P38 and JNK have opposing effects on persistence of in vivo leukocyte migration in zebrafish.

The recruitment and migration of macrophages and neutrophils is an important process during the early stages of the innate immune system in response to acute injury. Transgenic pu.1:EGFP zebrafish permit the acquisition of leukocyte migration trajectories during inflammation. Currently, these high-quality live-imaging data are mainly analysed using general statistics, for example, cell velocity. Here, we present a spatio-temporal analysis of the cell dynamics using transition matrices, which provide information of the type of cell migration. We find evidence that leukocytes exhibit types of migratory behaviour, which differ from previously described random walk processes. Dimethyl sulfoxide treatment decreased the level of persistence at early time points after wounding and ablated temporal dependencies observed in untreated embryos. We then use pharmacological inhibition of p38 and c-Jun N-terminal kinase mitogen-activated protein kinases to determine their effects on in vivo leukocyte migration patterns and discuss how they modify the characteristics of the cell migration process. In particular, we find that their respective inhibition leads to decreased and increased levels of persistent motion in leukocytes following wounding. This example shows the high level of information content, which can be gained from live-imaging data if appropriate statistical tools are used.

Apoptotic human cells inhibit migration of granulocytes via release of lactoferrin.

Apoptosis is a noninflammatory, programmed form of cell death. One mechanism underlying the non-phlogistic nature of the apoptosis program is the swift phagocytosis of the dying cells. How apoptotic cells attract mononuclear phagocytes and not granulocytes, the professional phagocytes that accumulate at sites of inflammation, has not been determined. Here, we show that apoptotic human cell lines of diverse lineages synthesize and secrete lactoferrin, a pleiotropic glycoprotein with known antiinflammatory properties. We further demonstrated that lactoferrin selectively inhibited migration of granulocytes but not mononuclear phagocytes, both in vitro and in vivo. Finally, we were able to attribute this antiinflammatory function of lactoferrin to its effects on granulocyte signaling pathways that regulate cell adhesion and motility. Together, our results identify lactoferrin as an antiinflammatory component of the apoptosis milieu and define what we believe to be a novel antiinflammatory property of lactoferrin: the ability to function as a negative regulator of granulocyte migration.

Selective inhibitors of Kv11.1 regulate IL-6 expression by macrophages in response to TLR/IL-1R ligands.

The mechanism by which the platelet-endothelial cell adhesion molecule PECAM-1 regulates leukodiapedesis, vascular endothelial integrity, and proinflammatory cytokine expression in vivo is not known. We recently identified PECAM-1 as a negative regulator of Kv11.1, a specific voltage-gated potassium channel that functioned in human macrophages to reset a resting membrane potential following depolarization. We demonstrate here that dofetilide (DOF), a selective inhibitor of the Kv11.1 current, had a profound inhibitory effect on neutrophil recruitment in mice following TLR/IL-1R-elicited peritonitis or intrascrotal injection of IL-1 Beta, but had no effect on responses seen with TNF alpha. Furthermore, inhibitors of Kv11.1 (DOF, E4031, and astemizole), but not Kv1.3 (margatoxin), suppressed the expression of IL-6 and MCP-1 cytokines by murine resident peritoneal macrophages, while again having no effect on TNF alpha. In contrast, IL-6 expression by peritoneal mesothelial cells was unaffected. Using murine P388 cells, which lack endogenous C/EBP Beta expression and are unresponsive to LPS for the expression of both IL-6 and MCP-1, we observed that DOF inhibited LPS-induced expression of IL-6 mRNA following ectopic expression of wild-type C/EBP Beta, but not a serine-64 point mutant. Finally, DOF inhibited the constitutive activation of cdk2 in murine peritoneal macrophages; cdk2 is known to phosphorylate C/EBP Beta at serine-64. Taken together, our results implicate a potential role for Kv11.1 in regulating cdk2 and C/EBP Beta activity, where robust transactivation of both IL-6 and MCP-1 transcription is known to be dependent on serine-64 of C/EBP Beta. Our data might also explain the altered phenotypes displayed by PECAM-1 knockout mice in several disease models.

Compartmentalized megakaryocyte death generates functional platelets committed to caspase-independent death.

Caspase-directed apoptosis usually fragments cells, releasing nonfunctional, prothrombogenic, membrane-bound apoptotic bodies marked for rapid engulfment by macrophages. Blood platelets are functional anucleate cells generated by specialized fragmentation of their progenitors, megakaryocytes (MKs), but committed to a constitutive caspase-independent death. Constitutive formation of the proplatelet-bearing MK was recently reported to be caspase-dependent, apparently involving mitochondrial release of cytochrome c, a known pro-apoptogenic factor. We extend those studies and report that activation of caspases in MKs, either constitutively or after Fas ligation, yields platelets that are functionally responsive and evade immediate phagocytic clearance, and retain mitochondrial transmembrane potential until constitutive platelet death ensues. Furthermore, the exclusion from the platelet progeny of caspase-9 present in the progenitor accounts for failure of mitochondrial release of cytochrome c to activate caspase-3 during platelet death. Thus, progenitor cell death by apoptosis can result in birth of multiple functional anucleate daughter cells.

