Apoptotic PMNs Research Articles

P120 catenin enhances macrophage efferocytosis and facilitates resolution of lung inflammatory injury

Abstract Efficient clearance of apoptotic neutrophils (PMNs) (efferocytosis) after sepsis plays a crucial role in facilitating tissue repair, resolving inflammation, and maintaining immune system balance. Here, we identified p120 catenin (p120) as a pivotal regulator in efferocytosis and subsequent recovery from inflammatory lung injury. We found that specific macrophage p120 deletion had a profound delay in the resolution of PMN infiltration, extracellular protein accumulation, and lung edema and injury following LPS challenge. p120 deletion increased the levels of TNF-α and IL-6, and reduced the levels of TGF-β1 and IL-10. Depletion of p120 also led to a marked decrease in the phagocytosis of apoptotic PMNs by macrophages. The number of p120-depleted alveolar macrophages containing apoptotic PMNs in bronchoalveolar lavage fluid was significantly lower. p120 deficiency caused a reduction in the expression of key efferocytosis-related molecules, namely AXL and CD36. p120-depleted macrophages exhibited a shift in polarization towards the M1 phenotype. Apoptotic cells triggered the association and co-localization of p120 and PPAR, and p120 deletion decreased the activity of PPARgamma in response to apoptotic PMNs. Inhibition of PPAR abolished p120-mediated efferocytosis and the resolution of lung inflammation. These findings underscore the critical role of p120 in facilitating the resolution of lung inflammation through modulation of macrophage polarization and PPAR signaling pathway.

Novel method to assess resident alveolar macrophage efferocytosis of apoptotic neutrophils by flow cytometry

Macrophage efferocytosis of apoptotic neutrophils (PMNs) plays a key role in the resolution of inflammation. In these studies, we describe a novel flow cytometric method to assess efferocytosis of apoptotic PMNs. Resident alveolar macrophages and PMNs were collected from lungs of mice exposed to inhaled ozone (0.8 ppm, 3 h) followed by lipopolysaccharide (3 mg/kg, i.v.) to induce acute lung injury. PMNs were labeled with PKH26 or DilC18(5)-DS (D12730) cell membrane dye and then incubated with resident alveolar macrophages at a ratio of 5:1. After 90 min, macrophage efferocytosis was analyzed by flow cytometry and confirmed by confocal microscopy. Whereas alveolar macrophages incubated with D12730-labeled PMNs could readily be identified as efferocytotic or non-efferocytotic, this was not possible with PKH26 labeled PMNs due to confounding macrophage autofluorescence. Using D12730 labeled PMNs, subsets of resident alveolar macrophages were identified with varying capacities to perform efferocytosis, which may be linked to the activation state of these cells. Future applications of this method will be useful in assessing the role of efferocytosis in the resolution of inflammation in response to toxicant exposure.

MFG-E8 promotes tendon-bone healing by regualting macrophage efferocytosis and M2 polarization after anterior cruciate ligament reconstruction.

BackgroundScar tissue formation at the tendon-bone interface caused by excessive inflammation leads to insufficient healing strength, while the phagocytic clearance of dying cells (efferocytosis) has profound consequences on macrophage polarisation and the inflammatory response. Modulating the inflammatory microenvironment may have satisfactory curative effects in patients with anterior cruciate ligament reconstruction (ACLR).MethodsBone marrow-derived macrophages (BMDMs) and polymorphonuclear leukocytes (PMNs) were harvested from bone marrow. The effects of milk fat globulin protein E8 (MFG-E8) on macrophage polarisation were compared with those of M1 and M2 macrophages induced by conventional methods. The BMDMs and apoptotic PMNs co-culture system was used to assess the efficiency of efferocytosis. The biological functions of MFG-E8 in tendon-bone healing by regulating macrophage efferocytosis and polarisation were further investigated using a rat ACLR model.ResultsBMDMs and PMNs were successfully isolated. Compared to conventional induction methods, MFG-E8 alone did not significantly induce macrophage M1 or M2 polarisation, but it could partially reverse the expression of inducible nitric oxide synthase (iNOS) in M1 macrophages. In vitro studies revealed that appropriate dosing of MFG-E8 could significantly promote the efficiency of macrophage efferocytosis and subsequently increase M2 polarisation. More importantly, significantly increased peri-tunnel new bone formation, tighter connected interface and better mechanical properties were observed after ACLR when treated with MFG-E8 in vivo. We further demonstrated that MFG-E8 remarkably facilitated the clearance of apoptotic cells and increased the number of M2 macrophages at the interface between the tendon graft and bone tunnel in the early postoperative stage.ConclusionMFG-E8 promoted tendon-bone healing histologically and biomechanically, probably by the regulation of inflammatory processes via macrophage efferocytosis and M2 polarisation.The translational potential of this article: Regulation of macrophage efferocytosis and M2 polarization by MFG-E8 is expected to be a therapeutic strategy for promoting tendon-bone healing in patients undergoing ACLR.

