Phosphorylation Regulation Mechanism of ß2 Integrin for the Binding of Filamin Revealed by Markov State Model.

Leukocyte adhesion deficiency-1 (LAD-1) disorder is a severe immunodeficiency syndrome caused by deficiency or mutation of ß2 integrin. The phosphorylation on threonine 758 of ß2 integrin acts as a molecular switch inhibiting the binding of filamin. However, the switch mechanism of site-specific phosphorylation at the atom level is still poorly understood. To resolve the regulation mechanism, all-atom molecular dynamics simulation and Markov state model were used to study the dynamic regulation pathway of phosphorylation. Wild type system possessed lower binding free energy and fewer number of states than the phosphorylated system. Both systems underwent local disorder-to-order conformation conversion when achieving steady states. To reach steady states, wild type adopted less number of transition paths/shortest path according to the transition path theory than the phosphorylated system. The underlying phosphorylated regulation pathway was from P1 to P0 and then P4 state, and the main driving force should be hydrogen bond and hydrophobic interaction disturbing the secondary structure of phosphorylated states. These studies will shed light on the pathogenesis of LAD-1 disease and lay a foundation for drug development.

The integrin-linked kinase is required for chemokine-triggered high-affinity conformation of the neutrophil ß2-integrin LFA-1.

Neutrophil adhesion and extravasation into tissue at sites of injury or infection depend on binding of the integrin lymphocyte function-associated antigen 1 (LFA-1) to ICAM-1 expressed on activated endothelial cells. The activation-dependent conformational change of LFA-1 to the high-affinity conformation (H+) requires kindlin-3 binding to the ß2-integrin cytoplasmic domain. Here we show that genetic deletion of the known kindlin interactor integrin-linked kinase (ILK) impaired neutrophil adhesion and extravasation in the cremaster muscle and in a clinically relevant model of renal ischemia reperfusion injury. Using in vitro microfluidic adhesion chambers and conformation-specific antibodies, we show that knockdown of ILK in HL-60 cells reduced the conformational change of ß2-integrins to the H+ conformation. Mechanistically, we found that ILK was required for protein kinase C (PKC) membrane targeting and chemokine-induced upregulation of its kinase activity. Moreover, PKC-α deficiency also resulted in impaired leukocyte adhesion in bone marrow chimeric mice. Mass spectrometric and western blot analyses revealed stimulation- and ILK-dependent phosphorylation of kindlin-3 upon activation. In summary, our data indicate an important role of ILK in kindlin-3-dependent conformational activation of LFA-1.

Regulation of neutrophil function by selective targeting of glycan epitopes expressed on the integrin CD11b/CD18.

Polymorphonuclear neutrophils (PMNs) play a critical role in the innate immune response to invading pathogens. However, dysregulated mucosal trafficking of PMNs and associated epithelial tissue damage is a pathological hallmark of numerous inflammatory conditions including inflammatory bowel disease. The glycoprotein CD11b/CD18 plays a well-described role in regulating PMN transepithelial migration and PMN inflammatory functions. Previous studies have demonstrated that targeting of the N-linked glycan Lewis X on CD11b blocks PMN transepithelial migration (TEpM). Given evidence of glycosylation-dependent regulation of CD11b/CD18 function, we performed MALDI TOF Mass Spectrometry (MS) analyses on CD11b/CD18 purified from human PMNs. Unusual glycan epitopes identified on CD11b/CD18 included high Mannose oligosaccharides recognized by the Galanthus Nivalis lectin and biantennary galactosylated N-glycans recognized by the Phaseolus Vulgaris erythroagglutinin lectin. Importantly, we show that selective targeting of glycans on CD11b with such lectins results in altered intracellular signaling events that inhibit TEpM and differentially affect key PMN inflammatory functions including phagocytosis, superoxide release and apoptosis. Taken together, these data demonstrate that discrete glycan motifs expressed on CD11b/CD18 such as biantennary galactose could represent novel targets for selective manipulation of CD11b function and reduction of PMN-associated tissue damage in chronic inflammatory diseases.

A pivotal role for a conserved bulky residue at the α1-helix of the αI integrin domain in ligand binding.

