Analysis of Clinical, Immunological and Molecular Features of Leukocyte Adhesion Deficiency Type I in Egyptian Children.

PURPOSE: Leukocyte adhesion deficiency (LAD) represents a rare group of inherited inborn errors of immunity (IEI) characterized by bacterial infections, delayed umbilical stump separation, and autoimmunity. This single-center study aimed at describing the clinical, immunological, and molecular characterizations of 34 LAD-I Egyptian pediatric patients. METHODS: Details of 34 patients' personal medical history, clinical and laboratory findings were recorded; Genetic material from 28 patients was studied. Mutational analysis was done by Sanger sequencing. RESULTS: Omphalitis, skin and soft tissue infections with poorly healing ulcers, delayed falling of the umbilical stump, and recurrent or un-resolving pneumonia were the most common presentations, followed by chronic otitis media, enteropathy, periodontitis; and recurrent oral thrush. Persistent leukocytosis and neutrophilia were reported in all patients, as well as CD18 and CD11b deficiency. CD18 expression was < 2% in around 90% of patients. Sixteen different pathological gene variants were detected in 28 patients who underwent ITGß2 gene sequencing, of those, ten were novel and six were previously reported. Three families received a prenatal diagnosis. Patients were on antimicrobials according to culture's results whenever available, and on prophylactic Trimethoprim-Sulfamethoxazole 5 mg/kg once daily, with regular clinical follow up. Hematopoietic stem cell transplantation (HSCT) was offered for 4 patients. However due to severity of the disease and delay in diagnosis, 58% of the patients passed away in the first 2 years of life. CONCLUSION: This study highlights the importance of early diagnosis and distribution of ITGß2 gene mutation in Egyptian children. Further molecular studies, however, remain a challenging necessity for better disease characterization in the region.

SKAP2 acts downstream of CD11b/CD18 and regulates neutrophil effector function.

Background: The importance of CD11b/CD18 expression in neutrophil effector functions is well known. Beyond KINDLIN3 and TALIN1, which are involved in the induction of the high-affinity binding CD11b/CD18 conformation, the signaling pathways that orchestrate this response remain incompletely understood. Method: We performed an unbiased screening method for protein selection by biotin identification (BioID) and investigated the KINDLIN3 interactome. We used liquid chromatography with tandem mass spectrometry as a powerful analytical tool. Generation of NB4 CD18, KINDLIN3, or SKAP2 knockout neutrophils was achieved using CRISPR-Cas9 technology, and the cells were examined for their effector function using flow cytometry, live cell imaging, microscopy, adhesion, or antibody-dependent cellular cytotoxicity (ADCC). Results: Among the 325 proteins significantly enriched, we identified Src kinase-associated phosphoprotein 2 (SKAP2), a protein involved in actin polymerization and integrin-mediated outside-in signaling. CD18 immunoprecipitation in primary or NB4 neutrophils demonstrated the presence of SKAP2 in the CD11b/CD18 complex at a steady state. Under this condition, adhesion to plastic, ICAM-1, or fibronectin was observed in the absence of SKAP2, which could be abrogated by blocking the actin rearrangements with latrunculin B. Upon stimulation of NB4 SKAP2-deficient neutrophils, adhesion to fibronectin was enhanced whereas CD18 clustering was strongly reduced. This response corresponded with significantly impaired CD11b/CD18-dependent NADPH oxidase activity, phagocytosis, and cytotoxicity against tumor cells. Conclusion: Our results suggest that SKAP2 has a dual role. It may restrict CD11b/CD18-mediated adhesion only under resting conditions, but its major contribution lies in the regulation of dynamic CD11b/CD18-mediated actin rearrangements and clustering as required for cellular effector functions of human neutrophils.

Gigantol ameliorates DSS-induced colitis via suppressing ß2 integrin mediated adhesion and chemotaxis of macrophage.

