Leukocyte Integrin Mac-1 Research Articles

An Activation-Specific Anti-Mac-1 Designed-Ankyrin-Repeat-Protein Attenuates Colitis in Mice.

(1) Background: Inflammatory bowel diseases are complex and multifactorial disorders of unknown etiology. The extravasation of activated leukocytes is a critical step in the pathogenesis of these diseases. Leukocyte integrin Mac-1 (αMβ2; CD11b/CD18) is crucial for the extravasation of myeloid cells, and a novel activation-specific anti-Mac-1 Designed Ankyrin Repeat protein (DARPin F7) is a promising therapeutic agent for inflammatory diseases. In its activated conformation, Mac-1 expresses the high-affinity binding site I-domain, which the DARPin F7 selectively targets. In our study, we aimed to explore the therapeutic potential of anti-Mac-1 DARPin F7 in murine dextrane sodium sulfate (DSS)-induced colitis. (2) Methods: C57BL/6J mice received 3% DSS drinking water for five days, followed by normal drinking water for one week. The mice were treated with DARPin F7 or a control substance daily via intraperitoneal injections. Disease activity index (DAI), colon length, myeloperoxidase (MPO) activity measurements, H&E staining, and qRT-PCR were conducted after euthanizing the mice on day 12. (3) Results: Treatment with DARPin F7 resulted in less pronounced colon shortening and significantly lower histological scores. The DARPin F7-treated animals experienced substantially less disease and myeloperoxidase (MPO) activity. Animals that received DARPin F7 treatment suffered less weight loss and recovered from the weight loss more efficiently. Treatment with DARPin F7 also led to significantly reduced mRNA expression of inflammatory cytokines. (4) Conclusion: Anti-Mac-1 treatment markedly reduced disease activity and inflammatory reaction accompanying DSS-induced colitis in mice.

Guanxinning tablet inhibits the interaction between leukocyte integrin Mac-1 and platelet GPIbα for antithrombosis without increased bleeding risk.

Recent studies have showed that thrombosis is closely related to leucocytes involved in immunity. Interfering with the binding of leukocyte integrin Mac-1 and platelet GPIbα can inhibit thrombosis without affecting physiological coagulation. Mac-1-GPIbα is proposed as a potential safety target for antithrombotic agents. Guanxinning tablet (GXNT) is an oral Chinese patent medicine used for the treatment of angina pectoris, which contains phenolic acid active ingredients, such as salvianolic acids, ferulic acid, chlorogenic acid, caffeic acid, rosmarinic acid, tanshinol, and protocatechualdehyde. Our previous studies demonstrated that GXN exhibited significant antithrombotic effects, and clinical studies suggested that it did not increase bleeding risk. In addition, GXN exerted a significantly regulatory effect on immune inflammation. In the current study, we intended to evaluate the effects of GXN on bleeding events and explore the safety antithrombotic mechanism of GXN based on leukocyte-platelet interaction. First, we established a gastric ulcer model induced by acetic acid in rats and found that GXN not only did not increase the degree of gastrointestinal bleeding when gastric ulcer occurred, but also had a certain promoting effect on the healing of gastric ulcer. Second, in vitroexperiments showed that after pretreatment with GXN and activation by phorbol 12-myristate-13-acetate (PMA), the adhesion and aggregation of leukocytes with human platelets were reduced. It was also found that GXN reduced the expression and activation of Mac-1 in leucocytes, and inhibited platelet activation due to leukocyte engagement via Mac-1. Overall, the results suggest that GXN may be a safe antithrombotic agent, and its low bleeding risk mechanism is probably related to inhibited leukocyte-platelet aggregation and its interaction target Mac-1-GPIbα.

Monocyte Dysfunction Detected by the Designed Ankyrin Repeat Protein F7 Predicts Mortality in Patients Receiving Veno-Arterial Extracorporeal Membrane Oxygenation.