The ligand-binding ßI and αI domains of integrin are the best-studied von Willebrand factor A domains undergoing significant conformational changes for affinity regulation. In both ßI and αI domains, the α1- and α7-helixes work in concert to shift the metal-ion-dependent adhesion site between the resting and active states. An absolutely conserved Gly in the middle of the α1-helix of ßI helps maintain the resting ßI conformation, whereas the homologous position in the αI α1-helix contains a conserved Phe. A functional role of this Phe is structurally unpredictable. Using αLß2 integrin as a model, we found that the residue volume at the Phe position in the α1-helix is critical for αLß2 activation because trimming the Phe by small amino acid substitutions abolished αLß2 binding with soluble and immobilized intercellular cell adhesion molecule 1. Similar results were obtained for αMß2 integrin. Our experimental and molecular dynamics simulation data suggested that the bulky Phe acts as a pawl that stabilizes the downward ratchet-like movement of ß6-α7 loop and α7-helix, required for high-affinity ligand binding. This mechanism may apply to other von Willebrand factor A domains undergoing large conformational changes. We further demonstrated that the conformational cross-talk between αL αI and ß2 ßI could be uncoupled because the ß2 extension and headpiece opening could occur independently of the αI activation. Reciprocally, the αI activation does not inevitably lead to the conformational changes of the ß2 subunit. Such loose linkage between the αI and ßI is attributed to the αI flexibility and could accommodate the αLß2-mediated rolling adhesion of leukocytes.

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.

P4H9-detected molecule expression on spindle-shaped fibroblasts indicates malignant phenotype of colorectal cancer.

BACKGROUND: Our previous study using a mammary fat pad mouse model showed that P4H9, produced by the ß2 integrin epitope, detected a molecule on fibroblasts in response to carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1)-expressing cancer cells. P4H9-detected molecule (PDM) expression appeared to be associated with myofibroblast differentiation. In this study, we investigated whether PDM is expressed on fibroblasts and cancer cells in clinical tissue samples, and whether the presence of PDM-expressing colorectal cancer cells is correlated with clinicopathological features of patients. METHODS: Immunohistochemistry was conducted to detect P4H9 on clinical tissue samples from 156 patients with colorectal cancer. Risk factors for metastases and survival were calculated for clinical implication of PDM-expressing spindle-shaped fibroblasts. RESULTS: Multivariate analysis showed that PDM-expressing spindle-shaped fibroblasts were an independent risk factor for lymph node metastasis, hematogenous metastasis, and poor survival. A Kaplan-Meier survival curve indicated that PDM-expressing spindle-shaped fibroblasts were associated with shorter survival time (P<0.0001). Immunofluorescence showed PDM expression on CCD-18Co fibroblasts and two colorectal cancer cell lines (HCT116 and HCT-15). CONCLUSIONS: PDM-expressing spindle-shaped fibroblasts are associated with metastasis and shorter survival in colorectal cancer patients. PDM-expressing spindle-shaped fibroblasts may have a role in eliciting the malignant phenotype of colorectal cancer.

Characterization of single amino acid substitutions in the ß2 integrin subunit of patients with leukocyte adhesion deficiency (LAD)-1.

Leukocyte adhesion deficiency 1 (LAD-1) is caused by defects in the ß2 integrin subunit. We studied 18 missense mutations, 14 of which fail to support the surface expression of the ß2 integrins. Integrins with the ß2-G150D mutation fail to bind ligands, possibly due to the failure of the α1 segment of the ßI domain to assume an α-helical structure. Integrins with the ß2-G716A mutation are not maintained in their resting states, and the patient has the severe phenotype of LAD-1. The ß2-S453N and ß2-P648L mutants support the expression of integrins and adhesion functions. They should be re-classified as polymorphic variants.

α7 helix region of αI domain is crucial for integrin binding to endoplasmic reticulum chaperone gp96: a potential therapeutic target for cancer metastasis.

Integrins play important roles in regulating a diverse array of cellular functions crucial to the initiation, progression, and metastasis of tumors. Previous studies have shown that a majority of integrins are folded by the endoplasmic reticulum chaperone gp96. Here, we demonstrate that the dimerization of integrin αL and ß2 is highly dependent on gp96. The αI domain (AID), a ligand binding domain shared by seven integrin α-subunits, is a critical region for integrin binding to gp96. Deletion of AID significantly reduced the interaction between integrin αL and gp96. Overexpression of AID intracellularly decreased surface expression of gp96 clients (integrins and Toll-like receptors) and cancer cell invasion. The α7 helix region is crucial for AID binding to gp96. A cell-permeable α7 helix peptide competitively inhibited the interaction between gp96 and integrins and blocked cell invasion. Thus, targeting the binding site of α7 helix of AID on gp96 is potentially a new strategy for treatment of cancer metastasis.