Baicalin clears inflammation by enhancing macrophage efferocytosis via inhibition of RhoA/ROCK signaling pathway and regulating macrophage polarization

Effective efferocytosis of apoptotic polymorphonuclear leukocytes (PMNs) in the initiation and resolution of inflammation iscrucial for preventing progression to chronicinflammatorydisease. Baicalin, a bioactive flavonoid drug, exerts multiple anti-inflammatory effects; however, its underlying mechanism remains to be elucidated. Here, we investigated the role of baicalin in efferocytosis in vitro and in a Porphyromonas gingivalis lipopolysaccharide (LPS)-inducedmurinemodelofpleurisy. We found that the macrophage engulfment of apoptotic PMNs was significantly promoted by baicalin in an inflammatory environment with an effectiveness similar to that of N-acetylcysteine. Meanwhile, the production of reactive oxygen species was significantly blocked in P. gingivalis LPS-stimulated J774A.1 macrophages by baicalin, and the RhoA/ROCK signaling pathway was inhibited in the same way as the ROCK inhibitor, Y-27632. In addition, the M1/M2macrophage ratio and the proinflammatory cytokine TNF-α were downregulated, whereas the anti-inflammatory cytokine IL-10 was upregulated both in vitro and in vivo. Therefore, we propose that baicalin may increase efferocytosis by acting as an antioxidant via a RhoA-dependent pathway and regulate macrophage polarization, thus promoting inflammatory resolution.

STAT6 induces expression of Gas6 in macrophages to clear apoptotic neutrophils and resolve inflammation

Efferocytosis of apoptotic neutrophils (PMNs) by alveolar macrophages (AMФs) is vital for resolution of inflammation and tissue injury. Here, we investigated the role of AMФ polarization and expression of the efferocytic ligand Gas6 in restoring homeostasis. In the murine model of lipopolysaccharide (LPS)-induced acute lung injury (ALI), we observed augmented temporal generation of cytokines IL-4 and TSG6 in bronchoalveolar fluid (BALF). Interestingly, we also observed increased expression of antiinflammatory markers consistent with a phenotype shift in AMФs. In particular, AMФs expressed the efferocytic ligand Gas6. In vitro priming of bone marrow-derived macrophages (BMMФs) with IL-4 or TSG6 also induced MФ transition and expression of Gas6. TSG6- or IL-4-primed BMMФs induced efferocytosis of apoptotic PMNs compared with control BMMФs. Adoptive transfer of TSG6- or IL-4-primed BMMФs i.t. into LPS-challenged mice more rapidly and effectively cleared PMNs in lungs compared with control BMMФs. We demonstrated that expression of Gas6 during AMФ transition was due to activation of the transcription factor signal transducer and activator of transcription-6 (STAT6) downstream of IL-4 or TSG6 signaling. Adoptive transfer of Gas6-depleted BMMФs failed to clear PMNs in lungs following LPS challenge and mice showed severely defective resolution of lung injury. Thus, activation of STAT6-mediated Gas6 expression during macrophage phenotype transition resulting in efferocytosis of PMNs plays a crucial role in the resolution of inflammatory lung injury.