ETHNOPHARMACOLOGICAL RELEVANCE: Dendrobium, recognized as "Shihu" in traditional Chinese medicine, holds a rich history of medicinal utilization documented in the Chinese Pharmacopoeia. Ancient texts like "Shen Nong Ben Cao Jing" extol Dendrobium's virtues as a superior herbal medicine fortifying "Yin" and invigorating the five viscera. Dendrobium is extensively employed for the treatment of gastrointestinal inflammatory disorders, showcasing significant therapeutic efficacy, particularly against ulcerative colitis (UC), within the realm of Chinese ethnopharmacology. Dendrobium plays crucial pharmacological roles due to its rich content of polysaccharides, alkaloids, phenanthrenes, and bibenzyls. Gigantol, a prominent bibenzyl compound, stands out as one of the most vital active constituents within Dendrobium, the gigantol content of Dendrobium leaves can reach approximately 4.79 µg/g. Its significance lies in being recognized as a noteworthy anti-inflammatory compound derived from Dendrobium. AIM OF THE STUDY: Given the pivotal role of gigantol as a primary active substance in Dendrobium, the therapeutic potential of gigantol for gastrointestinal diseases remains enigmatic. Our present investigation aimed to evaluate the therapeutic effects of gigantol on dextran sulfate sodium (DSS)-induced colitis and reveal its potential mechanism in countering UC activity. MATERIALS AND METHODS: The protective efficacy of gigantol against colitis was assessed by examining the histopathological changes and conducting biochemical analyses of colon from DSS-challenged mice. Assessments focused on gigantol's impact on improving the intestinal epithelial barrier and its anti-inflammatory effects in colonic tissues of colitis mice. Investigative techniques included the exploration of the macrophage inflammatory signaling pathway via qPCR and Western blot analyses. In vitro studies scrutinized macrophage adhesion, migration, and chemotaxis utilizing transwell and Zigmond chambers. Furthermore, F-actin and Rac1 activation assays detailed cellular cytoskeletal remodeling. The potential therapeutic target of gigantol was identified and validated through protein binding analysis, competitive enzyme-linked immunosorbent assay (ELISA), cellular thermal shift assay (CETSA), and drug affinity responsive target stability (DARTS) assay. The binding sites between gigantol and its target were predicted via molecular docking. RESULTS: Gigantol ameliorated symptoms of DSS-induced colitis, rectified damage to the intestinal barrier, and suppressed the production of pro-inflammatory cytokines in colonic tissues. Intriguingly, gigantol significantly curtailed NF-κB signaling activation in the colons of DSS-induced colitis mice. Notably, gigantol impaired the ß2 integrin-dependent adhesion and migratory capacity of RAW264.7 cells. Moreover, gigantol notably influenced the cytoskeleton remodeling of RAW264.7 cells by suppressing Vav1 phosphorylation and Rac1 activation. Mechanistically, gigantol interacted with ß2 integrin, subsequently diminishing binding affinity with intercellular adhesion molecule-1 (ICAM-1). CONCLUSIONS: In conclusion, these findings elucidate that gigantol ameliorates DSS-induced colitis by antagonizing ß2 integrin-mediated macrophage adhesion, migration, and chemotaxis, thus it may impede macrophage recruitment and infiltration into colonic tissues. This study suggests that gigantol shows promise as a viable candidate for clinical colitis therapy.

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.

Decreased toll-like receptor 4 and CD11b/CD18 expression on peripheral monocytes of hypertensive patients correlates with a lesser extent of endothelial damage: a preliminary study.

BACKGROUND: Low-grade chronic inflammation is recognized to contribute to the physiopathology of arterial hypertension. Therefore, this study aimed to assess the pro-inflammatory phenotype of peripheral monocytes of hypertensive patients by analyzing Toll-like receptor 4 (TLR4) and CD11b/CD18 surface expression. In the second part, the influence of phenotypic alterations of monocytes on the endothelial status reflected by circulating endothelial cells (CECs) was evaluated. PATIENTS: The study included 60 patients with arterial hypertension, who were divided into two subgroups based on the disease severity according to the applicable criteria. The mild hypertension and resistant hypertension groups included 30 patients each. The control group consisted of 33 normotensive volunteers matched for age and sex. RESULTS: Both in the entire group of patients and individual subgroups, reduced surface expression of TLR4 and CD11b/CD18 was found compared to normotensive volunteers. A reduced percentage of monocytes with the CD14 + TLR4 + immunophenotype was correlated with a lower MFI level of CD18 and CD11b in the entire group of patients and after division only in the mild hypertension group. Reduced surface expression of TLR4 in hypertensive patients correlated with a lower number of CECs. This relationship was not observed in the resistant hypertension group; instead, an independent effect of reduced CD11b/CD18 expression on the reduction of CEC number was demonstrated. CONCLUSION: Our preliminary study showed for the first time that hypertension of varying severity is accompanied by phenotypic changes in monocytes, manifested by reduced surface expression of both TLR4 and CD11b/CD18. These phenotypic changes were associated with a reduced degree of endothelial injury. Our study opens a new, unexplored area of research on the protective features of peripheral monocytes in hypertension.