Monodisperse and LPS-free Aggregatibacter actinomycetemcomitans leukotoxin: interactions with human ß2 integrins and erythrocytes.

Aggregatibacter actinomycetemcomitans is a gram-negative, facultatively anaerobic cocco-bacillus and a frequent member of the human oral flora. It produces a leukotoxin, LtxA, belonging to the repeats-in-toxin (RTX) family of bacterial cytotoxins. LtxA efficiently kills neutrophils and mononuclear phagocytes. The known receptor for LtxA on leukocytes is integrin α(L)ß(2) (LFA-1 or CD11a/CD18). However, the molecular mechanisms involved in LtxA-mediated cytotoxicity are poorly understood, partly because LtxA has proven difficult to prepare for experiments as free of contaminants and with its native structure. Here, we describe a protocol for the purification of LtxA from bacterial culture supernatant, which does not involve denaturing procedures. The purified LtxA was monodisperse, well folded as judged by the combined use of synchrotron radiation circular dichroism spectroscopy (SRCD) and in silico prediction of the secondary structure content, and free of bacterial lipopolysaccharide. The analysis by SRCD and similarity to a lipase from Pseudomonas with a known three dimensional structure supports the presence of a so-called beta-ladder domain in the C-terminal part of LtxA. LtxA rapidly killed K562 target cells transfected to express ß(2) integrin. Cells expressing α(M)ß(2) (CD11b/CD18) or α(X)ß(2) (CD11c/CD18) were killed as efficiently as cells expressing α(L)ß(2). Erythrocytes, which do not express ß(2) integrins, were lysed more slowly. In ligand blotting experiments, LtxA bound only to the ß(2) chain (CD18). These data support a previous suggestion that CD18 harbors the major binding site for LtxA as well as identifies integrins α(M)ß(2) and α(X)ß(2) as novel receptors for LtxA.

A Flow Cytometry-Based High-Throughput Technique for Screening Integrin-Inhibitory Drugs.

This protocol aims to establish a method for identifying small molecular antagonists of ß2 integrin activation, utilizing conformational-change-reporting antibodies and high-throughput flow cytometry. The method can also serve as a guide for other antibody-based high-throughput screening methods. ß2 integrins are leukocyte-specific adhesion molecules that are crucial in immune responses. Neutrophils rely on integrin activation to exit the bloodstream, not only to fight infections but also to be involved in multiple inflammatory diseases. Controlling ß2 integrin activation presents a viable approach for treating neutrophil-associated inflammatory diseases. In this protocol, a monoclonal antibody, mAb24, which specifically binds to the high-affinity headpiece of ß2 integrins, is utilized to quantify ß2 integrin activation on isolated primary human neutrophils. N-formylmethionyl-leucyl-phenylalanine (fMLP) is used as a stimulus to activate neutrophil ß2 integrins. A high-throughput flow cytometer capable of automatically running 384-well plate samples was used in this study. The effects of 320 chemicals on ß2 integrin inhibition are assessed within 3 h. Molecules that directly target ß2 integrins or target molecules in the G protein-coupled receptor-initiated integrin inside-out activation signaling pathway can be identified through this approach.

MMGBSA as a tool to understand the binding affinities of filamin-peptide interactions.

Filamins (FLN) are large dimeric proteins that cross-link actin and work as important scaffolds in human cells. FLNs consist of an N-terminal actin-binding domain followed by 24 immunoglobulin-like domains (FLN1-24). FLN domains are divided into four subgroups based on their amino acid sequences. One of these subgroups, including domains 4, 9, 12, 17, 19, 21, and 23, shares a similar ligand-binding site between the ß strands C and D. Several proteins, such as integrins ß2 and ß7, glycoprotein Ibα (GPIbα), and migfilin, have been shown to bind to this site. Here, we computationally estimated the binding free energies of filamin A (FLNa) subunits with bound peptides using the molecular mechanics-generalized Born surface area (MMGBSA) method. The obtained computational results correlated well with the experimental data, and they ranked efficiently both the binding of one ligand to all used FLNa-domains and the binding of all used ligands to FLNa21. Furthermore, the steered molecular dynamics (SMD) simulations pinpointed the binding hot spots for these complexes. These results demonstrate that molecular dynamics combined with free energy calculations are applicable to estimating the energetics of protein-protein interactions and can be used to direct the development of novel FLN function modulators.