Transcriptional and functional diversity of human macrophage repolarization

Macrophage plasticity allows cells to adopt different phenotypes, a property with important implications in disorders such as cystic fibrosis (CF) and asthma. We sought to examine the transcriptional and functional significance of macrophage repolarization from an M1 to an M2 phenotype and assess the role of a common human genetic disorder (CF) and a prototypical allergic disease (asthma) in this transformation. Monocyte-derived macrophages were collected from healthy subjects and patients with CF and polarized to an M2 state by using IL-4, IL-10, glucocorticoids, apoptotic PMNs, or azithromycin. We performed transcriptional profiling and pathway analysis for each stimulus. We assessed the ability of M2-repolarized macrophages to respond to LPS rechallenge and clear apoptotic neutrophils and used murine models to determine conserved functional responses to IL-4 and IL-10. We investigated whether M2 signatures were associated with alveolar macrophage phenotypes in asthmatic patients. We found that macrophages exhibit highly diverse responses to distinct M2-polarizing stimuli. Specifically, IL-10 activated proinflammatory pathways and abrogated LPS tolerance, allowing rapid restoration of LPS responsiveness. In contrast, IL-4 enhanced LPS tolerance, dampening proinflammatory responses after repeat LPS challenge. Acommon theme observed across all M2 stimuli was suppression of interferon-associated pathways. We found that CF macrophages had intact reparative and transcriptional responses, suggesting that macrophage contributions to CF-related lung disease are primarily shaped by their environment. Finally, we leveraged invitro-derived signatures to show that allergen provocation induces distinct M2 state transcriptional patterns in alveolar macrophages. Our findings highlight the diversity of macrophage polarization, attribute functional consequences to different M2 stimuli, and provide a framework to phenotype macrophages in disease states.

Unified nexus of macrophages and maresins in cardiac reparative mechanisms

Macrophages are immune-sensing "big eater" phagocytic cells responsible for an innate, adaptive, and regenerative response. After myocardial infarction, macrophages predominantly clear the deceased cardiomyocyte apoptotic or necrotic neutrophils to develop a regenerative and reparative program with the activation of the lipoxygenase-mediated maresin (MaR) metabolome at the site of ischemic injury. The specialized proresolving molecule and macrophage mediator in resolving inflammation, MaR-1, produced by human macrophages, has potent defining effects that limit polymorphonuclear neutrophil infiltration, enhance uptake of apoptotic PMNs, regulate inflammation resolution and tissue regeneration, and reduce pain. In addition to proresolving and anti-inflammatory actions, MaR-1 displays potent tissue regenerative effects in stroke and is an antinociceptive. Macrophages actively participate in the biosynthesis of bioactive MaR-2, which exhibits anti-inflammatory, proresolving, and atherosclerotic effects. A new class of macrophage-derived molecules, MaR conjugates in tissue regeneration, is identified that regulates phagocytosis and the repair and regeneration of damaged tissue. The presented review provides a current summary of the effect of MaR in resolution pathophysiology, with relevance to a cardiac repair program.-Jadapalli, J. K., Halade, G. V. Unified nexus of macrophages and maresins in cardiac reparative mechanisms.

Macrophage Proresolving Mediators-the When and Where.

Macrophages and neutrophils orchestrate acute inflammation and host defense as well as the resolution phase and return to homeostasis. In this article, we review the contribution of macrophages to local lipid mediator (LM) levels and the regulation of macrophage LM profiles by polymorphonuclear neutrophils and neutrophil-derived microparticles. We carried out LM metabololipidomics, profiling distinct phagocytes: neutrophils (PMNs), apoptotic PMNs, and macrophages. Efferocytosis increased specialized proresolving mediator (SPM) biosynthesis, including resolvin D1 (RvD1), RvD2, and RvE2, which were further elevated by PMN microparticles. In studies using deuterium-labeled precursors (d8-arachidonic acid, d5-eicosapentaenoic acid, and d5-docosahexaenoic acid), apoptotic PMNs and microparticles contributed to SPM biosynthesis during efferocytosis. Assessment of macrophage LM profiles in M2 macrophages demonstrated higher SPM levels in this macrophage subset, including maresin 1 (MaR1), and lower amounts of leukotriene B4 (LTB4) and prostaglandins than in M1. Apoptotic PMN uptake by both macrophage subtypes led to modulation of their LM profiles. LTB4 was downregulated in M2, whereas SPMs including lipoxin A4 were increased. Conversely, uptake of apoptotic PMNs by M2 macrophages reduced (∼25%) overall LMs. MaR1 displays potent tissue-regenerative and antinociceptive actions in addition to its proresolving and anti-inflammatory actions. In addition, the MaR1 biosynthetic intermediate 13S,14S-epoxy-maresin is also bioactive, inhibiting LTB4 biosynthesis and switching macrophage phenotypes from M1 to M2. Together, these results establish LM signature profiles of human phagocytes and related subpopulations. They demonstrate microparticle regulation of macrophage-specific endogenous LMs during defined stages of acute inflammation and their dynamic changes in human primary phagocytes.