The Rac-GEF Tiam1 controls integrin-dependent neutrophil responses.

Rac GTPases are required for neutrophil adhesion and migration, and for the neutrophil effector responses that kill pathogens. These Rac-dependent functions are impaired when neutrophils lack the activators of Rac, Rac-GEFs from the Prex, Vav, and Dock families. In this study, we demonstrate that Tiam1 is also expressed in neutrophils, governing focal complexes, actin cytoskeletal dynamics, polarisation, and migration, in a manner depending on the integrin ligand to which the cells adhere. Tiam1 is dispensable for the generation of reactive oxygen species but mediates degranulation and NETs release in adherent neutrophils, as well as the killing of bacteria. In vivo, Tiam1 is required for neutrophil recruitment during aseptic peritonitis and for the clearance of Streptococcus pneumoniae during pulmonary infection. However, Tiam1 functions differently to other Rac-GEFs. Instead of promoting neutrophil adhesion to ICAM1 and stimulating ß2 integrin activity as could be expected, Tiam1 restricts these processes. In accordance with these paradoxical inhibitory roles, Tiam1 limits the fMLP-stimulated activation of Rac1 and Rac2 in adherent neutrophils, rather than activating Rac as expected. Tiam1 promotes the expression of several regulators of small GTPases and cytoskeletal dynamics, including αPix, Psd4, Rasa3, and Tiam2. It also controls the association of Rasa3, and potentially αPix, Git2, Psd4, and 14-3-3ζ/δ, with Rac. We propose these latter roles of Tiam1 underlie its effects on Rac and ß2 integrin activity and on cell responses. Hence, Tiam1 is a novel regulator of Rac-dependent neutrophil responses that functions differently to other known neutrophil Rac-GEFs.

Clinical manifestations and expression of CD18 to guide the diagnosis of leukocyte adhesion deficiency type 1: Mexico experience.

BACKGROUND: Leukocyte adhesion deficiency type 1 (LAD-1) is an inborn error of immunity characterized by a defect in leukocyte trafficking. METHODS: Patients with clinical suspicion of LAD-1 were referred to our institution. Complete blood count and flow cytometric analysis, to identify the expression of CD18, CD11b, and the lymphocyte population phenotyping, were performed, and statistical analysis was completed. RESULTS: We report clinical manifestations and immunological findings of six Mexican patients diagnosed with LAD-1. The diagnosis was based on typical clinical presentation, combined with laboratory demonstration of leukocytosis, and significant reduction or near absence of CD18 and its associated molecules CD11a, CD11b, and CD11c on leukocytes. We found atypical manifestations, not described in other countries, such as early-onset autoimmunity or infections caused by certain microorganisms. CONCLUSIONS: Patients with LAD-1 may present with atypical manifestations, making flow cytometry an indispensable tool to confirm the diagnosis. We present the first report of LAD-1 patients in a Latin American country.

Aminopeptidase N/CD13 Crosslinking Promotes the Activation and Membrane Expression of Integrin CD11b/CD18.

The ß2 integrin CD11b/CD18, also known as complement receptor 3 (CR3), and the moonlighting protein aminopeptidase N (CD13), are two myeloid immune receptors with overlapping activities: adhesion, migration, phagocytosis of opsonized particles, and respiratory burst induction. Given their common functions, shared physical location, and the fact that some receptors can activate a selection of integrins, we hypothesized that CD13 could induce CR3 activation through an inside-out signaling mechanism and possibly have an influence on its membrane expression. We revealed that crosslinking CD13 on the surface of human macrophages not only activates CR3 but also influences its membrane expression. Both phenomena are affected by inhibitors of Src, PLCγ, Syk, and actin polymerization. Additionally, after only 10 min at 37 °C, cells with crosslinked CD13 start secreting pro-inflammatory cytokines like interferons type 1 and 2, IL-12p70, and IL-17a. We integrated our data with a bioinformatic analysis to confirm the connection between these receptors and to suggest the signaling cascade linking them. Our findings expand the list of features of CD13 by adding the activation of a different receptor via inside-out signaling. This opens the possibility of studying the joint contribution of CD13 and CR3 in contexts where either receptor has a recognized role, such as the progression of some leukemias.

