Leukocyte Integrin Research Articles

Phosphorylation of the α-chain in the integrin LFA-1 enables β2-chain phosphorylation and α-actinin binding required for cell adhesion

The integrin leukocyte function-associated antigen-1 (LFA-1) plays a pivotal role in leukocyte adhesion and migration, but the mechanism(s) by which this integrin is regulated has remained incompletely understood. LFA-1 integrin activity requires phosphorylation of its β2-chain and interactions of its cytoplasmic tail with various cellular proteins. The α-chain is constitutively phosphorylated and necessary for cellular adhesion, but how the α-chain regulates adhesion has remained enigmatic. We now show that substitution of the α-chain phosphorylation site (S1140A) in T cells inhibits the phosphorylation of the functionally important Thr-758 in the β2-chain, binding of α-actinin and 14-3-3 protein, and expression of an integrin-activating epitope after treatment with the stromal cell-derived factor-1α. The presence of this substitution resulted in a loss of cell adhesion and directional cell migration. Moreover, LFA-1 activation through the T-cell receptor in cells expressing the S1140A LFA-1 variant resulted in less Thr-758 phosphorylation, α-actinin and talin binding, and cell adhesion. The finding that the LFA-1 α-chain regulates adhesion through the β-chain via specific phosphorylation at Ser-1140 in the α-chain has not been previously reported and emphasizes that both chains are involved in the regulation of LFA-1 integrin activity.

JAK2-V617F promotes venous thrombosis through β1/β2 integrin activation.

JAK2-V617F-positive chronic myeloproliferative neoplasia (CMN) commonly displays dysfunction of integrins and adhesion molecules expressed on platelets, erythrocytes, and leukocytes. However, the mechanism by which the 2 major leukocyte integrin chains, β1 and β2, may contribute to CMN pathophysiology remained unclear. β1 (α4β1; VLA-4) and β2 (αLβ2; LFA-1) integrins are essential regulators for attachment of leukocytes to endothelial cells. We here showed enhanced adhesion of granulocytes from mice with JAK2-V617F knockin (JAK2+/VF mice) to vascular cell adhesion molecule 1- (VCAM1-) and intercellular adhesion molecule 1-coated (ICAM1-coated) surfaces. Soluble VCAM1 and ICAM1 ligand binding assays revealed increased affinity of β1 and β2 integrins for their respective ligands. For β1 integrins, this correlated with a structural change from the low- to the high-affinity conformation induced by JAK2-V617F. JAK2-V617F triggered constitutive activation of the integrin inside-out signaling molecule Rap1, resulting in translocation toward the cell membrane. Employing a venous thrombosis model, we demonstrated that neutralizing anti-VLA-4 and anti-β2 integrin antibodies suppress pathologic thrombosis as observed in JAK2+/VF mice. In addition, aberrant homing of JAK2+/VF leukocytes to the spleen was inhibited by neutralizing anti-β2 antibodies and by pharmacologic inhibition of Rap1. Thus, our findings identified cross-talk between JAK2-V617F and integrin activation promoting pathologic thrombosis and abnormal trafficking of leukocytes to the spleen.

Integrin αDβ2 (CD11d/CD18) Modulates Leukocyte Accumulation, Pathogen Clearance, and Pyroptosis in Experimental Salmonella Typhimurium Infection.

β2 integrins are critical in host defense responses to invading pathogens and inflammation. Previously, we reported that genetic deficiency of integrin αDβ2 in mice altered outcomes in experimental systemic infections including accelerated mortality in animals infected with Salmonella enterica serovar Typhimurium. Here, we show that deficiency of αDβ2 results in impaired accumulation of leukocytes in response to peritoneal infection by S. Typhimurium, impaired pathogen clearance in vivo, defective bacterial elimination by cultured peritoneal macrophages, and enhanced pyroptosis, a cell death process triggered by Salmonella. Salmonella-infected animals deficient in αDβ2 had increased levels of peritoneal cytokines in addition to other markers of pyroptosis, which may contribute to inflammatory injury and increased mortality in the context of impaired bacterial killing. These observations indicate important contributions of leukocyte integrins to the host response in experimental Salmonella infection and reveal previous activities of αDβ2 in bacterial infection.