Background: Veno-arterial extracorporeal membrane oxygenation (VA-ECMO) is used for critically ill patients requiring hemodynamic support but has been shown to induce an inflammatory response syndrome potentially leading to severe complications and poor outcome. Monocytes are comprised of different subsets and play a central role in the innate immune system. The unique small binding proteins, Designed Ankyrin Repeat Protein “F7” and single chain variable fragment “MAN-1,” specifically detect the activated conformation of the leukocyte integrin Mac-1 enabling the highly sensitive detection of monocyte activation status. The aim of this study was to characterize monocyte function and heterogeneity and their association with outcome in VA-ECMO patients.Methods: VA-ECMO patients were recruited from the ICUs of the University Hospital in Freiburg, Germany. Blood was sampled on day 0 and day 3 after VA-ECMO placement, after VA-ECMO explantation and from healthy controls. Monocyte subset distribution, baseline activation and stimulability were analyzed by flow cytometry using the unique small binding proteins F7 and MAN-1 and the conventional activation markers CD163, CD86, CD69, and CX3CR1. Furthermore, expression of monocyte activation markers in survivors and non-survivors on day 0 was compared. Simple logistic regression was conducted to determine the association of monocyte activation markers with mortality.Results: Twenty two patients on VA-ECMO and 15 healthy controls were recruited. Eleven patients survived until discharge from the ICU. Compared to controls, baseline monocyte activation was significantly increased, whereas stimulability was decreased. The percentage of classical monocytes increased after explantation, while the percentage of intermediate monocytes decreased. Total, classical, and intermediate monocyte counts were significantly elevated compared to controls. On day 0, baseline binding of F7 was significantly lower in non-survivors than survivors. The area under the ROC curve associated with mortality on day 0 was 0.802 (p = 0.02).Conclusions: Distribution of monocyte subsets changes during VA-ECMO and absolute classical and intermediate monocyte counts are significantly elevated compared to controls. Monocytes from VA-ECMO patients showed signs of dysfunction. Monocyte dysfunction, as determined by the unique tool F7, could be valuable for predicting mortality in patients receiving VA-ECMO and may be used as a novel biomarker guiding early clinical decision making in the future.

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

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

SUN-380 CLINICAL IMPACTS OF URINARY CD11B AND CD163 ON PATIENT OUTCOMES IN ANCA-ASSOCIATED GLOMERULONEPHRITIS

The leukocyte-derived molecules, CD11b (α subunit of leukocyte integrin Mac-1) and scavenger receptor CD163, undergo ectodomain cleavage upon inflammation-related cell activation; therefore, the presence of these molecules in urine may reflect the renal inflammation with glomerular crescents containing robust extra-capillary leukocytes in anti-neutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis (ANCA-GN). This study was undertaken to evaluate the clinical significance of urinary CD11b (U-CD11b) and CD163 (U-CD163) in ANCA-GN. We examined ANCA-GN urine samples by enzyme-linked immunosorbent assay in our institutional cohort (88 patients at diagnosis) and in a nation-wide ANCA vasculitis (AAV) cohort (138 patients at diagnosis; 56 patients after 6 months of treatment) and analyzed the associations of the results with renal histology related to ANCA-GN, and with remission status and patient outcome evaluated by the renal Vasculitis Damage Index (VDI) score at 6 months after remission induction therapy. Both U-CD11b and U-CD163 at diagnosis were significantly elevated in patients with ANCA-GN compared to those in with nephrosclerosis (n=20) and IgG4-related disease (n=12). Those in tubulointerstitial nephritis (n=15) demonstrated a mild increase of U-CD11b, but not of U-CD163, at levels statistically comparable to that of AAV. Histological analysis in tubulointerstitial area demonstrated moderate correlation of U-CD11b with interstitial CD11b+ infiltrates. When focusing on glomerular injury, U-CD11b and U-CD163 levels were increased particularly in patients with ANCA-GN in the crescentic category according to the European Vasculitis Study Group classification. Detailed histological analyses evidenced the significant associations of U-CD11b and U-CD163 levels with cellular crescent formation and accumulation of leukocytes expressing the surface markers in glomerular crescents. Although the levels of U-CD163, but not of U-CD11b, in identical patients at diagnosis were significantly reduced after the 6-month follow-up period, patients with unachieved remission at 6 months showed significant elevation of U-CD163 at diagnosis, but not at 6 months. In contrast, baseline U-CD11b levels did not affect the remission status after 6 months of treatment, but the levels at 6 months tended to be decreased in patients with unachieved remission. Analyses of U-CD11b and U-CD163 levels according to the renal VDI score revealed significant elevation of U-CD11b at 6 months and U-CD163 at diagnosis in ANCA-GN patients with a renal VDI score ≥ 2 compared to those with score of 0 at 6 months. Receiver operating characteristic curves for U-CD11b and U-CD163 at diagnosis demonstrated the superiority of U-CD163 to U-CD11b as a biomarker to predict the remission status (AUC; U-CD11b 0.517, U-CD163 0.723) and renal VDI score ≥ 2 (AUC; U-CD11b 0.647, U-CD163 0.785) at 6 months after remission induction therapy. U-CD11b and U-CD163 have different clinical values: U-CD11b reflects the ANCA-GN inflammatory status in the glomerulus and tubulointerstitium, and is associated with persistent renal damages, whereas U-CD163 specifically reflects glomerular inflammation and is associated with susceptibility to immunosuppressive treatment.