CD31 signaling promotes the detachment at the uropod of extravasating neutrophils allowing their migration to sites of inflammation.

Effective neutrophil migration to sites of inflammation is crucial for host immunity. A coordinated cascade of steps allows intravascular leukocytes to counteract the shear stress, transmigrate through the endothelial layer, and move toward the extravascular, static environment. Those events are tightly orchestrated by integrins, but, while the molecular mechanisms leading to their activation have been characterized, the regulatory pathways promoting their detachment remain elusive. In light of this, it has long been known that platelet-endothelial cell adhesion molecule (Pecam1, also known as CD31) deficiency blocks leukocyte transmigration at the level of the outer vessel wall, yet the associated cellular defects are controversial. In this study, we combined an unbiased proteomic study with in vitro and in vivo single-cell tracking in mice to study the dynamics and role of CD31 during neutrophil migration. We found that CD31 localizes to the uropod of migrating neutrophils along with closed ß2-integrin and is required for essential neutrophil actin/integrin polarization. Accordingly, the uropod of Pecam1-/- neutrophils is unable to detach from the extracellular matrix, while antagonizing integrin binding to extracellular matrix components rescues this in vivo migratory defect. Conversely, we showed that sustaining CD31 co-signaling actively favors uropod detachment and effective migration of extravasated neutrophils to sites of inflammation in vivo. Altogether, our results suggest that CD31 acts as a molecular rheostat controlling integrin-mediated adhesion at the uropod of egressed neutrophils, thereby triggering their detachment from the outer vessel wall to reach the inflammatory sites.

A myristoylated alanine-rich C-kinase substrate (MARCKS) inhibitor peptide attenuates neutrophil outside-in ß2-integrin activation and signaling.

MARCKS is an actin and PIP2-binding protein that plays an essential role in neutrophil migration and adhesion; however, the molecular details regarding MARCKS function in these processes remains unclear. Neutrophil adhesion and migration also require the cell surface receptors ß2-integrins. We hypothesized that MARCKS inhibition would alter neutrophil ß2-integrin activation and signaling. We utilized a MARCKS-targeting peptide to inhibit MARCKS in inside-out and outside-in ß2-integrin activation in neutrophils. MANS-mediated MARCKS inhibition had no significant effect on inside-out ß2-integrin activation. MANS treatment significantly attenuated ICAM-1/Mn2+-stimulated static adhesion, cell spreading and ß2-integrin clustering, suggesting a role for MARCKS function in outside-in ß2-integrin activation. Additional work is needed to better understand the molecular mechanisms of MARCKS role in outside-in ß2-integrin activation and signaling.

Endothelial CCRL2 induced by disturbed flow promotes atherosclerosis via chemerin-dependent ß2 integrin activation in monocytes.

AIMS: Chemoattractants and their cognate receptors are essential for leucocyte recruitment during atherogenesis, and atherosclerotic plaques preferentially occur at predilection sites of the arterial wall with disturbed flow (d-flow). In profiling the endothelial expression of atypical chemoattractant receptors (ACKRs), we found that Ackr5 (CCRL2) was up-regulated in an endothelial subpopulation by atherosclerotic stimulation. We therefore investigated the role of CCRL2 and its ligand chemerin in atherosclerosis and the underlying mechanism. METHODS AND RESULTS: By analysing scRNA-seq data of the left carotid artery under d-flow and scRNA-seq datasets GSE131776 of ApoE-/- mice from the Gene Expression Omnibus database, we found that CCRL2 was up-regulated in one subpopulation of endothelial cells in response to d-flow stimulation and atherosclerosis. Using CCRL2-/-ApoE-/- mice, we showed that CCRL2 deficiency protected against plaque formation primarily in the d-flow areas of the aortic arch in ApoE-/- mice fed high-fat diet. Disturbed flow induced the expression of vascular endothelial CCRL2, recruiting chemerin, which caused leucocyte adhesion to the endothelium. Surprisingly, instead of binding to monocytic CMKLR1, chemerin was found to activate ß2 integrin, enhancing ERK1/2 phosphorylation and monocyte adhesion. Moreover, chemerin was found to have protein disulfide isomerase-like enzymatic activity, which was responsible for the interaction of chemerin with ß2 integrin, as identified by a Di-E-GSSG assay and a proximity ligation assay. For clinical relevance, relatively high serum levels of chemerin were found in patients with acute atherothrombotic stroke compared to healthy individuals. CONCLUSIONS: Our findings indicate that d-flow-induced CCRL2 promotes atherosclerotic plaque formation via a novel CCRL2-chemerin-ß2 integrin axis, providing potential targets for the prevention or therapeutic intervention of atherosclerosis.