Abstract 031: A Ligand-specific Blockade of the Integrin Mac-1 Selectively Targets Pathologic Vascular Inflammation While Maintaining Protective Host-defense

Background: Integrins, such as Mac-1 (alphaMbeta2, CD11b/CD18), drive myeloid cell recruitment and inflammation in cardiovascular disease. Although integrins have generated interest as therapeutic targets in acute and chronic inflammation, they also contribute to host defense, regeneration, and haemostasis. To overcome these limitations, we have designed a novel antibody that targets a distinct region within the Mac-1 major ligand-binding (I-) domain required for binding of its ligand CD40L. Here, we describe the consequences of a ligand-specific inhibition and its efficacy in mice. Methods and Results: To generate an antibody specifically targeting the interaction of Mac-1 with CD40L, mice were immunized with a peptide containing the CD40L-binding motif EQLKKSKTL. Antibody clones were screened and tested for specificity. One clone, termed anti-M7, showed specific peptide binding and blocked the binding of the activated, open conformation of Mac-1 to CD40L, but not to alternative Mac-1 ligands suggesting ligand specificity. Anti-M7 highly effectively limited leukocyte recruitment in vitro and to the mesentery and the omentum in intravital confocal microscopy. While conventional anti-Mac-1 antibodies induced outside-in activation of the integrin, anti-M7 did not provoke MAP-kinase dependent activation and expression of inflammatory cytokines in macrophages. Following cecal-ligation and puncture (CLP) sepsis in mice, a conventional anti-Mac-1 antibody inhibited leukocyte accumulation, but potentiated bacteremia and limited overall survival. In contrast, anti-M7 limited peritoneal leukocyte accumulation, reduced bacterial titers, and improved survival during CLP. Conclusion: We present proof-of-concept demonstrating selective inhibition of the interaction between the leukocyte integrin Mac-1 and CD40L by a novel monoclonal antibody. This antibody shows several advantages over conventional anti-integrin therapy, and prevented inflammation and sepsis-related mortality in mice. This novel approach gives new insight into ligand-specific integrin pathways and merits further evaluation as a selective anti-inflammatory therapy in cardiovascular disease.

Leukocyte integrin Mac-1 (CD11b/CD18, αMβ2, CR3) acts as a functional receptor for platelet factor 4

Platelet factor 4 (PF4) is one of the most abundant cationic proteins secreted from α-granules of activated platelets. Based on its structure, PF4 was assigned to the CXC family of chemokines and has been shown to have numerous effects on myeloid leukocytes. However, the receptor for PF4 remains unknown. Here, we demonstrate that PF4 induces leukocyte responses through the integrin Mac-1 (αMβ2, CD11b/CD18). Human neutrophils, monocytes, U937 monocytic and HEK293 cells expressing Mac-1 strongly adhered to immobilized PF4 in a concentration-dependent manner. The cell adhesion was partially blocked by anti-Mac-1 mAb and inhibition was enhanced when anti-Mac-1 antibodies were combined with glycosaminoglycans, suggesting that cell-surface proteoglycans act cooperatively with Mac-1. PF4 also induced Mac-1-dependent migration of human neutrophils and murine WT, but not Mac-1-deficient macrophages. Coating of Escherichia coli bacteria or latex beads with PF4 enhanced their phagocytosis by macrophages by ∼4-fold, and this process was blocked by different Mac-1 antagonists. Furthermore, PF4 potentiated phagocytosis by WT, but not Mac-1-deficient macrophages. As determined by biolayer interferometry, PF4 directly bound the αMI-domain, the major ligand-binding region of Mac-1, and this interaction was governed by a Kd of 1.3 ± 0.2 μm Using the PF4-derived peptide library, synthetic peptides duplicating the αMI-domain recognition sequences and recombinant mutant PF4 fragments, the binding sites for αMI-domain were identified in the PF4 segments Cys12-Ser26 and Ala57-Ser70 These results identify PF4 as a ligand for the integrin Mac-1 and suggest that many immune-modulating effects previously ascribed to PF4 are mediated through its interaction with Mac-1.