Stable coordination of the inhibitory Ca2+ ion at the metal ion-dependent adhesion site in integrin CD11b/CD18 by an antibody-derived ligand aspartate: implications for integrin regulation and structure-based drug design.

A central feature of integrin interaction with physiologic ligands is the monodentate binding of a ligand carboxylate to a Mg(2+) ion hexacoordinated at the metal ion-dependent adhesion site (MIDAS) in the integrin A domain. This interaction stabilizes the A domain in the high-affinity state, which is distinguished from the default low-affinity state by tertiary changes in the domain that culminate in cell adhesion. Small molecule ligand-mimetic integrin antagonists act as partial agonists, eliciting similar activating conformational changes in the A domain, which has contributed to paradoxical adhesion and increased patient mortality in large clinical trials. As with other ligand-mimetic integrin antagonists, the function-blocking mAb 107 binds MIDAS of integrin CD11b/CD18 A domain (CD11bA), but in contrast, it favors the inhibitory Ca(2+) ion over the Mg(2+) ion at MIDAS. We determined the crystal structures of the Fab fragment of mAb 107 complexed to the low- and high-affinity states of CD11bA. Favored binding of the Ca(2+) ion at MIDAS is caused by the unusual symmetric bidentate ligation of a Fab-derived ligand Asp to a heptacoordinated MIDAS Ca(2+) ion. Binding of the Fab fragment of mAb 107 to CD11bA did not trigger the activating tertiary changes in the domain or in the full-length integrin. These data show that the denticity of the ligand Asp/Glu can modify the divalent cation selectivity at MIDAS and hence integrin function. Stabilizing the Ca(2+) ion at MIDAS by bidentate ligation to a ligand Asp/Glu may provide one approach for designing pure integrin antagonists.

The C-terminal rod 2 fragment of filamin A forms a compact structure that can be extended.

Filamins are large proteins that cross-link actin filaments and connect to other cellular components. The C-terminal rod 2 region of FLNa (filamin A) mediates dimerization and interacts with several transmembrane receptors and intracellular signalling adaptors. SAXS (small-angle X-ray scattering) experiments were used to make a model of a six immunoglobulin-like domain fragment of the FLNa rod 2 (domains 16-21). This fragment had a surprising three-branched structural arrangement, where each branch was made of a tightly packed two-domain pair. Peptides derived from transmembrane receptors and intracellular signalling proteins induced a more open structure of the six domain fragment. Mutagenesis studies suggested that these changes are caused by peptides binding to the CD faces on domains 19 and 21 which displace the preceding domain A-strands (18 and 20 respectively), thus opening the individual domain pairs. A single particle cryo-EM map of a nine domain rod 2 fragment (domains 16-24), showed a relatively compact dimeric particle and confirmed the three-branched arrangement as well as the peptide-induced conformation changes. These findings reveal features of filamin structure that are important for its interactions and mechanical properties.

Ser756 of ß2 integrin controls Rap1 activity during inside-out activation of αMß2.

During αMß2-mediated phagocytosis, the small GTPase Rap1 activates the ß2 integrin by binding to a region between residues 732 and 761. Using COS-7 cells transfected with αMß2, we show that αMß2 activation by the phorbol ester PMA involves Ser(756) of ß2. This residue is critical for the local positioning of talin and biochemically interacts with Rap1. Using the CaM (calmodulin) antagonist W7, we found Rap1 recruitment and the inside-out activation of αMß2 to be affected. We also report a role for CaMKII (calcium/CaM-dependent kinase II) in the activation of Rap1 during integrin activation. These results demonstrate a distinct physiological role for Ser(756) of ß2 integrin, in conjunction with the actions of talin and Rap1, during αMß2 activation in macrophages.

MMP-9 sheds the beta2 integrin subunit (CD18) from macrophages.

