is as a biological process dependent on cell viability, energy metabolism, and temperature, receptor clustering can only be quantified by gating on viable cells under physiological conditions. In summary, with this novel method, receptor clustering on nonadherent cells can easily be monitored by high-throughput conventional flow cytometry.

Anchorage-dependent binding of integrin I-domain to adhesion ligands.

The dynamic interactions between leukocyte integrin receptors and ligands in the vascular endothelium, extracellular matrix, or invading pathogens result in leukocyte adhesion, extravasation, and phagocytosis. This work examined the mechanical strength of the connection between iC3b, a complement component that stimulates phagocytosis, and the ligand-binding domain, the I-domain, of integrin αMß2. Single-molecule force measurements of αM I-domain-iC3b complexes were conducted by atomic force microscope. Strikingly, depending on loading rates, immobilization of the I-domain via its C-terminus resulted in a 1.3-fold to 1.5-fold increase in unbinding force compared with I-domains immobilized via the N-terminus. The force spectra (unbinding force versus loading rate) of the I-domain-iC3b complexes revealed that the enhanced mechanical strength is due to a 2.4-fold increase in the lifetime of the I-domain-iC3b bond. Given the structural and functional similarity of all integrin I-domains, our result supports the existing allosteric regulatory model by which the ligand binding strength of integrin can be increased rapidly when a force is allowed to stretch the C-terminus of the I-domain. This type of mechanism may account for the rapid ligand affinity adjustment during leukocyte migration.

Ligand recognition specificity of leukocyte integrin αMß2 (Mac-1, CD11b/CD18) and its functional consequences.

The broad recognition specificity exhibited by integrin α(M)ß2 (Mac-1, CD11b/CD18) has allowed this adhesion receptor to play innumerable roles in leukocyte biology, yet we know little about how and why α(M)ß2 binds its multiple ligands. Within α(M)ß2, the α(M)I-domain is responsible for integrin's multiligand binding properties. To identify its recognition motif, we screened peptide libraries spanning sequences of many known protein ligands for α(M)I-domain binding and also selected the α(M)I-domain recognition sequences by phage display. Analyses of >1400 binding and nonbinding peptides derived from peptide libraries showed that a key feature of the α(M)I-domain recognition motif is a small core consisting of basic amino acids flanked by hydrophobic residues. Furthermore, the peptides selected by phage display conformed to a similar pattern. Identification of the recognition motif allowed the construction of an algorithm that reliably predicts the α(M)I-domain binding sites in the α(M)ß2 ligands. The recognition specificity of the α(M)I-domain resembles that of some chaperones, which allows it to bind segments exposed in unfolded proteins. The disclosure of the α(M)ß2 binding preferences allowed the prediction that cationic host defense peptides, which are strikingly enriched in the α(M)I-domain recognition motifs, represent a new class of α(M)ß2 ligands. This prediction has been tested by examining the interaction of α(M)ß2 with the human cathelicidin peptide LL-37. LL-37 induced a potent α(M)ß2-dependent cell migratory response and caused activation of α(M)ß2 on neutrophils. The newly revealed recognition specificity of α(M)ß2 toward unfolded protein segments and cationic proteins and peptides suggests that α(M)ß2 may serve as a previously proposed "alarmin" receptor with important roles in innate host defense.