The neuroimmune guidance cue netrin-1 controls resolution programs and promotes liver regeneration.

Hepatic ischemia/reperfusion (I/R) is a major adverse reaction to liver transplantation, hemorrhagic shock, or resection. Recently, the anti-inflammatory properties of the axonal guidance cue netrin-1 were reported. Here, we demonstrate that netrin-1 also impacts the resolution of inflammation and promotes hepatic repair and regeneration during liver I/R injury. In initial studies, we investigated the induction of netrin-1 and its receptors in murine liver tissues after I/R injury. Hepatic I/R injury was performed in mice with a partial genetic netrin-1 deficiency (Ntn1(+/-) ) or wild-type C57BL/6 treated with exogenous netrin-1 to examine the endogenous and therapeutically administered impact of netrin-1. These investigations were corroborated by studies determining the characteristics of intravascular leukocyte flow, clearance of apoptotic neutrophils (polymorphonuclear cells [PMNs]), production of specialized proresolving lipid mediators (SPMs), generation of specific growth factors contributing to the resolution of inflammation, and liver repair. Hepatic I/R was associated with a significant reduction of netrin-1 transcript and protein in murine liver tissue. Subsequent studies in netrin-1-deficient mice revealed lower efficacies in reducing PMN infiltration, proinflammatory cytokine levels, and hepatic-specific injury enzymes. Conversely, mice treated with exogenous netrin-1 exhibited increased liver protection and repair, reducing neutrophil influx into the injury site, decreasing proinflammatory mediators, increasing efferocytosis of apoptotic PMNs, and stimulating local endogenous biosynthesis of SPMs and the generation of specific growth factors. Finally, genetic studies implicated the A2B adenosine receptor in netrin-1-mediated protection during hepatic I/R injury. The present study indicates a previously unrecognized role for netrin-1 in liver protection and its contribution to tissue homeostasis and regeneration.

Prolonged cultures of unstimulated human neutrophils lead to the apparition and persistence of rest-in-plate structures (RIPs) recognized by professional phagocytes in vitro and in vivo

Polymorphonuclear neutrophil cells (PMNs) are known to spontaneously undergo apoptosis and then eliminated by professional phagocytes to prevent inflammation, a process called efferocytosis. However, when efferocytosis is impaired, PMNs will fall into secondary necrosis. Whether this state can persist for a certain period of time is unclear, since most of the studies investigating secondary necrosis are performed within 24h following induction by a proapoptotic agent. In this study, freshly isolated human PMNs were incubated without addition of exogenous agents in order to force them to undergo apoptosis and then secondary necrosis, an ideal experimental condition to study the behavior of secondary necrotic PMNs in absence of efferocytosis. By monitoring PMN cell morphology over time, we observed that an increasing proportion of cells harbored a ghost-like phenotype. Because these cellular remnants persist in plates for several days, we introduce here the terminology RIPs for ‘rest-in-plate’ structure. Heating of freshly isolated PMNs for 5min did not lead to the apparition of RIPs over time. In vivo administration of 7-days old RIPs in the murine air pouch model induced a slight inflammation resorbed within 24h. PKH26-stained RIPs were found to be ingested by professional phagocytes in vitro and in vivo in the murine air pouch and peritonitis models. Therefore, aged-PMNs have the potential to become RIPs in absence of efficient efferocytosis. Fortunately RIPs are recognized by professional phagocytes and, therefore, the concept of resolution of inflammation based on elimination of apoptotic and secondary necrotic PMNs could also be applied to RIPs.

Impact of anemia treatment with methoxy polyethylene glycol-epoetin beta on polymorphonuclear cells apoptosis in predialysis patients with chronic kidney disease.