Calcineurin positively regulates TCR-induced LFA-1-dependent cell adhesion via dephosphorylation of Lck Ser-59.

Abstract T cell adhesion via the interaction of the integrin leukocyte function-associated antigen-1 (LFA-1) with its ligand ICAM-1 is essential for the initiation of immune response. Upon ligation of T cell receptor (TCR), activation-induced LFA-1 conformational change occurs via phosphorylation of Thr-758 resulting in the enhancement of binding to ICAM-1. Recently, we reported that dephosphorylation of Lck Ser-59 (LckS59) by calcineurin results in enhanced TCR-proximal signaling, indicating that phosphorylated LckS59 negatively regulates T cell activation. Here we find that inhibition of calcineurin activity by immunosuppressive drugs such as cyclosporin A (CsA) rapidly reverses existing TCR-induced LFA-1-dependent cell adhesion as well as the phosphorylation of LFA-1 Thr-758 in Jurkat and primary human T cells. In addition, TCR-induced cell adhesion of T cells from LckS59A knock-in mice was stronger than T cells from LckWT mice. Consistent with enhanced cell adhesion, some of the signaling events downstream of TCR proximal signaling were also enhanced in LckS59A mouse T cells. Notably, LckS59A T cell adhesion was resistant to CsA inhibition. These observations provide new insights into a previously unrecognized role for calcineurin inhibitors in reversing ligand-dependent adhesion independently of its effects on NFAT activation.

Ligand- and cation-induced structural alterations of the leukocyte integrin LFA-1

In αI integrins, including leukocyte function-associated antigen 1 (LFA-1), ligand-binding function is delegated to the αI domain, requiring extra steps in the relay of signals that activate ligand binding and coordinate it with cytoplasmic signals. Crystal structures reveal great variation in orientation between the αI domain and the remainder of the integrin head. Here, we investigated the mechanisms involved in signal relay to the αI domain, including whether binding of the ligand intercellular adhesion molecule-1 (ICAM-1) to the αI domain is linked to headpiece opening and engenders a preferred αI domain orientation. Using small-angle X-ray scattering and negative-stain EM, we define structures of ICAM-1, LFA-1, and their complex, and the effect of activation by Mn2+ Headpiece opening was substantially stabilized by substitution of Mg2+ with Mn2+ and became complete upon ICAM-1 addition. These agents stabilized αI-headpiece orientation, resulting in a well-defined orientation of ICAM-1 such that its tandem Ig-like domains pointed in the opposite direction from the β-subunit leg of LFA-1. Mutations in the integrin βI domain α1/α1' helix stabilizing either the open or the closed βI-domain conformation indicated that α1/α1' helix movements are linked to ICAM-1 binding by the αI domain and to the extended-open conformation of the ectodomain. The LFA-1-ICAM-1 orientation described here with ICAM-1 pointing anti-parallel to the LFA-1 β-subunit leg is the same orientation that would be stabilized by tensile force transmitted between the ligand and the actin cytoskeleton and is consistent with the cytoskeletal force model of integrin activation.

Transmission of integrin β7 transmembrane domain topology enables gut lymphoid tissue development.

Integrin activation regulates adhesion, extracellular matrix assembly, and cell migration, thereby playing an indispensable role in development and in many pathological processes. A proline mutation in the central integrin β3 transmembrane domain (TMD) creates a flexible kink that uncouples the topology of the inner half of the TMD from the outer half. In this study, using leukocyte integrin α4β7, which enables development of gut-associated lymphoid tissue (GALT), we examined the biological effect of such a proline mutation and report that it impairs agonist-induced talin-mediated activation of integrin α4β7, thereby inhibiting rolling lymphocyte arrest, a key step in transmigration. Furthermore, the α4β7(L721P) mutation blocks lymphocyte homing to and development of the GALT. These studies show that impairing the ability of an integrin β TMD to transmit talin-induced TMD topology inhibits agonist-induced physiological integrin activation and biological function in development.

Anti-inflammatory therapies in myocardial infarction: failures, hopes and challenges.