Activated macrophages are essential effectors of immunity and a rich source of matrix metalloproteinase-9 (MMP-9; gelatinase B). To search for cellular substrates of the enzyme, we subjected wild-type macrophages and macrophages expressing an autoactivating form of pro-MMP-9 (M9A macrophages) to proteomics analysis. Two-dimensional liquid chromatography together with tandem mass spectrometry identified 467 proteins in medium conditioned by M9A and/or wild-type macrophages. Subtractive proteomics identified 18 candidate MMP-9 substrates. Biochemical studies confirmed that two transmembrane proteins, beta(2) integrin subunit (CD18) and amyloid protein precursor (APP), were enriched in the medium of M9A macrophages. To identify potential cleavage sites, we synthesized an overlapping library of peptides that spanned 60 residues of the ectodomain and transmembrane domain of beta(2) integrin. Active MMP-9 cleaved a single peptide, ECVKGPNVAAIVGGT, at residues corresponding to Ala(705) and Ile(706) of the beta(2) integrin. Peptides corresponding to this cleavage site were detected by tandem mass spectrometric analysis only in medium from M9A macrophages, strongly supporting the proposal that beta(2) integrin is shed by autoactivating MMP-9. Our observations indicate that subtractive proteomics in concert with peptide substrate mapping is a powerful approach for identifying proteolytic substrates and suggest that MMP-9 plays previously unsuspected roles in the regulation and shedding of beta(2) integrin.

ß2 Integrin CD11d/CD18: From Expression to an Emerging Role in Staged Leukocyte Migration.

CD11d/CD18 is the most recently discovered and least understood ß2 integrin. Known CD11d adhesive mechanisms contribute to both extravasation and mesenchymal migration - two key aspects for localizing peripheral leukocytes to sites of inflammation. Differential expression of CD11d induces differences in monocyte/macrophage mesenchymal migration including impacts on macrophage sub-set migration. The participation of CD11d/CD18 in leukocyte localization during atherosclerosis and following neurotrauma has sparked interest in the development of CD11d-targeted therapeutic agents. Whereas the adhesive properties of CD11d have undergone investigation, the signalling pathways induced by ligand binding remain largely undefined. Underlining each adhesive and signalling function, CD11d is under unique transcriptional control and expressed on a sub-set of predominately tissue-differentiated innate leukocytes. The following review is the first to capture the nearly three decades of CD11d research and discusses the emerging role of CD11d in leukocyte migration and retention during the progression of a staged immune response.

Molecular Insights Into Neutrophil Biology From the Zebrafish Perspective: Lessons From CD18 Deficiency.

Neutrophils are key players in innate immunity and originate from the bone marrow of the adult mammalian organism. In mammals, mature neutrophils are released from the bone marrow into the peripheral blood where they circulate until their recruitment to sites of inflammation in a multistep adhesion cascade. Here, adhesion molecules of the ß2 integrin family (CD11/CD18) are critically required for the initial neutrophil adhesion to the inflamed endothelium and several post-adhesion steps allowing their extravasation into the inflamed tissue. Within the mammalian tissue, interstitial neutrophil migration can occur widely independent of ß2 integrins. This is in sharp contrast to neutrophil recruitment in zebrafish larvae (Danio rerio) where neutrophils originate from the caudal hematopoietic tissue and mainly migrate interstitially to sites of lesion upon the early onset of inflammation. However, neutrophils extravasate from the circulation to the inflamed tissue in zebrafish larvae at later-time points. Although zebrafish larvae are a widely accepted model system to analyze neutrophil trafficking in vivo, the functional impact of ß2 integrins for neutrophil trafficking during acute inflammation is completely unknown in this model. In this study, we generated zebrafish with a genetic deletion of CD18, the ß subunit of ß2 integrins, using CRISPR/Cas9 technology. Sequence alignments demonstrated a high similarity of the amino acid sequences between zebrafish and human CD18 especially in the functionally relevant I-like domain. In addition, the cytoplasmic domain of CD18 harbors two highly conserved NXXF motifs suggesting that zebrafish CD18 may share functional properties of human CD18. Accordingly, CD18 knock-out (KO) zebrafish larvae displayed the key symptoms of patients suffering from leukocyte adhesion deficiency (LAD) type I due to defects in ITGB2, the gene for CD18. Importantly, CD18 KO zebrafish larvae showed reduced neutrophil trafficking to sites of sterile inflammation despite the fact that an increased number of neutrophils was detectable in the circulation. By demonstrating the functional importance of CD18 for neutrophil trafficking in zebrafish larvae, our findings shed new light on neutrophil biology in vertebrates and introduce a new model organism for studying LAD type I.