Class III antiarrhythmic methanesulfonanilides inhibit leukocyte recruitment in zebrafish.

Understanding fundamental molecular mechanisms that govern the transmigration and interstitial migration of leukocytes to sites of tissue damage and infection is of potential significance in identifying novel therapeutic targets for the management of chronic inflammatory disorders. CD31 is a mammalian cell adhesion molecule that regulates the recruitment of leukocytes from the circulation. Our recent unpublished work has suggested that homophilic ligation of CD31 can negatively regulate the ether-à-go-go-related gene (ERG) current within leukocytes to regulate cell-cell adhesion. To validate and probe the functional significance of ERG in leukocytes, we developed an infected wound model of inflammation in zebrafish and assessed the efficacy of two ERG-specific inhibitors, dofetilide and E4031, as well as an ERG-specific antisense RNA morpholino on neutrophil recruitment. Our data confirm a hitherto undescribed role for ERG in leukocytes, where inhibition or translational knockdown of ERG resulted in significant attenuation of the inflammatory response to an infectious stimulus. Inhibition of ERG was verified independently by a decrease in the ventricular heart rate, where ERG also functions in the repolarization of the cardiac action potential. Our results suggest that ERG-specific Class III antiarrhythmic drugs can modulate inflammatory responses to infection.

CD31 delays phagocyte membrane repolarization to promote efficient binding of apoptotic cells.

Homophilic ligation of CD31, a member of the Ig superfamily of adhesion receptors, promotes macrophage clearance of apoptotic leukocytes by a mechanism hitherto not described. In studying CD31-dependent regulation of beta1-integrin binding of fibronectin-coated Latex beads, we discovered a role for the voltage-gated potassium channel ether-à-go-go-related gene (ERG) as a downstream effector of CD31 signaling. ERG was identified by tandem mass spectrometry as a 140-kDa protein, which was selectively modified with biotin following the targeted delivery of a biotin-transfer reagent to CD31 using Fab fragments of an anti-CD31 mAb. Similar results were obtained with macrophages but not K562 cells, expressing a truncated cytoplasmic tail of CD31, which failed to regulate bead binding. Colocalization of CD31 with ERG was confirmed by immunofluorescence for K562 cells and macrophages. We now demonstrate that the resting membrane potential of macrophages is depolarized on contact with apoptotic cells and that CD31 inhibits the ERG current, which would otherwise function to repolarize. Sustained depolarization favored the firm binding of phagocytic targets, a prerequisite for efficient engulfment. Our results identify ERG as a downstream effector of CD31 in the regulation of integrin-dependent binding of apoptotic cells by macrophages.

Electric fields and inflammation: may the force be with you.

Integrins are a family of ubiquitous cell surface receptors comprising heterodimers of alpha and beta chains that are required for cell adhesion and motility. Integrin-dependent adhesion and signaling is associated with major conformational changes in the ectodomain as it shifts from a low-affinity "bent" to a high-affinity "extended" structure. The ability of a cell to regulate dynamically the affinity or activation state of an integrin, and hence its binding to extracellular matrix or cell adhesion molecules, is assumed to be driven by intracellular signaling events transmitted by protein binding to the cytoplasmic tail. The binding of an integrin to its ligand can then transmit signals back into the cell to regulate the formation of a macromolecular focal adhesion complex that effectively anchors the cytoskeleton to the adhesion site. Many proteins have been reported to associate physically and functionally with integrins, leading to altered signaling events. A particularly intriguing molecular association exists between integrins and transmembrane proteins that gate the movement of charge, especially voltage-gated potassium channels, although the significance of this interaction is not understood. Although ample evidence indicates that the engagement of integrins can promote potassium efflux by both excitable and nonexcitable cells, we speculate the converse, that the activation state of integrins is dynamically regulated by changes in a transmembrane potential. In this way, direct-current electric fields generated at a site of tissue injury can promote the galvanotaxis or directed migration of cells involved in tissue repair and inflammation.

CD31 promotes beta1 integrin-dependent engulfment of apoptotic Jurkat T lymphocytes opsonized for phagocytosis by fibronectin.