In the infarcted heart, the damage-associated molecular pattern proteins released by necrotic cells trigger both myocardial and systemic inflammatory responses. Induction of chemokines and cytokines and up-regulation of endothelial adhesion molecules mediate leukocyte recruitment in the infarcted myocardium. Inflammatory cells clear the infarct of dead cells and matrix debris and activate repair by myofibroblasts and vascular cells, but may also contribute to adverse fibrotic remodelling of viable segments, accentuate cardiomyocyte apoptosis and exert arrhythmogenic actions. Excessive, prolonged and dysregulated inflammation has been implicated in the pathogenesis of complications and may be involved in the development of heart failure following infarction. Studies in animal models of myocardial infarction (MI) have suggested the effectiveness of pharmacological interventions targeting the inflammatory response. This article provides a brief overview of the cell biology of the post-infarction inflammatory response and discusses the use of pharmacological interventions targeting inflammation following infarction. Therapy with broad anti-inflammatory and immunomodulatory agents may also inhibit important repair pathways, thus exerting detrimental actions in patients with MI. Extensive experimental evidence suggests that targeting specific inflammatory signals, such as the complement cascade, chemokines, cytokines, proteases, selectins and leukocyte integrins, may hold promise. However, clinical translation has proved challenging. Targeting IL-1 may benefit patients with exaggerated post-MI inflammatory responses following infarction, not only by attenuating adverse remodelling but also by stabilizing the atherosclerotic plaque and by inhibiting arrhythmia generation. Identification of the therapeutic window for specific interventions and pathophysiological stratification of MI patients using inflammatory biomarkers and imaging strategies are critical for optimal therapeutic design.

P017 CD47 REGULATES CD11B-DEPENDENT NEUTROPHIL TRANSEPITHELIAL MIGRATION DURING INTESTINAL INFLAMMATION

Neutrophil (PMN) migration across the intestinal mucosa correlates with disease flares in individuals with IBD. Despite this correlation, molecular events regulating PMN transepithelial migration (TEpM) are still elusive. However, it is clear that the leukocyte integrin CD11b/CD18 and the ubiquitously expressed membrane glycoprotein CD47 play crucial roles in this process. We hypothesized that CD47 interacts with CD11b/CD18 to regulate PMN trafficking in the gut. In vivo ileal loop assays showed reduced PMN migration into the lumen of CD47-/- mice in response to LTB4 (>60% reduction vs WT; p<0.05). This reduction is not due to the loss of CD47 on intestinal epithelial cells, as there were no differences in PMN migration between VillinCreCD47fl/fl mice and control CD47fl/fl mice. In vitro assays demonstrated a 25% reduction in LTB4 driven TEpM with CD47-/- PMN. In addition, blocking CD11b/CD18 antibody (mAb) reduced WT PMN migration to levels similar to those observed with CD47-/- PMN. However, CD11b/CD18 mAb did not reduce migration of CD47-/- PMN further, suggesting that CD47 plays a role in regulating integrin-dependent chemotaxis. In co-immunoprecipitation assays, CD47 was observed to associate with CD11b in PMN. Furthermore, we observed that upregulation of CD11b upon stimulation was reduced in CD47-/- PMN compared to WT, despite expression of similar levels at baseline. Consistent with these results, we observed reduced upregulation of CD11b upon chemoattractant stimulation in CD47 deficient human neutrophil like HL60 cells. Lastly, using antibodies specific for high affinity conformation of CD11b/CD18, we observed reduced CD11b/CD18 activation in CD47 deficient HL60 cells. These data support a role for CD47 in regulating PMN TEpM through effects on CD11b/CD18 functions. Targeting CD47 may provide a new therapeutic approach aimed at reducing pathologic intestinal inflammation in IBD.

A Functional Analysis on the Interspecies Interaction between Mouse LFA-1 and Human Intercellular Adhesion Molecule-1 at the Cell Level.