Some data in literature indicate increased apoptosis of polymorphonuclear cells (PMNs) in chronic kidney disease (CKD), what seems to be connected with anemia. Erythropoiesis-stimulating agents, used in anemia treatment in CKD may affect cells apoptosis. Aim of this study was to investigate impact of anemia treatment with methoxy polyethylene glycol-epoetin beta (CERA) on PMNs apoptosis in predialysis patients with CKD. Percentage of early and late apoptotic PMNs was measured by flow cytometry based on annexin V and propidium iodide binding. CD90 (Fas), CD95L (FasL), CD16 and CD11b expression on PMNs were evaluated by flow cytometry after incubation with respective monoclonal antibody. Percentage of PMNs in early and late apoptosis in CKD patients before CERA treatment was significantly higher to control group, which was accompanied by significantly higher Fas and Fas-L expression and significantly lower expression of CD16. CERA treatment downregulated significantly percentage of early, apoptotic PMNs but percentage of late apoptotic cells did not change and was still significantly higher to control group. In all investigated groups we observed a significant negative correlation between hemoglobin concentration and percentage of apoptotic PMNs, as well as Fas and FasL expression and significant positive correlation between Hb and CD16 expression. Our results indicate that PMNs apoptosis is increased in predialysis patients with CKD and anemia treatment with CERA may diminish readiness of PMNs to undergo apoptosis. This antiapoptotic impact of anemia treatment with CERA seems to concern early apoptotic PMNs before they undergo to late, irreversible stage of apoptosis.

Resolution of acute inflammation bridges the gap between innate and adaptive immunity

AbstractAcute inflammation is traditionally characterized by polymorphonuclear leukocytes (PMN) influx followed by phagocytosing macrophage (Mφs) that clear injurious stimuli leading to resolution and tissue homeostasis. However, using the peritoneal cavity, we found that although innate immune-mediated responses to low-dose zymosan or bacteria resolve within days, these stimuli, but not hyperinflammatory stimuli, trigger a previously overlooked second wave of leukocyte influx into tissues that persists for weeks. These cells comprise distinct populations of tissue-resident Mφs (resMφs), Ly6chi monocyte-derived Mφs (moMφs), monocyte-derived dendritic cells (moDCs), and myeloid-derived suppressor cells (MDSCs). Postresolution mononuclear phagocytes were observed alongside lymph node expansion and increased numbers of blood and peritoneal memory T and B lymphocytes. The resMφs and moMφs triggered FoxP3 expression within CD4 cells, whereas moDCs drive T-cell proliferation. The resMφs preferentially clear apoptotic PMNs and migrate to lymph nodes to bring about their contraction in an inducible nitric oxide synthase-dependent manner. Finally, moMφs remain in tissues for months postresolution, alongside altered numbers of T cells collectively dictating the magnitude of subsequent acute inflammatory reactions. These data challenge the prevailing idea that resolution leads back to homeostasis and asserts that resolution acts as a bridge between innate and adaptive immunity, as well as tissue reprogramming.

Characterization of the impairment of the uptake of apoptotic polymorphonuclear cells by monocyte subpopulations in systemic lupus erythematosus.

Efficient removal of apoptotic polymorphonuclear leukocytes (PMNs) is an important step in the resolution of inflammation, which protects tissues from the noxious contents of dying cells. While the impairment of apoptotic PMNs removal has been demonstrated for macrophages in systemic lupus erythematosus (SLE), recent studies show that monocytes are also capable of such phagocytosis, although their involvement in SLE is not clear. Therefore, we characterized phagocytosis of apoptotic PMNs by monocytes in 22 patients with SLE and 22 healthy controls. Using flow cytometry we demonstrate that in SLE peripheral blood monocytes show impaired phagocytosis of autologous apoptotic PMNs, while they efficiently engulf apoptotic PMNs isolated from healthy subjects. Monocytes CD14highCD16+ and CD14dimCD16+ more efficiently interacted with apoptotic neutrophils than CD16- cells both in SLE and healthy subjects. Monocytes in SLE showed modestly decreased expression of CD35 and CD91 and increased expression of T Cell Ig- and mucin-domain-containing molecule-3 (TIM-3); however, these differences were evident mainly in selected subsets of monocytes (CD16+) while defects in phagocytosis were observed in all monocyte subsets. Apoptotic cell-dependent induction of lipopolysaccharide (LPS) stimulated production of anti-inflammatory cytokine IL-10 by peripheral blood mononuclear cells (PBMC) was blunted in SLE while the production of pro-inflammatory cytokine TNF-α was unchanged.

