Mechano-Redox Control of Mac-1 De-Adhesion by PDI Promotes Directional Movement Under Flow.

BACKGROUND: Neutrophil migration is critical to the initiation and resolution of inflammation. Macrophage-1 antigen (Mac-1; CD11b/CD18, αMß2) is a leukocyte integrin essential for firm adhesion to endothelial ICAM-1 (intercellular adhesion molecule 1) and migration of neutrophils in the shear forces of the circulation. PDI (protein disulfide isomerase) has been reported to influence neutrophil adhesion and migration. We aimed to elucidate the molecular mechanism of PDI control of Mac-1 affinity for ICAM-1 during neutrophil migration under fluid shear. METHODS: Neutrophils isolated from whole blood were perfused over microfluidic chips coated with ICAM-1. Colocalization of Mac-1 and PDI on neutrophils was visualized by fluorescently labeled antibodies and confocal microscopy. The redox state of Mac-1 disulfide bonds was mapped by differential cysteine alkylation and mass spectrometry. Wild-type or disulfide mutant Mac-1 was expressed recombinantly in Baby Hamster Kidney cells to measure ligand affinity. Mac-1 conformations were measured by conformation-specific antibodies and molecular dynamics simulations. Neutrophils crawling on immobilized ICAM-1 were measured in presence of oxidized or reduced PDI, and the effect of PDI inhibition using isoquercetin on neutrophil crawling on inflamed endothelial cells was examined. Migration indices in the X- and Y-direction were determined and the crawling speed was calculated. RESULTS: PDI colocalized with high-affinity Mac-1 at the trailing edge of stimulated neutrophils when crawling on ICAM-1 under fluid shear. PDI cleaved 2 allosteric disulfide bonds, C169-C176 and C224-C264, in the ßI domain of the ß2 subunit, and cleavage of the C224-C264 disulfide bond selectively controls Mac-1 disengagement from ICAM-1 under fluid shear. Molecular dynamics simulations and conformation-specific antibodies reveal that cleavage of the C224-C264 bond induces conformational change and mechanical stress in the ßI domain. This allosterically alters the exposure of an αI domain epitope associated with a shift of Mac-1 to a lower-affinity state. These molecular events promote neutrophil motility in the direction of flow at high shear stress. Inhibition of PDI by isoquercetin reduces neutrophil migration in the direction of flow on endothelial cells during inflammation. CONCLUSIONS: Shear-dependent PDI cleavage of the neutrophil Mac-1 C224-C264 disulfide bond triggers Mac-1 de-adherence from ICAM-1 at the trailing edge of the cell and enables directional movement of neutrophils during inflammation.

Complement receptor 3 mediates ruffle-like, actin-rich aggregates during phagocytosis of Leishmania infantum metacyclics.

The parasitic protozoan Leishmania infantum resides primarily in macrophages throughout mammalian infection. Infection is initiated by deposition of the metacyclic promastigote into the dermis of a mammalian host by the sand fly vector. Promastigotes enter macrophages by ligating surface receptors such as complement receptor 3 (CR3), inducing phagocytosis of the parasite. At the binding site of metacyclic promastigotes, we observed large asymmetrical aggregates of macrophage membrane with underlying actin, resembling membrane ruffles. Actin accumulation was observed at the point of initial contact, before phagosome formation and accumulation of peri-phagosomal actin. Ruffle-like structures did not form during phagocytosis of attenuated promastigotes or during phagocytosis of the intracellular amastigote form of L. infantum. Entry of promastigotes through massive actin accumulation was associated with a subsequent delay in fusion of the parasitophorous vacuole (PV) with the lysosomal markers LAMP-1 and Cathepsin D. Actin accumulation was also associated with entry through CR3, since macrophages from CD11b knockout (KO) mice did not form massive aggregates of actin during phagocytosis of metacyclic promastigotes. Furthermore, intracellular survival of L. infantum was significantly decreased in CD11b KO compared to wild type macrophages, although entry rates were similar. We conclude that both promastigote virulence and host cell CR3 are needed for the formation of ruffle-like membrane structures at the site of metacyclic promastigote phagocytosis, and that formation of actin-rich aggregates during entry correlates with the intracellular survival of virulent promastigotes.