The interaction between intercellular adhesion molecules (ICAM) and the integrin leukocyte function-associated antigen-1 (LFA-1) is crucial for the regulation of several physiological and pathophysiological processes like cell-mediated elimination of tumor or virus infected cells, cancer metastasis, or inflammatory and autoimmune processes. Using purified proteins it was reported a species restriction for the interaction of ICAM-1 and LFA-1, being mouse ICAM-1 able to interact with human LFA-1 but not human ICAM-1 with mouse LFA-1. However, in vivo results employing tumor cells transfected with human ICAM-1 suggest that functionally mouse LFA-1 can recognize human ICAM-1. In order to clarify the interspecies cross-reactivity of the ICAM-1/LFA-1 interaction, we have performed functional studies analyzing the ability of human soluble ICAM-1 and human/mouse LFA-1 derived peptides to inhibit cell aggregation and adhesion as well as cell-mediated cytotoxicity in both mouse and human systems. In parallel, the affinity of the interaction between mouse LFA-1-derived peptides and human ICAM-1 was determined by calorimetry assays. According to the results obtained, it seems that human ICAM-1 is able to interact with mouse LFA-1 on intact cells, which should be taking into account when using humanized mice and xenograft models for the study of immune-related processes.

Role of Structural Dynamics in Leukocyte Integrins Function: Interplay between the Shape-Shifting Mechanism and Allostery

Role of Structural Dynamics in Leukocyte Integrins Function: Interplay between the Shape-Shifting Mechanism and Allostery

Leukocyte integrin Mac-1 regulates thrombosis via interaction with platelet GPIb\u03b1

Inflammation and thrombosis occur together in many diseases. The leukocyte integrin Mac-1 (also known as integrin αMβ2, or CD11b/CD18) is crucial for leukocyte recruitment to the endothelium, and Mac-1 engagement of platelet GPIbα is required for injury responses in diverse disease models. However, the role of Mac-1 in thrombosis is undefined. Here we report that mice with Mac-1 deficiency (Mac-1−/−) or mutation of the Mac-1-binding site for GPIbα have delayed thrombosis after carotid artery and cremaster microvascular injury without affecting parameters of haemostasis. Adoptive wild-type leukocyte transfer rescues the thrombosis defect in Mac-1−/− mice, and Mac-1-dependent regulation of the transcription factor Foxp1 contributes to thrombosis as evidenced by delayed thrombosis in mice with monocyte-/macrophage-specific overexpression of Foxp1. Antibody and small-molecule targeting of Mac-1:GPIbα inhibits thrombosis. Our data identify a new pathway of thrombosis involving leukocyte Mac-1 and platelet GPIbα, and suggest that targeting this interaction has anti-thrombotic therapeutic potential with reduced bleeding risk.

A role for leukocyte integrins and extracellular matrix remodeling of adipose tissue in the risk of weight regain after weight loss ,

Background: Weight loss (WL) is often followed by weight regain after an energy-restricted dietary intervention (DI). When people are following a diet, the volume of an adipocyte decreases by loss of triglycerides, which creates stress between the cell contents and the surrounding extracellular matrix (ECM). Previously, we observed that genetic variations in ECM genes are associated with an increased risk of weight regain.Objective: We investigated the relation between the expression of ECM genes during WL and a period of weight stabilization (WS) and the risk of weight regain.Design: In this randomized controlled trial, 61 healthy overweight or obese participants followed either a 5-wk very-low-calorie diet (VLCD; 500 kcal/d) or a 12-wk low-calorie diet (1250 kcal/d) (WL period) with a subsequent 4-wk WS period and a 9-mo follow-up. The WL and WS periods combined were considered the DI. Abdominal subcutaneous adipose tissue biopsy samples were collected for microarray analysis. Gene expression changes for a broad set of ECM-related genes were correlated with the weight-regain percentage (WR%).Results: A total of 26 of the 277 genes were significantly correlated with WR% during WL, WS, or the DI periods. Most correlations were observed in the VLCD group during the WS period. Four genes code for leukocyte-specific receptors. These and other genes belong to a group of 26 genes, among which the expression changes were highly correlated (r ≥ 0.7, P ≤ 0.001). This group could be divided into 3 subclusters linking to 2 biological processes—leukocyte integrin gene activity and ECM remodeling—and a link to insulin sensitivity was also apparent.Conclusions: Our present findings indicate the importance of adipose tissue leukocytes for the risk of weight regain. ECM modification also seems to be involved, and we observed a link to insulin sensitivity. This trial was registered at clinicaltrials.gov as NCT01559415.