Functional capacity of alveolar neutrophils in acute respiratory distress syndrome

BackgroundInappropriate accumulation or persistence of neutrophils (polymorphonuclear leucocytes [PMNs]), within the alveolar airspace is considered to be crucial to the pathogenesis of acute respiratory distress syndrome (ARDS). Previous work has established that alveolar PMNs from patients with ARDS have a pro-survival phenotype, and that bronchoalveolar lavage fluid (BALF) in ARDS suppresses constitutive PMN apoptosis in vitro. However, the functional activity of alveolar-sequestered PMNs in ARDS is unknown, partly due to the technical challenges in obtaining and purifying alveolar PMNs. Proinflammatory mediators in the ARDS alveolar milieu conceivably prime or activate key PMN functions in vivo, including the generation of toxic reactive oxygen species (ROS). This research evaluates agonist-stimulated ROS generation in alveolar and blood PMNs from the same patient with ARDS compared with healthy volunteer blood PMNs. MethodsAlveolar and blood PMNs from mechanically ventilated patients who fulfilled the Berlin criteria of ARDS were studied in parallel. Patients underwent fibreoptic bronchoscopy and bronchoalveolar lavage within 36 h of ARDS onset, and the recovered BALF was processed immediately. Alveolar PMNs were isolated to 90% (SE 2·5) purity with a rapid semi-automated negative-selection immunomagnetic technique (Robosep, Stemcell Technologies, Vancouver, Canada). BALF supernatants and serum were stored at −80°C for subsequent cytokine analysis. Patients undergoing bronchoalveolar lavage for other clinical indications (sarcoidosis, tracheomalacia, and haemoptysis) served as controls. Blood PMNs were isolated using dextran sedimentation and plasma Percoll gradients. Neutrophil apoptosis was detected by flow cytometry using annexin V and propidium iodide. Kinetics of the PMN oxidative burst in response to ex-vivo stimulation with N-formyl-methionyl-leucylphenylalanine was determined with real-time horseradish peroxidase–luminol-dependent chemiluminescence detection. FindingsThe BALF from patients with ARDS consisted predominantly of alveolar PMNs (65%, SE 5), whereas alveolar macrophages constituted the major cell type (92%, SE 5) in BALF from control individuals. Nearly 70% of ARDS alveolar PMNs showed hypersegmented nuclear morphology and had a reduced rate of apoptosis after 20 h in culture (22% apoptotic PMNs, SE 10) compared with blood PMNs from control individuals (68% apoptotic PMNs, SE 7·2). ROS production by untreated alveolar neutrophils was 1·5 times higher compared with optimally (tumour necrosis factor α) ex vivo-primed ARDS blood PMNs from the same ARDS patient and control blood PMNs. This finding could reflect the alveolar cytokine milieu since concentrations of the proinflammatory mediators interleukin (IL) 1, IL6, IL8, IL10, IL12, and tumour necrosis factor α were significantly increased in BALF and serum from patients with ARDS compared with controls. InterpretationThese findings indicate that ARDS alveolar PMNs have altered morphology, enhanced survival, and augmented capacity for ROS generation, which might contribute to PMN-dependent tissue injury. FundingNIHR Cambridge Biomedical Research Centre, GlaxoSmithKline.

Cigarette Smoke Inhibits Engulfment of Apoptotic Cells by Macrophages through Inhibition of Actin Rearrangement

Exposure to cigarette smoke (CS) was shown to impair the capacity of macrophages to clear bacteria and apoptotic cells. Here, we show that both the exposure of macrophages to cigarette smoke extract (CSE) in vitro and an acute single exposure to CS in vivo impair the macrophage clearance of apoptotic polymorphonuclear leukocytes (PMNs). Upon longer periods of exposure to smoke in vivo (4-12 weeks), the impaired capacity of macrophages to clear apoptotic cells persisted after the cessation of smoking, with slow recovery to normality observed 4 weeks later. With respect to the mechanism by which CS impairs the macrophage uptake of apoptotic PMNs, we did not detect altered surface expression of receptors associated with apoptotic cell clearance. We did observe the impaired phosphorylation of the guanine nucleotide exchange factor Vav1 and the downstream inhibition of Ras-related C3 botulinum toxin substrate 1 (Rac1) activation. Consistent with these findings, CS impaired the macrophage cytoskeletal changes observed after stimulation with apoptotic cells. A loss of actin occurred at the leading edge, manifested as impaired ruffling of the cell membrane and a decreased capacity to engulf apoptotic cells. The inability to clear PMNs would lead to a greater release of destructive PMN products, and would diminish the reparative phenotype induced by the macrophage clearance of apoptotic cells.