Molecular underpinnings of integrin binding to collagen-mimetic peptides containing vascular Ehlers–Danlos syndrome–associated substitutions

Collagens carry out critical extracellular matrix (ECM) functions by interacting with numerous cell receptors and ECM components. Single glycine substitutions in collagen III, which predominates in vascular walls, result in vascular Ehlers-Danlos syndrome (vEDS), leading to arterial, uterine, and intestinal rupture and an average life expectancy of <50 years. Collagen interactions with integrin α2β1 are vital for platelet adhesion and activation; however, how these interactions are impacted by vEDS-associated mutations and by specific amino acid substitutions is unclear. Here, we designed collagen-mimetic peptides (CMPs) with previously reported Gly → Xaa (Xaa = Ala, Arg, or Val) vEDS substitutions within a high-affinity integrin α2β1-binding motif, GROGER. We used these peptides to investigate, at atomic-level resolution, how these amino acid substitutions affect the collagen III-integrin α2β1 interaction. Using a multitiered approach combining biological adhesion assays, CD, NMR, and molecular dynamics (MD) simulations, we found that these substitutions differentially impede human mesenchymal stem cell spreading and integrin α2-inserted (α2I) domain binding to the CMPs and were associated with triple-helix destabilization. Although an Ala substitution locally destabilized hydrogen bonding and enhanced mobility, it did not significantly reduce the CMP-integrin interactions. MD simulations suggested that bulkier Gly → Xaa substitutions differentially disrupt the CMP-α2I interaction. The Gly → Arg substitution destabilized CMP-α2I side-chain interactions, and the Gly → Val change broke the essential Mg2+ coordination. The relationship between the loss of functional binding and the type of vEDS substitution provides a foundation for developing potential therapies for managing collagen disorders.

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

AbstractCell-surface receptor interactions between leukocyte integrin macrophage-1 antigen (Mac-1, also known as CR3, αMβ2, CD11b/CD18) and platelet glycoprotein Ibα (GPIbα) are critical to vascular inflammation. To define the key residues at the binding interface, we used nuclear magnetic resonance (NMR) to assign the spectra of the mouse Mac-1 I-domain and mapped the residues contacting the mouse GPIbα N-terminal domain (GPIbαN) to the locality of the integrin metal ion-dependant adhesion site (MIDAS) surface. We next determined the crystal structures of the mouse GPIbαN and Mac-1 I-domain to 2 Å and 2.5 Å resolution, respectively. The mouse Mac-1 I-domain crystal structure reveals an active conformation that is stabilized by a crystal contact from the α7-helix with a glutamate side chain completing the octahedral coordination sphere of the MIDAS Mg2+ ion. The amino acid sequence of the α7-helix and disposition of the glutamic acid matches the C-terminal capping region α-helix of GPIbα effectively acting as a ligand mimetic. Using these crystal structures in combination with NMR measurements and docking analysis, we developed a model whereby an acidic residue from the GPIbα leucine-rich repeat (LRR) capping α-helix coordinates directly to the Mac-1 MIDAS Mg2+ ion. The Mac-1:GPIbαN complex involves additional interactions consolidated by an elongated pocket flanking the GPIbαN LRR capping α-helix. The GPIbαN α-helix has an HxxxE motif, which is equivalent by homology to RxxxD from the human GPIbαN. Subsequent mutagenesis of residues at this interface, coupled with surface plasmon resonance studies, confirmed the importance of GPIbαN residues H218, E222, and the Mac-1 MIDAS residue T209 to formation of the complex.