From leukocyte recruitment to resolution of inflammation: the cardinal role of integrins.

Integrins constitute a large group of adhesion receptors that are formed as heterodimers of α and β subunits. Their presence and activation status on the surface of leukocytes modulate a broad spectrum of processes in inflammation and immunity. This mini review critically outlines research advances with regard to the function of leukocyte integrins in regulating and integrating the onset and resolution of acute inflammation. Specifically, we summarize and discuss relevant, current literature that supports the multifunctional role of integrins and their partners. The latter include molecules that physically associate with integrins or regulate their activity in the context of the following: 1) leukocyte recruitment to an inflamed tissue, 2) recognition and phagocytosis of apoptotic neutrophils (efferocytosis), and 3) egress of efferocytic macrophages from the inflamed site to lymphoid tissues. The understanding of the fine-tuning mechanisms of the aforementioned processes by integrins and their functional partners may enable the design of therapeutic tools to counteract destructive inflammation and promote more efficient resolution of inflammation.

Distinct recognition of complement iC3b by integrins αXβ2 and αMβ2

Recognition by the leukocyte integrins αXβ2 and αMβ2 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 αXβ2 and αMβ2 in complex with iC3b. Despite high homology, the two integrins bind iC3b at multiple distinct sites. αXβ2 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, αMβ2 uses its αI domain to bind iC3b at the thioester domain and simultaneously interacts through a region near the αM β-propeller and β2 βI domain with a region of the C3c moiety near the C345C domain. Remarkably, there is no overlap between the primary binding site of αXβ2 and the binding site of αMβ2 on iC3b. Distinctive binding sites on iC3b by integrins αXβ2 and αMβ2 may be biologically beneficial for leukocytes to more efficiently capture opsonized pathogens and to avoid subversion by pathogen factors.

ICAM-1-Targeted Nanocarriers Attenuate Endothelial Release of Soluble ICAM-1, an Inflammatory Regulator.

Targeting of drug nanocarriers (NCs) to intercellular adhesion molecule‐1 (ICAM‐1), an endothelial‐surface protein overexpressed in many pathologies, has shown promise for therapeutic delivery into and across this lining. However, due to the role of ICAM‐1 in inflammation, the effects of targeting this receptor need investigation. Since ICAM‐1 binding by natural ligands (leukocyte integrins) results in release of the “soluble ICAM‐1” ectodomain (sICAM‐1), an inflammatory regulator, we investigated the influence of targeting ICAM‐1 with NCs on this process. For this, sICAM‐1 was measured by ELISA from cell‐medium supernatants, after incubation of endothelial cell (EC) monolayers in the absence versus presence of anti‐ICAM NCs. In the absence of NCs, ECs released sICAM‐1 when treated with a pro‐inflammatory cytokine. This was reduced by inhibiting matrix metalloproteinases MMP‐9 or MMP‐2, yet inhibiting both did not render additive effects. Release of sICAM‐1 mainly occurred at the basolateral versus apical side, and both MMP‐9 and MMP‐2 influenced apical release, while basolateral release depended on MMP‐9. Interestingly, anti‐ICAM NCs reduced sICAM‐1 to a greater extent than MMP inhibition, both at the apical and basolateral sides. This effect was enhanced with time, although NCs had been removed after binding to cells, ruling out a “trapping” effect of NCs. Instead, inhibiting anti‐ICAM NC endocytosis counteracted their inhibition on sICAM‐1 release. Hence, anti‐ICAM NCs inhibited sICAM‐1 release by mobilizing ICAM‐1 from the cell‐surface into intracellular vesicles. Since elevated levels of sICAM‐1 associate with numerous diseases, this effect represents a secondary benefit of using ICAM‐1‐targeted NCs for drug delivery.

The Systemic Lupus Erythematosus-Associated Single Nucleotide Polymorphism rs1143678 in Integrin αM Cytoplasmic Tail Generates a 14-3-3ζ Binding Site That Is Proinflammatory.