Interaction of hypochlorous acid and myeloperoxidase with phosphatidylserine in the presence of ammonium ions

The close association of the heme enzyme myeloperoxidase to phosphatidylserine epitopes on the surface of non-vital polymorphonuclear leukocytes (PMNs) and other apoptotic cells at inflammatory sites favours modifications of this phospholipid by myeloperoxidase products. As detected by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, ammonium ions inhibit in a concentration-dependent manner the hypochlorous acid-mediated formation of aldehyde and nitrile products from 1,2-dipalmitoyl- sn-glycero-3-phosphoserine (DPPS). Concomitantly, the formation of monochloramine (NH 2Cl) raises with increasing NH 4 + concentrations. A transchlorination from monochlorinated O-phospho- l-serine to NH 4 + with the formation of NH 2Cl occurs only when extraordinary high NH 4 + concentrations are applied. Due to the low rate of 0.044 M − 1 s − 1 for this process, a transhalogenation reaction from transient chlorinated intermediates of the serine moiety to NH 4 + can be ruled out as an important process contributing to the HOCl-mediated formation of NH 2Cl. A significant formation of NH 2Cl by myeloperoxidase interacting with DPPS in the presence of ammonium ions takes only place at acidic pH values around 5, a scenario that may occur in phagosomes of macrophages after the uptake of apoptotic PMNs.

Dendritic cell activation by sensing Mycobacterium tuberculosis–induced apoptotic neutrophils via DC-SIGN

Mycobacterium tuberculosis (Mtb) manipulates cells of the innate immune system to provide the bacteria with a sustainable intracellular niche. Mtb spread through aerosol carrying them deep into the lungs, where they are internalized by phagocytic cells, such as neutrophils (PMNs), dendritic cells (DCs), and macrophages. PMNs undergo accelerated apoptosis after interaction with the bacterium, and apoptotic cells are sequestered by neighboring phagocytes. Removal of aged apoptotic cells because of natural tissue turnover is described as an immunologically silent process facilitating resolution of inflammation and inhibition of DC maturation. Silencing of immune cells could be favorable for intracellular bacteria. The aim of this study was to clarify the interaction between Mtb-induced apoptotic PMNs and DCs, and evaluate whether this interaction follows the proposed anti-inflammatory pathway. In contrast to aged apoptotic cells, Mtb-induced apoptotic PMNs induced functional DC maturation. We found that the cell fraction from Mtb-induced apoptotic PMNs contained almost all stimulatory capacity, suggesting that cell–cell interaction is crucial for DC activation. Inhibitory studies showed that this cell contact–dependent activation required binding of the PMN Mac-1 (CD11b/CD18) to the DC via DC-SIGN and endocytic activity involving the α vβ 5 but did not involve the scavenger receptor CD36. Taken together, this study demonstrates that the DCs can distinguish between normal and infected apoptotic PMNs via cellular crosstalk, where the DCs can sense the presence of danger on the Mtb-infected PMNs and modulate their response accordingly.

Resolvin D1 binds human phagocytes with evidence for proresolving receptors

Endogenous mechanisms that act in the resolution of acute inflammation are essential for host defense and the return to homeostasis. Resolvin D1 (RvD1), biosynthesized during resolution, displays potent and stereoselective anti-inflammatory actions, such as limiting neutrophil infiltration and proresolving actions. Here, we demonstrate that RvD1 actions on human polymorphonuclear leukocytes (PMNs) are pertussis toxin sensitive, decrease actin polymerization, and block LTB(4)-regulated adhesion molecules (beta2 integrins). Synthetic [(3)H]-RvD1 was prepared, which revealed specific RvD1 recognition sites on human leukocytes. Screening systems to identify receptors for RvD1 gave two candidates--ALX, a lipoxin A(4) receptor, and GPR32, an orphan--that were confirmed using a beta-arrestin-based ligand receptor system. Nuclear receptors including retinoid X receptor-alpha and peroxisome proliferator-activated receptor-alpha, -delta, -gamma were not activated by either resolvin E1 or RvD1 at bioactive nanomolar concentrations. RvD1 enhanced macrophage phagocytosis of zymosan and apoptotic PMNs, which increased with overexpression of human ALX and GPR32 and decreased with selective knockdown of these G-protein-coupled receptors. Also, ALX and GPR32 surface expression in human monocytes was up-regulated by zymosan and granulocyte-monocyte-colony-stimulating factor. These results indicate that RvD1 specifically interacts with both ALX and GPR32 on phagocytes and suggest that each plays a role in resolving acute inflammation.