Leukocyte integrin Mac-1 (CD11b/CD18, αMß2, 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 (αMß2, 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.

Dermatophagoides farinae Upregulates the Effector Functions of Eosinophils through αMß2-Integrin and Protease-Activated Receptor-2.

BACKGROUND: Even in subjects who are not sensitized to house dust mite (HDM), allergic symptoms can be aggravated by exposure to dust, suggesting that innate immune responses may be involved in these processes. Since eosinophils express pattern recognition receptors, HDM may directly upregulate eosinophil functions through these re ceptors. The objective of this study was to examine whether Dermatophagoides farinae (Df), a representative HDM, or Der f 1, a major allergen of Df, modifies the effector functions of eosinophils. METHODS: Eosinophils isolated from the blood of healthy donors or allergic patients were stimulated with Df extract or Der f 1, and their adhesion to recombinant human intercellular adhesion molecule (ICAM)-1 was measured using eosinophil peroxidase assays. Generation of the eosinophil superoxide anion (O2-) was examined based on the superoxide dismutase-inhibitable reduction of cytochrome C. Eosinophil-derived neurotoxin (EDN) concentrations in cell media were measured by ELISA as a marker of degranulation. RESULTS: Df extract or Der f 1 directly induced eosinophil adhesion to ICAM-1, O2- generation, and EDN release. Anti-αM- or anti-ß2-integrin antibodies or protease-activated receptor (PAR)-2 antagonists suppressed the eosinophil adhesion, O2- generation, and EDN release induced by Df extract or Der f 1. Eosinophils from allergic patients showed higher adhesion to ICAM-1 than those from healthy donors. CONCLUSIONS: These findings suggested that Df extract and Der f 1 directly activate eosinophil functions through αMß2-integrin and PAR-2. Eosinophil activation by HDM may play roles in the aggravation of allergic symptoms, not only in HDM-sensitized patients, but also in nonsensitized patients.

Microvascular Mechanisms of Polyphosphate-Induced Neutrophil-Endothelial Cell Interactions in vivo.

BACKGROUND: Polyphosphates (PolyPs) have been reported to exert pro-inflammatory effects. However, the molecular mechanisms regulating PolyP-provoked tissue accumulation of leukocytes are not known. The aim of the present investigation was to determine the role of specific adhesion molecules in PolyP-mediated leukocyte recruitment. METHODS: PolyPs and TNF-α were intrascrotally administered, and anti-P-selectin, anti-E-selectin, anti-P-selectin glycoprotein ligand-1 (PSGL-1), anti-membrane-activated complex-1 (Mac-1), anti-lymphocyte function antigen-1 (LFA-1), and neutrophil depletion antibodies were injected intravenously or intraperitoneally. Intravital microscopy of the mouse cremaster microcirculation was used to examine leukocyte-endothelium interactions and recruitment in vivo. RESULTS: Intrascrotal injection of PolyPs increased leukocyte accumulation. Depletion of neutrophils abolished PolyP-induced leukocyte-endothelium interactions, indicating that neutrophils were the main leukocyte subtype responding to PolyP challenge. Immunoneutralization of P-selectin and PSGL-1 abolished PolyP-provoked neutrophil rolling, adhesion, and emigration. Moreover, immunoneutralization of Mac-1 and LFA-1 had no impact on neutrophil rolling but markedly reduced neutrophil adhesion and emigration evoked by PolyPs. CONCLUSION: These results suggest that P-selectin and PSGL-1 exert important roles in PolyP-induced inflammatory cell recruitment by mediating neutrophil rolling. In addition, our data show that Mac-1 and LFA-1 are necessary for supporting PolyP-triggered firm adhesion of neutrophils to microvascular endothelium. These novel findings define specific molecules as potential targets for pharmacological intervention in PolyP-dependent inflammatory diseases.

Adhesive Mechanisms of Histone-Induced Neutrophil-Endothelium Interactions in the Muscle Microcirculation.