Beta2 integrin phosphorylation on Thr758 acts as a molecular switch to regulate 14-3-3 and filamin binding.

Leukocyte integrins of the beta2 family are essential for immune cell-cell adhesion. In activated cells, beta2 integrins are phosphorylated on the cytoplasmic Thr758, leading to 14-3-3 protein recruitment to the beta2 integrin. The mutation of this phosphorylation site impairs cell adhesion, actin reorganization, and cell spreading. Thr758 is contained in a Thr triplet of beta2 that also mediates binding to filamin. Here, we investigated the binding of filamin, talin, and 14-3-3 proteins to phosphorylated and unphosphorylated beta2 integrins by biochemical methods and x-ray crystallography. 14-3-3 proteins bound only to the phosphorylated integrin cytoplasmic peptide, with a high affinity (K(d), 261 nM), whereas filamin bound only the unphosphorylated integrin cytoplasmic peptide (K(d), 0.5 mM). Phosphorylation did not regulate talin binding to beta2 directly, but 14-3-3 was able to outcompete talin for the binding to phosphorylated beta2 integrin. X-ray crystallographic data clearly explained how phosphorylation eliminated filamin binding and induced 14-3-3 protein binding. Filamin knockdown in T cells led to an increase in stimulated cell adhesion to ICAM-1-coated surfaces. Our results suggest that the phosphorylation of beta2 integrins on Thr758 acts as a molecular switch to inhibit filamin binding and allow 14-3-3 protein binding to the integrin cytoplasmic domain, thereby modulating T-cell adhesion.

Frontline Science: A flexible kink in the transmembrane domain impairs ß2 integrin extension and cell arrest from rolling.

ß2 integrins are the main adhesion molecules in neutrophils and other leukocytes and are rapidly activated by inside-out signaling, which results in conformational changes that are transmitted through the transmembrane domain (TMD). Here, we investigated the biologic effect of introducing a proline mutation in the ß2 integrin TMD to create a flexible kink that uncouples the topology of the inner half of the TMD from the outer half and impairs integrin activation. The ß2 integrin alpha chains, αL, αM, αX, and αD, all contain an inserted (I) domain with homology to von Willebrand factor A domain. ß2 activation was monitored in a homogenous binding assay of 2 reporter monoclonal antibodies: KIM127 reporting extension (E+ ) and mAb24 reporting the high-affinity (H+ ) conformation of the ß2 I-like domain. The proline mutation partially diminished chemokine-induced extension, but not the high-affinity conformation. The proline mutation in the TMD of ß2 completely inhibited arrest of rolling HL-60 cells in response to the chemokine IL-8. TMD mutant HL-60 cells rolling on P-selectin and ICAM-1 were unable to reduce their rolling velocity in response to IL-8. Quantitative dynamic footprinting live-cell imaging showed that blocking TMD topology transmission impaired the chemokine-induced activation of ß2, limiting the appearance of extended high-affinity (E+ H+ ) ß2. This also resulted in a defect in early spreading (3 min after arrest), which could be overcome by forced integrin activation using Mn2+ . We conclude that the TMD proline mutation severely impairs ß2 integrin extension, cell arrest, and early spreading.

ß2 Integrin Signaling Cascade in Neutrophils: More Than a Single Function.

Neutrophils are the most prevalent leukocytes in the human body. They have a pivotal role in the innate immune response against invading bacterial and fungal pathogens, while recent emerging evidence also demonstrates their role in cancer progression and anti-tumor responses. The efficient execution of many neutrophil effector responses requires the presence of ß2 integrins, in particular CD11a/CD18 or CD11b/CD18 heterodimers. Although extensively studied at the molecular level, the exact signaling cascades downstream of ß2 integrins still remain to be fully elucidated. In this review, we focus mainly on inside-out and outside-in signaling of these two ß2 integrin members expressed on neutrophils and describe differences between various neutrophil stimuli with respect to integrin activation, integrin ligand binding, and the pertinent differences between mouse and human studies. Last, we discuss how integrin signaling studies could be used to explore the therapeutic potential of targeting ß2 integrins and the intracellular signaling cascade in neutrophils in several, among other, inflammatory conditions in which neutrophil activity should be dampened to mitigate disease.