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.

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.

Inflammation, but not recruitment, of adipose tissue macrophages requires signalling through Mac-1 (CD11b/CD18) in diet-induced obesity (DIO).

Cell accumulation is a prerequisite for adipose tissue inflammation. The leukocyte integrin Mac-1 (CD11b/CD18, αMβ2) is a classic adhesion receptor critically regulating inflammatory cell recruitment. Here, we tested the hypothesis that a genetic deficiency and a therapeutic modulation of Mac-1 regulate adipose tissue inflammation in a mouse model of diet-induced obesity (DIO). C57Bl6/J mice genetically deficient (Mac-1-/-) or competent for Mac-1 (WT) consumed a high fat diet for 20 weeks. Surprisingly, Mac-1-/- mice presented with increased diet-induced weight gain, decreased insulin sensitivity in skeletal muscle and in the liver in insulin-clamps, insulin secretion deficiency and elevated glucose levels in fasting animals, and dyslipidaemia. Unexpectedly, accumulation of adipose tissue macrophages (ATMs) was unaffected, while gene expression indicated less inflamed adipose tissue and macrophages in Mac-1-/- mice. In contrast, inflammatory gene expression at distant locations, such as in skeletal muscle, was not changed. Treatment of ATMs with an agonistic anti-Mac-1 antibody, M1/70, induced pro-inflammatory genes in cell culture. In vivo, treatment with M1/70 induced a hyper-inflammatory phenotype with increased expression of IL-6 and MCP-1, whereas accumulation of ATMs did not change. Finally, inhibition of Mac-1's adhesive interaction to CD40L by the peptide inhibitor cM7 did not affect myeloid cell accumulation in adipose tissue. We present the surprising finding that adhesive properties of the leukocyte integrin Mac-1 are not required for macrophage accumulation in adipose tissue. Instead, Mac-1 modulates inflammatory gene expression in macrophages. These findings question the net effect of integrin blockade in cardio-metabolic disease.

INFLAMMATION, BUT NOT RECRUITMENT, OF ADIPOSE TISSUE MACROPHAGES REQUIRES SIGNALING THROUGH MAC-1 IN DIET-INDUCED OBESITY (DIO)

Cell accumulation is a prerequisite for adipose tissue inflammation. The leukocyte integrin Mac-1 (CD11b/CD18, αMβ2) is a classic adhesion receptor critically regulating inflammatory cell recruitment. Here, we tested the hypothesis that genetic deficiency and therapeutic modulation of Mac-1

Interruption of classic CD40L-CD40 signalling but not of the novel CD40L-Mac-1 interaction limits arterial neointima formation in mice

The co-stimulatory immune molecule CD40L figures prominently in a variety of inflammatory conditions including arterial disease. Recently, we made the surprising finding that CD40L mediates atherogenesis independently of its classic receptor CD40 via a novel interaction with the leukocyte integrin Mac-1. Here, we hypothesised that selective blockade of the CD40L-Mac-1 interaction may also retard restenosis. We induced neointima formation in C57/BL6 mice by ligation of the left carotid artery. Mice were randomised to daily intraperitoneal injections of either cM7, a small peptide selectively inhibiting the CD40L-Mac-1 interaction, scM7, a scrambled control peptide, or saline for 28 days. Interestingly, cM7-treated mice developed neointima of similar size compared with mice receiving the control peptide or saline as assessed by computer-assisted analysis of histological cross sections. These data demonstrate that the CD40L-Mac-1 interaction is not required for the development of restenosis. In contrast, CD40-deficient mice subjected to carotid ligation in parallel, developed significantly reduced neointimal lesions compared with respective wild-type controls (2872 ± 843 µm² vs 35469 ± 11870 µm²). Flow cytometry in CD40-deficient mice revealed reduced formation of platelet-granulocyte and platelet-inflammatory monocyte- aggregates. In vitro, supernatants of CD40-deficient platelet-leukocyte aggregates attenuated proliferation and increased apoptosis of smooth muscle cells. Unlike in the setting of atherosclerosis, CD40L mediates neointima formation via its classic receptor CD40 rather than via its recently described novel interaction with Mac-1. Therefore, selective targeting of CD40L-Mac-1 binding does not appear to be a favorable strategy to fight restenosis.