BACKGROUND: Extracellular histones released during cell damage have the capacity to cause tissue injury associated with increased leukocyte accumulation. However, the molecular mechanisms regulating histone-induced leukocyte recruitment remain elusive. The objective of this study was to examine the role of adhesion molecules in histone-dependent leukocyte accumulation by use of intravital microscopy of the mouse cremaster microcirculation. METHODS: Histone 3 and TNF-α were intrascrotally administered, and anti-P-selectin, anti-P-selectin glycoprotein ligand-1 (PSGL-1), anti-membrane-activated complex-1 (Mac-1), anti-lymphocyte function antigen-1 (LFA-1) antibody and neutrophil depletion antibody were injected intravenously or intraperitoneally. RESULTS: Intrascrotal injection of histone 3 dose-dependently increased leukocyte recruitment. Neutrophil depletion abolished intravascular and extravascular leukocytes after histone 3 challenge, suggesting that neutrophils were the dominating leukocyte subtype responding to histone stimulation. Pretreatment with an anti-P-selectin and an anti-PSGL-1 antibody abolished histone-stimulated neutrophil rolling, adhesion and emigration. When the anti-P-selectin or the anti-PSGL-1 antibody was administrated after histone 3 stimulation, neutrophil rolling was reduced, whereas the number of firmly adherent and emigrated neutrophils were unchanged, suggesting that the inhibitory effect of blocking P-selectin and PSGL-1 on neutrophil adhesion and recruitment was due to the reduction in neutrophil rolling. Moreover, pretreatment with antibodies against Mac-1 and LFA-1 had no effect of neutrophil rolling but abolished adhesion and emigration evoked by histone 3. Thus, our data demonstrate that P-selectin and PSGL-1 play an important role in histone-induced inflammatory cell recruitment by mediating neutrophil rolling as a precondition for histone-provoked firm adhesion and emigration in vivo. Moreover, we conclude that both Mac-1 and LFA-1 are critical in supporting histone-provoked firm adhesion of neutrophils to endothelial cells. CONCLUSION: These novel findings define specific selectins and integrins as potential targets for pharmacological intervention in histone-dependent inflammatory diseases.

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

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

Genetic elimination of the binding motif on fibrinogen for the S. aureus virulence factor ClfA improves host survival in septicemia.

Fibrinogen can support host antimicrobial containment/clearance mechanisms, yet selected pathogens appear to benefit from host procoagulants to drive bacterial virulence. Here, we explored the hypothesis that host fibrin(ogen), on balance, supports Staphylococcus aureus infection in the context of septicemia. Survival studies following intravenous infection in control and fibrinogen-deficient mice established the overall utility of host fibrin(ogen) to S. aureus virulence. Complementary studies in mice expressing mutant forms of fibrinogen-retaining clotting function, but lacking either the bacterial ClfA (Fibγ(Δ5)) binding motif or the host leukocyte integrin receptor αMß2 (Fibγ(390-396A)) binding motif, revealed the preeminent importance of the bacterial ClfA-fibrin(ogen) interaction in determining host survival. Studies of mice lacking platelets or the platelet integrin receptor subunit αIIb established that the survival benefits observed in Fibγ(Δ5) mice were largely independent of platelet αIIbß3-mediated engagement of fibrinogen. Fibγ(Δ5) mice exhibited reduced bacterial burdens in the hearts and kidneys, a blunted host proinflammatory cytokine response, diminished microscopic tissue damage, and significantly diminished plasma markers of cardiac and other organ damage. These findings indicate that host fibrin(ogen) and bacterial ClfA are dual determinants of virulence and that therapeutic interventions at the level of fibrinogen could be advantageous in S. aureus septicemia.

C3a modulates IL-1ß secretion in human monocytes by regulating ATP efflux and subsequent NLRP3 inflammasome activation.