A conserved tryptophan in the acylated segment of RTX toxins controls their ß2 integrin-independent cell penetration.

The acylated Repeats in ToXins (RTX) leukotoxins, the adenylate cyclase toxin (CyaA) or α-hemolysin (HlyA), bind ß2 integrins of leukocytes but also penetrate cells lacking these receptors. We show that the indoles of conserved tryptophans in the acylated segments, W876 of CyaA and W579 of HlyA, are crucial for ß2 integrin-independent membrane penetration. Substitutions of W876 by aliphatic or aromatic residues did not affect acylation, folding, or the activities of CyaA W876L/F/Y variants on cells expressing high amounts of the ß2 integrin CR3. However, toxin activity of CyaA W876L/F/Y on cells lacking CR3 was strongly impaired. Similarly, a W579L substitution selectively reduced HlyA W579L cytotoxicity towards cells lacking ß2 integrins. Intriguingly, the W876L/F/Y substitutions increased the thermal stability (Tm) of CyaA by 4 to 8 °C but locally enhanced the accessibility to deuteration of the hydrophobic segment and of the interface of the two acylated loops. W876Q substitution (showing no increase in Tm), or combination of W876F with a cavity-filling V822M substitution (this combination decreasing the Tm closer to that of CyaA), yielded a milder defect of toxin activity on erythrocytes lacking CR3. Furthermore, the activity of CyaA on erythrocytes was also selectively impaired when the interaction of the pyrrolidine of P848 with the indole of W876 was ablated. Hence, the bulky indoles of residues W876 of CyaA, or W579 of HlyA, rule the local positioning of the acylated loops and enable a membrane-penetrating conformation in the absence of RTX toxin docking onto the cell membrane by ß2 integrins.

Targeting Neutrophil ß2-Integrins: A Review of Relevant Resources, Tools, and Methods.

Neutrophils are important innate immune cells that respond during inflammation and infection. These migratory cells utilize ß2-integrin cell surface receptors to move out of the vasculature into inflamed tissues and to perform various anti-inflammatory responses. Although critical for fighting off infection, neutrophil responses can also become dysregulated and contribute to disease pathophysiology. In order to limit neutrophil-mediated damage, investigators have focused on ß2-integrins as potential therapeutic targets, but so far these strategies have failed in clinical trials. As the field continues to move forward, a better understanding of ß2-integrin function and signaling will aid the design of future therapeutics. Here, we provide a detailed review of resources, tools, experimental methods, and in vivo models that have been and will continue to be utilized to investigate the vitally important cell surface receptors, neutrophil ß2-integrins.

Decrypting Integrins by Mixed-Solvent Molecular Dynamics Simulations.

Integrins are a family of α/ß heterodimeric cell surface adhesion receptors which are capable of transmitting signals bidirectionally across membranes. They are known for their therapeutic potential in a wide range of diseases. However, the development of integrin-targeting medications has been impacted by unexpected downstream effects including unwanted agonist-like effects. Allosteric modulation of integrins is a promising approach to potentially overcome these limitations. Applying mixed-solvent molecular dynamics (MD) simulations to integrins, the current study uncovers hitherto unknown allosteric sites within the integrin α I domains of LFA-1 (αLß2; CD11a/CD18), VLA-1 (α1ß1; CD49a/CD29), and Mac-1 (αMß2, CD11b/CD18). We show that these pockets are putatively accessible to small-molecule modulators. The findings reported here may provide opportunities for the design of novel allosteric integrin inhibitors lacking the unwanted agonism observed with earlier as well as current integrin-targeting drugs.

The Role of LFA-1 for the Differentiation and Function of Regulatory T Cells-Lessons Learned from Different Transgenic Mouse Models.