Combined single cell AFM manipulation and TIRFM for probing the molecular stability of multilayer fibrinogen matrices

Adsorption of fibrinogen on various surfaces produces a nanoscale multilayer matrix, which strongly reduces the adhesion of platelets and leukocytes with implications for hemostasis and blood compatibility of biomaterials. The nonadhesive properties of fibrinogen matrices are based on their extensibility, ensuing the inability to transduce strong mechanical forces via cellular integrins and resulting in weak intracellular signaling. In addition, reduced cell adhesion may arise from the weaker associations between fibrinogen molecules in the superficial layers of the matrix. Such reduced stability would allow integrins to pull fibrinogen molecules out of the matrix with comparable or smaller forces than required to break integrin-fibrinogen bonds. To examine this possibility, we developed a method based on the combination of total internal reflection fluorescence microscopy, single cell manipulation with an atomic force microscope and microcontact printing to study the transfer of fibrinogen molecules out of a matrix onto cells. We calculated the average fluorescence intensities per pixel for wild-type HEK 293 (HEK WT) and HEK 293 cells expressing leukocyte integrin Mac-1 (HEK Mac-1) before and after contact with multilayered matrices of fluorescently labeled fibrinogen. For contact times of 500 s, HEK Mac-1 cells show a median increase of 57% of the fluorescence intensity compared to 6% for HEK WT cells. The results suggest that the integrin Mac-1-fibrinogen interactions are stronger than the intermolecular fibrinogen interactions in the superficial layer of the matrix. The low mechanical stability of the multilayer fibrinogen surface may contribute to the reduced cell adhesive properties of fibrinogen-coated substrates. We anticipate that the described method can be applied to various cell types to examine their integrin-mediated adhesion to the extracellular matrices with a variable protein composition.

Pharmacological control of platelet-leukocyte interactions by the human anti-P-selectin antibody inclacumab – preclinical and clinical studies

Background and ObjectiveElevated levels of platelet-leukocyte aggregates (PLAs) have been reported in several cardiovascular diseases and suggested to contribute to disease pathology. Our aim was to characterize the effects of inclacumab, a novel human anti-P-selectin antibody, on the interactions between leukocytes and platelets in preclinical and clinical studies. Experimental ApproachesDual-label flow cytometry was used to detect the effect of inclacumab on agonist-induced platelet-leukocyte/platelet-monocyte aggregates in cynomolgus monkeys and humans, following ex vivo and in vivo administration. Platelet-dependent leukocyte activation and leukocyte adhesion to a platelet monolayer were also investigated after ex vivo administration of inclacumab to human blood. ResultsTreatment of cynomolgus monkeys with inclacumab profoundly inhibited thrombin receptor-activating peptide (TRAP) or adenosine diphosphate (ADP)-induced PLAs with an IC50 (<2μg/mL) similar to the in vitro spiking experiments. Maximal inhibition of PLAs persisted for ≥28days following single dose of inclacumab. In human blood, inclacumab was about 2-fold more potent in inhibiting TRAP-induced PLAs (IC50: 0.7μg/mL) compared to monkeys. PLA formation was suppressed independently of the inducing platelet agonist. Inclacumab also inhibited the activation of the leukocyte integrin Mac-1 and leukocyte adhesion to a platelet monolayer under flow conditions.In clinical studies, inclacumab inhibited TRAP-induced PLA formation in a dose-dependent manner following single and multiple dose administration to healthy volunteers. It also reduced elevated circulating PLA levels in patients with peripheral arterial disease. ConclusionBy inhibiting platelet-leukocyte interactions, demonstrated in multiple preclinical and clinical studies, inclacumab may provide an effective treatment for cardiovascular diseases.