Interleukin-1ß (IL-1ß) is a proinflammatory cytokine and a therapeutic target in several chronic autoimmune states. Monocytes and macrophages are the major sources of IL-1ß. IL-1ß production by these cells requires Toll-like receptor (TLR) and adenosine triphosphate (ATP)-mediated P2X purinoceptor 7 (P2X7) signals, which together activate the inflammasome. However, how TLR signals and ATP availability are regulated during monocyte activation is unclear and the involvement of another danger signal system has been proposed. Here, we demonstrate that both lipopolysaccharide (LPS) and the anaphylatoxin C3a are needed for IL-1ß production in human macrophages and dendritic cells, while in monocytes, C3a enhanced the secretion of LPS-induced IL-1ß. C3a and LPS-stimulated monocytes increased T helper 17 (Th17) cell induction in vitro, and human rejecting, but not nonrejecting, kidney transplant biopsies were characterized by local generation of C3a and monocyte and Th17 cell infiltration. Mechanistically, C3a drives IL-1ß production in monocytes by controlling the release of intracellular ATP into the extracellular space via regulation of as-yet unidentified ATP-releasing channels in an extracellular signal-regulated kinase 1/2-dependent fashion. These data define a novel function for complement in inflammasome activation in monocytes and suggest that C3aR-mediated signaling is a vital component of the IL-1ß-Th17 axis.

Integrin activation by P-Rex1 is required for selectin-mediated slow leukocyte rolling and intravascular crawling.

Integrin activation is essential for the function of leukocytes. Impaired integrin activation on leukocytes is the hallmark of the leukocyte adhesion deficiency syndrome in humans, characterized by impaired leukocyte recruitment and recurrent infections. In inflammation, leukocytes collect different signals during the contact with the microvasculature, which activate signaling pathways leading to integrin activation and leukocyte recruitment. We report the role of P-Rex1, a Rac-specific guanine nucleotide exchanging factor, in integrin activation and leukocyte recruitment. We find that P-Rex1 is required for inducing selectin-mediated lymphocyte function-associated antigen-1 (LFA-1) extension that corresponds to intermediate affinity and induces slow leukocyte rolling, whereas P-Rex1 is not involved in the induction of the high-affinity conformation of LFA-1 obligatory for leukocyte arrest. Furthermore, we demonstrate that P-Rex1 is involved in Mac-1-dependent intravascular crawling. In vivo, both LFA-1-dependent slow rolling and Mac-1-dependent crawling are defective in P-Rex1(-/-) leukocytes, whereas chemokine-induced arrest and postadhesion strengthening remain intact in P-Rex1-deficient leukocytes. Rac1 is involved in E-selectin-mediated slow rolling and crawling. In vivo, in an ischemia-reperfusion-induced model of acute kidney injury, abolished selectin-mediated integrin activation contributed to decreased neutrophil recruitment and reduced kidney damage in P-Rex1-deficient mice. We conclude that P-Rex1 serves distinct functions in LFA-1 and Mac-1 activation.

Lipopolysaccharide-mediated enhancement of zymosan phagocytosis by RAW 264.7 macrophages is independent of opsonins, laminarin, mannan, and complement receptor 3.

BACKGROUND: Fungal and bacterial coinfections are common in surgical settings; however, little is known about the effects of polymicrobial interactions on the cellular mechanisms involved in innate immune recognition and phagocytosis. MATERIALS AND METHODS: Zymosan particles, cell wall derivatives of the yeast Saccharomyces cerevisiae, are used to model fungal interactions with host immune cells since they display carbohydrates, including beta-glucan, that are characteristic of fungal pathogens. Using in vitro cell culture, RAW 264.7 macrophages were challenged with zymosan, and phagocytosis determined via light microscopy. The effects of different concentrations of lipopolysaccharide (LPS) on zymosan phagocytosis were assessed. In addition, the transfer of supernatant from LPS-treated cells to naïve cells, the effects of soluble carbohydrates laminarin, mannan, or galactomannan, and the impact of complement receptor 3 (CR3) inhibition on phagocytosis were also determined. RESULTS: LPS enhanced phagocytosis of zymosan in a dose-dependent manner. Transfer of supernatants from LPS-primed cells to naïve cells had no effect on phagocytosis. Laminarin inhibited zymosan phagocytosis in naïve cells but not in LPS-primed cells. Neither mannan, galactomannan, nor CR3 inhibition had a significant effect on ingestion of unopsonized zymosan in naïve or LPS-treated cells. CONCLUSIONS: Zymosan recognition by naïve cells is inhibited by laminarin, but not mannan, galactomannan, or CR3 inhibition. LPS enhancement of phagocytosis is laminarin insensitive and not mediated by supernatant factors or zymosan engagement by the mannose or CR3 receptors. Our data suggest alternative mechanisms of zymosan recognition in the presence and absence of LPS.