Abnormalities in circulating plasmacytoid dendritic cells in patients with systemic lupus erythematosus

IntroductionDendritic cells (DCs) are capable of inducing immunity or tolerance. Previous studies have suggested plasmacytoid DCs (pDCs) are pathogenic in systemic lupus erythematosus (SLE). However, the functional characteristics of directly isolated peripheral circulating blood pDCs in SLE have not been evaluated previously.MethodsPeripheral blood pDCs from 62 healthy subjects and 58 SLE patients were treated with apoptotic cells derived from polymorphonuclear cells (PMNs). Antigen loaded or unloaded pDCs were then co-cultured with autologous or allogenous T cells. Changes in T cell proliferation, cell surface CD25 expression, intracellular Foxp3 expression and cytokine production were evaluated. pDCs that had captured apoptotic PMNs (pDCs + apoPMNs were also studied for their cytokine production (interferon (IFN)-alpha, interleukin (IL)-6, IL-10, IL-18) and toll like receptor (TLR) expression.ResultsCirculating pDCs from SLE patients had an increased ability to stimulate T cells when compared with control pDCs. Using allogenous T cells as responder cells, SLE pDCs induced T cell proliferation even in the absence of apoptotic PMNs. In addition, healthy pDCs + apoPMNs induced suppressive T regulatory cell features with increased Foxp3 expression in CD4 + CD25 + cells while SLE pDCs + apoPMNs did not. There were differences in the cytokine profile of pDCs that had captured apoptotic PMNs between healthy subjects and patients with SLE. Healthy pDCs + apoPMNs showed decreased production of IL-6 but no significant changes in IL-10 and IL-18. These pDCs + apoPMNs also showed increased mRNA transcription of TLR9. On the other hand, while SLE pDCs + apoPMNs also had decreased IL-6, there was decreased IL-18 mRNA expression and persistent IL-10 protein synthesis. In addition, SLE pDCs lacked TLR9 recruitment.ConclusionsWe have demonstrated that peripheral circulating pDCs in patients with SLE were functionally abnormal. They lacked TLR9 expression, were less capable of inducing regulatory T cell differentiation and had persistent IL-10 mRNA expression following the capture of apoptotic PMNs. We suggest circulating pDCs may be pathogenically relevant in SLE.

Disease-causing Mutations in the Cystic Fibrosis Transmembrane Conductance Regulator Determine the Functional Responses of Alveolar Macrophages

Alveolar macrophages (AMs) play a major role in host defense against microbial infections in the lung. To perform this function, these cells must ingest and destroy pathogens, generally in phagosomes, as well as secrete a number of products that signal other immune cells to respond. Recently, we demonstrated that murine alveolar macrophages employ the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel as a determinant in lysosomal acidification (Di, A., Brown, M. E., Deriy, L. V., Li, C., Szeto, F. L., Chen, Y., Huang, P., Tong, J., Naren, A. P., Bindokas, V., Palfrey, H. C., and Nelson, D. J. (2006) Nat. Cell Biol. 8, 933-944). Lysosomes and phagosomes in murine cftr(-/-) AMs failed to acidify, and the cells were deficient in bacterial killing compared with wild type controls. Cystic fibrosis is caused by mutations in CFTR and is characterized by chronic lung infections. The information about relationships between the CFTR genotype and the disease phenotype is scarce both on the organismal and cellular level. The most common disease-causing mutation, DeltaF508, is found in 70% of patients with cystic fibrosis. The mutant protein fails to fold properly and is targeted for proteosomal degradation. G551D, the second most common mutation, causes loss of function of the protein at the plasma membrane. In this study, we have investigated the impact of CFTR DeltaF508 and G551D on a set of core intracellular functions, including organellar acidification, granule secretion, and microbicidal activity in the AM. Utilizing primary AMs from wild type, cftr(-/-), as well as mutant mice, we show a tight correlation between CFTR genotype and levels of lysosomal acidification, bacterial killing, and agonist-induced secretory responses, all of which would be expected to contribute to a significant impact on microbial clearance in the lung.