The leukocyte integrin αMβ2 (CR3 or Mac-1) has both proinflammatory and immune regulatory functions. Genome-wide association studies have identified several ITGAM (αM subunit) single nucleotide polymorphisms that are associated with systemic lupus erythematosus. The single nucleotide polymorphism rs1143678 substitutes Pro1146 for Ser in the integrin αM cytoplasmic tail. A detailed functional characterization of this substitution is lacking. Using transfected human cell lines, reconstituted mouse bone marrow neutrophils, and bone marrow-derived macrophages (BMDMs), we showed that P1146S (PS) substitution promoted integrin αMβ2-mediated adhesion, spreading, and migration of cells on iC3b and fibrinogen. In the presence of LPS together with iC3b or fibrinogen, the expression levels of IL-6 and TNF-α in integrin αM(PS)β2 BMDMs were significantly higher than those of integrin αM(wild-type)β2 BMDMs, and they showed faster kinetics of Erk1/2 activation through the src family kinase(s)-Syk signaling pathway. Integrin αM(PS)β2 BMDMs also exhibited higher levels of active RhoA and phagocytic activity. Mechanistically, P1146S substitution in the αM cytoplasmic tail generates a noncanonical 14-3-3ζ binding site that modulates integrin αM(PS)β2 outside-in signaling.

Prophylactic orthosteric inhibition of leukocyte integrin CD11b/CD18 prevents long-term fibrotic kidney failure in cynomolgus monkeys

Ischaemic acute kidney injury (AKI), an inflammatory disease process, often progresses to chronic kidney disease (CKD), with no available effective prophylaxis. This is in part due to lack of clinically relevant CKD models in non-human primates. Here we demonstrate that inhibition of the archetypal innate immune receptor CD11b/CD18 prevents progression of AKI to CKD in cynomolgus monkeys. Severe ischaemia-reperfusion injury of the right kidney, with subsequent periods of the left ureter ligation, causes irreversible right kidney failure 3, 6 or 9 months after AKI. Moreover, prophylactic inactivation of CD11b/CD18, using the orthosteric CD11b/CD18 inhibitor mAb107, improves microvascular perfusion and histopathology, reduces intrarenal pro-inflammatory mediators and salvages kidney function long term. These studies reveal an important early role of CD11b+ leukocytes in post-ischaemic kidney fibrosis and failure, and suggest a potential early therapeutic intervention to mitigate progression of ischaemic AKI to CKD in humans.

Characterization of αX I-Domain Binding to Receptors for Advanced Glycation End Products (RAGE).

The β2 integrins are cell surface transmembrane proteins regulating leukocyte functions, such as adhesion and migration. Two members of β2 integrin, αMβ2 and αXβ2, share the leukocyte distribution profile and integrin αXβ2 is involved in antigen presentation in dendritic cells and transendothelial migration of monocytes and macrophages to atherosclerotic lesions. Receptor for advanced glycation end products (RAGE), a member of cell adhesion molecules, plays an important role in chronic inflammation and atherosclerosis. Although RAGE and αXβ2 play an important role in inflammatory response and the pathogenesis of atherosclerosis, the nature of their interaction and structure involved in the binding remain poorly defined. In this study, using I-domain as a ligand binding motif of αXβ2, we characterize the binding nature and the interacting moieties of αX I-domain and RAGE. Their binding requires divalent cations (Mg2+ and Mn2+) and shows an affinity on the sub-micro molar level: the dissociation constant of αX I-domains binding to RAGE being 0.49 μM. Furthermore, the αX I-domains recognize the V-domain, but not the C1 and C2-domains of RAGE. The acidic amino acid substitutions on the ligand binding site of αX I-domain significantly reduce the I-domain binding activity to soluble RAGE and the alanine substitutions of basic amino acids on the flat surface of the V-domain prevent the V-domain binding to αX I-domain. In conclusion, the main mechanism of αX I-domain binding to RAGE is a charge interaction, in which the acidic moieties of αX I-domains, including E244, and D249, recognize the basic residues on the RAGE V-domain encompassing K39, K43, K44, R104, and K107.