Natural IgM mediates complement-dependent uptake of Francisella tularensis by human neutrophils via complement receptors 1 and 3 in nonimmune serum.

A fundamental step in the life cycle of Francisella tularensis is bacterial entry into host cells. F. tularensis activates complement, and recent data suggest that the classical pathway is required for complement factor C3 deposition on the bacterial surface. Nevertheless, C3 deposition is inefficient and neither the specific serum components necessary for classical pathway activation by F. tularensis in nonimmune human serum nor the receptors that mediate infection of neutrophils have been defined. In this study, human neutrophil uptake of GFP-expressing F. tularensis strains live vaccine strain and Schu S4 was quantified with high efficiency by flow cytometry. Using depleted sera and purified complement components, we demonstrated first that C1q and C3 were essential for F. tularensis phagocytosis, whereas C5 was not. Second, we used purification and immunodepletion approaches to identify a critical role for natural IgM in this process, and then used a wbtA2 mutant to identify LPS O-Ag and capsule as prominent targets of these Abs on the bacterial surface. Finally, we demonstrate using receptor-blocking Abs that CR1 (CD35) and CR3 (CD11b/CD18) acted in concert for phagocytosis of opsonized F. tularensis by human neutrophils, whereas CR3 and CR4 (CD11c/CD18) mediated infection of human monocyte-derived macrophages. Altogether, our data provide fundamental insight into mechanisms of F. tularensis phagocytosis and support a model whereby natural IgM binds to surface capsular and O-Ag polysaccharides of F. tularensis and initiates the classical complement cascade via C1q to promote C3 opsonization of the bacterium and phagocytosis via CR3 and either CR1 or CR4 in a phagocyte-specific manner.

Human lupus serum induces neutrophil-mediated organ damage in mice that is enabled by Mac-1 deficiency.

Systemic lupus erythematosus (SLE) is a chronic, multiorgan inflammatory autoimmune disorder associated with high levels of circulating autoantibodies and immune complexes. We report that passive transfer of human SLE sera into mice expressing the uniquely human FcγRIIA and FcγRIIIB on neutrophils induces lupus nephritis and in some cases arthritis only when the mice additionally lack the CD18 integrin, Mac-1. The prevailing view is that Mac-1 on macrophages is responsible for immune complex clearance. However, disease permitted by the absence of Mac-1 is not related to enhanced renal immune complex deposition or in situ C1q/C3 complement activation and proceeds even in the absence of macrophages. Instead, disease is associated with increased FcγRIIA-induced neutrophil accumulation that is enabled by Mac-1 deficiency. Intravital microscopy in the cremasteric vasculature reveals that Mac-1 mitigates FcγRIIA-dependent neutrophil recruitment in response to deposited immune complexes. Our results provide direct evidence that human SLE immune complexes are pathogenic, demonstrate that neutrophils are primary mediators of end organ damage in a novel humanized lupus mouse model, and identify Mac-1 regulation of FcγRIIA-mediated neutrophil recruitment as a key step in development of target organ damage.

RIAM (Rap1-interacting adaptor molecule) regulates complement-dependent phagocytosis.