Regulatory T cells (Treg) are essential for the maintenance of peripheral tolerance. Treg dysfunction results in diverse inflammatory and autoimmune diseases with life-threatening consequences. ß2-integrins (CD11a-d/CD18) play important roles in the migration of leukocytes into inflamed tissues and cell signaling. Of all ß2-integrins, T cells, including Treg, only express CD11a/CD18, termed lymphocyte function-associated antigen 1 (LFA-1), on their surface. In humans, loss-of-function mutations in the common subunit CD18 result in leukocyte adhesion deficiency type-1 (LAD-1). Clinical symptoms vary depending on the extent of residual ß2-integrin function, and patients may experience leukocytosis and recurrent infections. Some patients can develop autoimmune diseases, but the immune processes underlying the paradoxical situation of immune deficiency and autoimmunity have been scarcely investigated. To understand this complex phenotype, different transgenic mouse strains with a constitutive knockout of ß2-integrins have been established. However, since a constitutive knockout affects all leukocytes and may limit the validity of studies focusing on their cell type-specific role, we established a Treg-specific CD18-floxed mouse strain. This mini-review aims to delineate the role of LFA-1 for the induction, maintenance, and regulatory function of Treg in vitro and in vivo as deduced from observations using the various ß2-integrin-deficient mouse models.

Trail Formation Alleviates Excessive Adhesion and Maintains Efficient Neutrophil Migration.

Migrating neutrophils are found to leave behind subcellular trails in vivo, but the underlying mechanisms remain unclear. Here, an in vitro cell migration test plus an in vivo observation was applied to monitor neutrophil migration on intercellular cell adhesion molecule-1 (ICAM-1) presenting surfaces. Results indicated that migrating neutrophils left behind long-lasting, chemokine-containing trails. Trail formation tended to alleviate excessive cell adhesion enhanced by the trans-binding antibody and maintain efficient cell migration, which was associated with differential instantaneous edge velocity between the cell front and rear. CD11a and CD11b worked differently in inducing trail formation with polarized distributions on the cell body and uropod. Trail release at the cell rear was attributed to membrane ripping, in which ß2-integrin was disrupted from the cell membrane through myosin-mediated rear contraction and integrin-cytoskeleton dissociation, potentiating a specialized strategy of integrin loss and cell deadhesion to maintain efficient migration. Moreover, neutrophil trails left on the substrate served as immune forerunners to recruit dendritic cells. These results provided an insight in elucidating the mechanisms of neutrophil trail formation and deciphering the roles of trail formation in efficient neutrophil migration.

The CIS association of CD47 with integrin Mac-1 regulates macrophage responses by stabilizing the extended integrin conformation.

CD47 is a ubiquitously expressed cell surface integrin-associated protein. Recently, we have demonstrated that integrin Mac-1 (αMß2, CD11b/CD18, CR3), the major adhesion receptor on the surface of myeloid cells, can be coprecipitated with CD47. However, the molecular basis for the CD47-Mac-1 interaction and its functional consequences remain unclear. Here, we demonstrated that CD47 regulates macrophage functions directly interacting with Mac-1. In particular, adhesion, spreading, migration, phagocytosis, and fusion of CD47-deficient macrophages were significantly decreased. We validated the functional link between CD47 and Mac-1 by coimmunoprecipitation analysis using various Mac-1-expressing cells. In HEK293 cells expressing individual αM and ß2 integrin subunits, CD47 was found to bind both subunits. Interestingly, a higher amount of CD47 was recovered with the free ß2 subunit than in the complex with the whole integrin. Furthermore, activating Mac-1-expressing HEK293 cells with phorbol 12-myristate 13-acetate (PMA), Mn2+, and activating antibody MEM48 increased the amount of CD47 in complex with Mac-1, suggesting CD47 has a greater affinity for the extended integrin conformation. Notably, on the surface of cells lacking CD47, fewer Mac-1 molecules could convert into an extended conformation in response to activation. Additionally, we identified the binding site in CD47 for Mac-1 in its constituent IgV domain. The complementary binding sites for CD47 in Mac-1 were localized in integrin epidermal growth factor-like domains 3 and 4 of the ß2 and calf-1 and calf-2 domains of the αM subunits. These results indicate that Mac-1 forms a lateral complex with CD47, which regulates essential macrophage functions by stabilizing the extended integrin conformation.

Hematologically important mutations: Leukocyte adhesion deficiency (second update).