ICAM-2 facilitates luminal neutrophil-endothelial cell interactionsin vivo

ABSTRACTIntercellular adhesion molecule 2 (ICAM-2) is expressed on endothelial cells (ECs) and supports neutrophil extravasation. However, the full details of its role remain unknown, and the present study investigates the functional mechanisms of ICAM-2 in neutrophil–endothelial-cell interactions. Our initial studies showed expression of ICAM-2 at both EC junctions and on the EC body. In line with the observed expression profile analysis of neutrophil–vessel-wall interactions using real-time in vivo confocal microscopy identified numerous functional roles for ICAM-2 within the vascular lumen and at the stage of neutrophil extravasation. Functional or genetic blockade of ICAM-2 significantly reduced neutrophil crawling velocity, increased frequency of crawling with a disrupted stop-start profile, and prolonged interaction of neutrophils with EC junctions prior to transendothelial cell migration (TEM), collectively resulting in significantly reduced extravasation. Pharmacological blockade of the leukocyte integrin MAC-1 indicated that some ICAM-2-dependent functions might be mediated through ligation of this integrin. These findings highlight novel roles for ICAM-2 in mediating luminal neutrophil crawling and the effect on subsequent levels of extravasation.

Abstract 9773: Phage Display of D esigned A nkyrin R epeat P roteins (DARPins) against the I Domain Of Mac-1 Results in Novel Diagnostic and Therapeutic Agents Specifically Acting on Activated Monocytes

Introduction: D esigned A nkyrin R epeat P roteins (DARPins) are a novel class of highly stable binding proteins (≈10-20kDa) and represent a valuable source for specific binders with picomolar affinities. The I domain is the activation-dependent binding site of the leukocyte integrin Mac-1 that is a central mediator of inflammation. Hypothesis: We assessed whether phage selection against the I domain results in DARPins that specifically bind to activated leukocytes without affecting non-activated ones. Methods: Selection was performed on purified mouse I domain using a DARPin phage library with a high diversity (10 10 ). Results: After 3 panning rounds, specific phages were selected and DARPins expressed with a high yield (≈ 200 mg/L). PMA activation revealed that DARPins bind stronger to activated than to non-activated mouse monocytes (Fig.1). Static and dynamic adhesion assays proved that selected DARPins inhibit binding of Mac-1 to its ligands (Fig.2). Using a peritonitis model, DARPins inhibited leukocyte migration into inflammatory areas, thereby indicating strong therapeutic, anti-inflammatory effects ( control vs DARPin: 940 vs. 230 cells, p&lt;0.02 ). DARPins showed strong crossreactivity to activated human Mac-1. Conclusion: Selected DARPins specifically bind to activated Mac-1 and can be used for direct monitoring of monocyte activation both in mice and humans. Thereby, based on the novel DARPin format we established a potential anti-monocyte drug that exerts strong anti-inflammatory effects in in vivo . Cross-reactivity with human Mac-1 promises translation into a broad spectrum of inflammation driven diseases.