Phagocytosis mediated by the complement receptor CR3 (also known as integrin αMß2 or Mac-1) is regulated by the recruitment of talin to the cytoplasmic tail of the ß2 integrin subunit. Talin recruitment to this integrin is dependent on Rap1 activation. However, the mechanism by which Rap1 regulates this event and CR3-dependent phagocytosis remains largely unknown. In the present work, we examined the role of the Rap1 effector RIAM, a talin-binding protein, in the regulation of complement-mediated phagocytosis. Using the human myeloid cell lines HL-60 and THP-1, we determined that knockdown of RIAM impaired αMß2 integrin affinity changes induced by stimuli fMLP and LPS. Phagocytosis of complement-opsonized RBC particles, but not of IgG-opsonized RBC particles, was impaired in RIAM knockdown cells. Rap1 activation via EPAC induced by 8-pCPT-2'-O-Me-cAMP resulted in an increase of complement-mediated phagocytosis that was abrogated by knockdown of RIAM in HL-60 and THP-1 cell lines and in macrophages derived from primary monocytes. Furthermore, recruitment of talin to ß2 integrin during complement-mediated phagocytosis was reduced in RIAM knockdown cells. These results indicate that RIAM is a critical component of the phagocytosis machinery downstream of Rap1 and mediates its function by recruiting talin to the phagocytic complement receptors.

Carbohydrate clearance receptors in transfusion medicine.

BACKGROUND: Complex carbohydrates play important functions for circulation of proteins and cells. They provide protective shields and refraction from non-specific interactions with negative charges from sialic acids to enhance circulatory half-life. For recombinant protein therapeutics carbohydrates are especially important to enhance size and reduce glomerular filtration loss. Carbohydrates are, however, also ligands for a large number of carbohydrate-binding lectins exposed to the circulatory system that serve as scavenger receptors for the innate immune system, or have more specific roles in targeting of glycoproteins and cells. SCOPE OF REVIEW: Here we provide an overview of the common lectin receptors that play roles for circulating glycoproteins and cells, and present a discussion of ways to engineer glycosylation of recombinant biologics and cells to improve therapeutic effects. MAJOR CONCLUSIONS: While the pharmaceutical industry has learned how to exploit carbohydrates to improve pharmacokinetic properties of recombinant therapeutics, our understanding of how to improve cell-based therapies by manipulation of complex carbohydrates is still at its infancy. Progress with the latter has recently been achieved with cold-stored platelets, where exposure of uncapped glycans lead to rapid clearance from circulation by several lectin-mediated pathways. GENERAL SIGNIFICANCE: Understanding lectin-mediated clearance pathways is essential for progress in development of biological pharmaceuticals.

The role of MAC1 in diesel exhaust particle-induced microglial activation and loss of dopaminergic neuron function.

Increasing reports support that air pollution causes neuroinflammation and is linked to central nervous system (CNS) disease/damage. Diesel exhaust particles (DEP) are a major component of urban air pollution, which has been linked to microglial activation and Parkinson's disease-like pathology. To begin to address how DEP may exert CNS effects, microglia and neuron-glia cultures were treated with either nanometer-sized DEP (< 0.22 µM; 50 µg/mL), ultrafine carbon black (ufCB, 50 µg/mL), or DEP extracts (eDEP; from 50 µg/mL DEP), and the effect of microglial activation and dopaminergic (DA) neuron function was assessed. All three treatments showed enhanced ameboid microglia morphology, increased H2 O2 production, and decreased DA uptake. Mechanistic inquiry revealed that the scavenger receptor inhibitor fucoidan blocked DEP internalization in microglia, but failed to alter DEP-induced H2 O2 production in microglia. However, pre-treatment with the MAC1/CD11b inhibitor antibody blocked microglial H2 O2 production in response to DEP. MAC1(-/-) mesencephalic neuron-glia cultures were protected from DEP-induced loss of DA neuron function, as measured by DA uptake. These findings support that DEP may activate microglia through multiple mechanisms, where scavenger receptors regulate internalization of DEP and the MAC1 receptor is mandatory for both DEP-induced microglial H2 O2 production and loss of DA neuron function.

The fibrin-derived gamma377-395 peptide inhibits microglia activation and suppresses relapsing paralysis in central nervous system autoimmune disease.