Leukocyte adhesion deficiency (LAD) is an immunodeficiency caused by defects in the adhesion of leukocytes (especially neutrophils) to the blood vessel wall. As a result, patients with LAD suffer from severe bacterial infections and impaired wound healing, accompanied by neutrophilia. In LAD-I, characterized directly after birth by delayed separation of the umbilical cord, mutations are found in ITGB2, the gene that encodes the ß subunit (CD18) of the ß2 integrins. In the rare LAD-II disease, the fucosylation of selectin ligands is disturbed, caused by mutations in SLC35C1, the gene that encodes a GDP-fucose transporter of the Golgi system. LAD-II patients lack the H and Lewis Lea and Leb blood group antigens. Finally, in LAD-III, the conformational activation of the hematopoietically expressed ß integrins is disturbed, leading to leukocyte and platelet dysfunction. This last syndrome is caused by mutations in FERMT3, encoding the kindlin-3 protein in all blood cells, involved in the regulation of ß integrin conformation. This article contains an update of the mutations that we consider to be relevant for the various forms of LAD.

A Novel Deletion in FERMT3 Causes LAD-III in a Turkish Family.

Leukocyte adhesion deficiency-III (LAD-III) is an extremely rare autosomal recessive syndrome caused by mutations in FERMT3, the gene encoding kindlin-3. The genetic alterations in this gene lead to abnormal expression or activity of kindlin-3 in leukocytes and platelets. Kindlin-3 acts as an important regulator of integrin activation. LAD-III has features of the bleeding syndrome of Glanzmann and also of leukocyte adhesion deficiency. In this study, we report on two families, one of Turkish and one of Syrian origin, with clinical features of LAD-III, loss of kindlin-3 protein expression, and a functional leukocyte defect. A novel, homozygous deletion in FERMT3 (c.921delC, p.Ser307Argfs*21) was found in the Turkish patient. The parents were carriers of the mutation, consistent with an autosomal recessive inheritance. A common c.1525C > T (p.Arg509*) mutation was found in the Syrian patient. In conclusion, beside the variant c.1525C > T in the FERMT3 gene, which was previously found in more than 15 patients in Anatolia, our study is the first to identify the novel homozygous variant c.921delC in the FERMT3 gene.

Neutrophil heterogeneity and emergence of a distinct population of CD11b/CD18-activated low-density neutrophils after trauma.

INTRODUCTION: Multiple large clinical trauma trials have documented an increased susceptibility to infection after injury. Although neutrophils (polymorphonuclear leukocytes [PMNs]) were historically considered a homogeneous cell type, we hypothesized that injury could alter neutrophil heterogeneity and predispose to dysfunction. To explore whether trauma modifies PMN heterogeneity, we performed an observational mass-spectrometry-based cytometry study on total leukocytes and low-density PMNs found in the peripheral blood mononuclear cell fraction of leukocytes from healthy controls and trauma patients. METHODS: A total of 74 samples from 12 trauma patients, each sampled at 1 or more time points, and matched controls were fractionated and profiled by mass-spectrometry-based cytometry using a panel of 44 distinct markers. After deconvolution and conservative gating on neutrophils, data were analyzed using Seurat, followed by clustering of principal components. RESULTS: Eleven distinct neutrophil populations were resolved in control and trauma neutrophils based on differential protein surface marker expression. Trauma markedly altered the basal heterogeneity of neutrophil subgroups seen in the control samples, with loss of a dominant population of resting neutrophils marked by high expression of C3AR and low levels of CD63, CD64, and CD177 (cluster 1), and expansion of two alternative neutrophil populations, one of which is marked by high expression of CD177 with suppression of CD10, CD16, C3AR, CD63, and CD64 (cluster 6). Remarkably, following trauma, a substantially larger percentage of neutrophils sediment in the monocyte fraction. These low-density neutrophils bear markers of functional exhaustion and form a unique trauma-induced population (cluster 9) with markedly upregulated expression of active surface adhesion molecules (activated CD11b/CD18), with suppression of nearly all other surface markers, including receptors for formyl peptides, leukotrienes, chemokines, and complement. CONCLUSION: Circulating neutrophils demonstrate considerable evidence of functional heterogeneity that is markedly altered by trauma. Trauma induces evolution of a novel, exhausted, low-density neutrophil population with immunosuppressive features.