Circulation Research Thematic Synopsis

<i>Circulation Research</i> Thematic Synopsis

Inflammation and Vascular Injury

The invited special lecture at the 76(th) Annual Scientific Meeting of the Japanese Circulation Society focused on the central role of inflammation in vascular injury and repair. Early studies pioneered the concept that mechanical injury, such as balloon angioplasty and endovascular stent deployment, elicits an inflammatory response from the vessel wall. This hypothesis was developed and substantiated at a time when the prevailing dogma viewed restenosis following angioplasty as a primarily proliferative smooth muscle cell disease. Antibody targeting of Mac-1 reduced leukocyte accumulation and limited neointimal formation following balloon injury or stent implantation. Genetic absence of Mac-1 resulted in diminished leukocyte accumulation and neointimal thickening after carotid artery injury in mice. In the course of those studies, our laboratory made fundamental discoveries regarding the mechanism of leukocyte recruitment at sites of vascular injury and identified platelet glycoprotein (GP) Ibα, a component of the GPIb-IX-V complex, as the previously unknown platelet counter-receptor for Mac-1. Follow-on studies have focused extensively on the structure, function, and signaling of the leukocyte integrin Mac-1. The binding site for GPIbα in Mac-1 has been mapped and subsequently showed that leukocyte engagement of platelet GPIbα via Mac-1 is critical not only for the biological response to vascular injury, but also for thrombosis, vasculitis, glomerulonephritis, and multiple sclerosis, thereby advancing the hypothesis that virtually all inflammation is platelet-dependent. Furthermore, ligand engagement of Mac-1 initiates a novel gene program that promotes inflammation by activating NFκB and downregulating the expression of the forkhead transcription factor Foxp1 that controls monocyte differentiation. Small molecule inhibitors of Mac-1 function have been pursued, including targeting of Mac-1-GPIbα binding or the downstream tyrosine kinase spleen tyrosine kinase. Drs Teruo Inoue, Koichi Node, Tatsuya Fukotomi, Masashi Sakuma, Toshifumi Morooka, and Kohsuke Nakajima, valued Japanese collaborators and post-doctoral fellows, have contributed enormously to these discoveries.

Host Fibrinogen and the S. Aureus-Encoded Procoagulant Vwbp Are Context-Dependent Determinants of Bacterial Virulence.

AbstractAbstract 1152Microbial pathogens frequently express bacterial products that are specifically designed to engage hemostatic system components in their vertebrate hosts. Staphylococcus aureus has evolved a particularly impressive repertoire of factors to control fibrin deposition, dissolution, and binding, including two bacterial products that form proteolytically active procoagulant complexes with prothrombin, staphylocoagulase (Coa) and von Willebrand factor binding protein (vWbp). These findings support the working hypothesis that hemostatic factors, in general, and fibrinogen, in particular, are likely to be important determinants of S. aureus virulence/host defense. To directly explore the role of fibrin(ogen) in host inflammatory/antimicrobial processes, a comparative analysis of bacterial clearance was done in control and fibrinogen-deficient mice in the context of S. aureus peritonitis. Control mice challenged with 109 CFU of S. aureus were found to efficiently clear S. aureus within the peritoneal cavity and eliminated ∼99.5% of bacterial CFUs within 20 minutes, whereas fibrin(ogen)-deficient mice exhibited little capacity to clear the microbe, even after several hours. Consistent with these findings, fibrinogen-deficient mice challenged with S. aureus peritonitis also exhibited a poor survival profile relative to control animals. More detailed studies to define the mechanism(s) underlying the rapid, fibrin(ogen)-dependent clearance of S. aureus have thus far excluded a critical contribution of host T cells, B cells, neutrophils, immunglobulins, complement, toll-like receptor signaling pathways, and the bacterial fibrinogen receptor clumping factor A (ClfA). However, mice carrying low levels of circulating prothrombin or expressing a mutant form of fibrinogen that cannot be converted to fibrin, exhibited a profound defect in S. aureus clearance following i.p. infection, suggesting a central role for fibrin polymer in the implementation of an effective antimicrobial program. Although fibrin formation is necessary, it is not sufficient for efficient bacterial clearance; Fibγ390-396A mice retaining full clotting function, but lacking the leukocyte integrin Mac-1 binding motif, also exhibit an impediment in bacterial clearance relative to wild-type mice. Complementary studies of S. aureus mutants deficient in bacterial procoagulants indicate that vWbp, but not Coa, is a fundamental determinant of bacterial clearance in this peritonitis model. Remarkably, bacterial procoagulant vWbp is distinctly counter-productive to the microbe in the context of peritonitis and supports the rapid, fibrin(ogen)-dependent clearance of intraperitoneal S. aureus. Based on findings indicating that host fibrinogen and bacterial factors that engage fibrin(ogen) support S. aureus virulence in other infection settings, the present findings suggest that host hemostatic factors and the bacterial procoagulant vWbp are likely to be context-dependent determinants of bacterial virulence. A better understanding of the interactions between bacterial proteins and host hemostatic factors, as well as interactions between the host hemostatic and inflammatory/immune systems, may well reveal novel therapeutic approaches for limiting microbial infections and sepsis. Disclosures:No relevant conflicts of interest to declare.