Perivascular microglia activation is a hallmark of inflammatory demyelination in multiple sclerosis (MS), but the mechanisms underlying microglia activation and specific strategies to attenuate their activation remain elusive. Here, we identify fibrinogen as a novel regulator of microglia activation and show that targeting of the interaction of fibrinogen with the microglia integrin receptor Mac-1 (alpha(M)beta(2), CD11b/CD18) is sufficient to suppress experimental autoimmune encephalomyelitis in mice that retain full coagulation function. We show that fibrinogen, which is deposited perivascularly in MS plaques, signals through Mac-1 and induces the differentiation of microglia to phagocytes via activation of Akt and Rho. Genetic disruption of fibrinogen-Mac-1 interaction in fibrinogen-gamma(390-396A) knock-in mice or pharmacologically impeding fibrinogen-Mac-1 interaction through intranasal delivery of a fibrinogen-derived inhibitory peptide (gamma(377-395)) attenuates microglia activation and suppresses relapsing paralysis. Because blocking fibrinogen-Mac-1 interactions affects the proinflammatory but not the procoagulant properties of fibrinogen, targeting the gamma(377-395) fibrinogen epitope could represent a potential therapeutic strategy for MS and other neuroinflammatory diseases associated with blood-brain barrier disruption and microglia activation.

The role of ß2-integrins and CD44 in intrahepatic leukocyte sequestration.

BACKGROUND: Intrahepatic leukocyte sequestration is a component of the systemic inflammatory response, and can be triggered by systemic immune dysfunction during sepsis. METHODS: To examine leukocyte sequestration over time during endotoxemia, its influence on liver function, and the role of specific cell adhesion molecules, endotoxemia was induced in mice by intraperitoneal application of lipopolysaccharides. Leukocyte sequestration was measured at different times after induction using fluorescence microscopy. Liver injury was evaluated by measuring liver enzymes and tissue histology. RESULTS: Endotoxin induces a strong leukocyte sequestration in the liver microvasculature. This was associated with an induction of liver injury, as reflected by an increase in enzyme levels and histomorphologic changes. Intrahepatic leukocyte sequestration was reduced in CD44(-/-), but not in intercellular adhesion molecule-1 (ICAM-1)(-/-), lymphocyte function-associated antigen-1(-/-), and macrophage-1(-/-) antigen mice. Leukocyte sequestration dropped in ICAM-1(-/-), lymphocyte function-associated antigen-1(-/-), and macrophage-1(-/-) mice in later stages, but remained stable in wild-type and CD44(-/-) animals. Reduced leukocyte sequestration in CD44(-/-) mice was accompanied by a significant decrease in transferase levels. CONCLUSIONS: Endotoxemia induces stable intra-sinusoidal leukocyte sequestration, which contributes to liver injury. At the initial stage of the endotoxemia, leukocyte sequestration depends on CD44 but is independent of ICAM-1 and ß2-integrins. Intercellular adhesion molecule-1 and ß2-integrins, but not CD44, stabilize leukocyte sequestration during the later stage of endotoxemia. The molecular modulation of intrahepatic leukocyte sequestration may have important therapeutic implications in sepsis, reducing liver injury, and improving immune defense capabilities.

Linkage between coenzyme a metabolism and inflammation: roles of pantetheinase.

Pantetheinase is an enzyme hydrolyzing pantetheine, an intermediate of the coenzyme A degradation pathway. Pantetheinase has long been considered as the enzyme that recycles pantothenic acid (vitamin B5) generated during coenzyme A breakdown. Genetic analyses showed that mammals have multiple genes known as vanin family genes. Recent studies using mice lacking the vanin-1 gene (pantetheinase gene) suggest that pantetheinase is actively involved in the progression of inflammatory reactions by generating cysteamine. Additional studies using human leukocytes demonstrate that human neutrophils have abundant pantetheinase proteins on the surface and inside the cells. The second pantetheinase protein, GPI-80/VNN2, is suggested to work as a modulator of the function of Mac-1 (CD11b/CD18), an adhesion molecule important to neutrophil functions. This review delineates the characteristics of the pantetheinase/vanin gene family and how they affect inflammation.