Phagocyte integrins, by binding "bridging" molecules, mediate the ingestion of late apoptotic cells and apoptotic bodies by mechanisms that remain obscure. We recently reported that human monocyte-derived macrophages capture viable and apoptotic human leukocytes through homophilic interactions involving CD31 and that CD31 then promotes the engulfment of apoptotic cells or the detachment of viable cells. We now report that CD31 homophilic interactions between phagocyte and target cells lead to activation of phagocyte alpha5beta1 integrin and the engulfment of apoptotic Jurkat T lymphocytes via a fibronectin (Fn) "bridge." Although Fn and serum served as an opsonin for beta1 integrin-dependent phagocytosis of apoptotic leukemic T cells, they failed to do so for neutrophils. Given the complexities and inherent variability of working with primary cells, we have refined our model to show that ligation of CD31 on THP-1 macrophages also regulates beta1 integrin-dependent phagocytosis of Fn-coated Latex beads. Thus, selective "tethering" of apoptotic leukocytes by phagocyte CD31 not only discriminates dying from viable cells but also selectively activates phagocyte integrins for the engulfment of apoptotic cells.

Feeding filaggrin: effects of l-histidine supplementation in atopic dermatitis.

Atopic dermatitis (AD), also known as eczema, is one of the most common chronic skin conditions worldwide, affecting up to 16% of children and 10% of adults. It is incurable and has significant psychosocial and economic impacts on the affected individuals. AD etiology has been linked to deficiencies in the skin barrier protein, filaggrin. In mammalian skin, l-histidine is rapidly incorporated into filaggrin. Subsequent filaggrin proteolysis releases l-histidine as an important natural moisturizing factor (NMF). In vitro studies were conducted to investigate the influence of l-histidine on filaggrin processing and barrier function in human skin-equivalent models. Our further aim was to examine the effects of daily oral l-histidine supplementation on disease severity in adult AD patients. We conducted a randomized, double-blind, placebo-controlled, crossover, nutritional supplementation pilot study to explore the effects of oral l-histidine in adult AD patients (n=24). In vitro studies demonstrated that l-histidine significantly increased both filaggrin formation and skin barrier function (P<0.01, respectively). Data from the clinical study indicated that once daily oral l-histidine significantly reduced (P<0.003) AD disease severity by 34% (physician assessment using the SCORingAD tool) and 39% (patient self-assessment using the Patient Oriented Eczema Measure tool) after 4 weeks of treatment. No improvement was noted with the placebo (P>0.32). The clinical effect of oral l-histidine in AD was similar to that of mid-potency topical corticosteroids and combined with its safety profile suggests that it may be a safe, nonsteroidal approach suitable for long-term use in skin conditions that are associated with filaggrin deficits such as AD.

The death of human platelets during incubation in citrated plasma involves shedding of CD42b and aggregation of dead platelets.

The ability to readily identify dead platelets is invaluable to studies examining the means of their death, factors affecting their lifespan and their means of clearance by phagocytes. The aim of the present work was to develop a vital staining procedure for the rapid and objective discrimination of live from dead platelets that accrued in citrated platelet rich plasma (cPRP) incubated at 37 degrees C for several days. By transmission electron microscopy it was noted that platelet death was morphologically similar to necrosis and associated with aggregate formation. The vital dyes calcein-AM and FM 4-64 were found to robustly report the death of platelets and indicated that the aggregates which formed during incubation were populated exclusively by dead platelets. Additionally, platelet death was associated with the shedding of CD42b. Microscopic and cytometric analyses of incubated cPRP indicated that shedding of CD42b and aggregate formation by dead platelets were completely inhibited by the metalloproteinase inhibitor GM6001. Automated counting of platelets incubated in the presence of GM6001 revealed that death did not lead to a loss in cellularity. It is proposed that calcein-AM and FM4-64 are effective as vital stains for the reliable assessment of platelet viability and that platelet aggregation can occur by a novel mechanism dependent upon platelet death and metalloproteinase activity.

Promoting apoptosis in disease management: a panacea or Trojan horse?

In our enthusiasm to advocate apoptosis as a therapeutic strategy for the management of disease we need to be mindful that the clearance of apoptotic cells is itself immunomodulatory and that it may not always be as benign or beneficial as we think. Indeed, the existence of free apoptotic cells in situ may potentially be pathological, and not necessarily physiological, and any attempt to promote apoptosis in the absence of an appropriate phagocytic response for the treatment of, for example, inflammation or cancer might exacerbate or initiate an autoimmune pathology.

Agonists of toll-like receptor (TLR)2 and TLR4 are unable to modulate platelet activation by adenosine diphosphate and platelet activating factor.

Inappropriate platelet activation is a feature of acute and chronic diseases such as disseminated intravascular coagulation (DIC) and atherosclerosis. Since proinflammatory microbial-derived agonists can be involved in the pathogenesis of these diseases, we examined the potential role of TLR4 (mediating responses to LPS) and TLR2 (which responds to bacterial lipopeptides) in platelet activation. Our data suggested low-level expression of TLR2 and TLR4 on platelets, determined by flow cytometry, and we also observed expression of TLR4 on a megakaryocytic cell line by both flow cytometry and immunohistochemistry. Stimulation of the platelets with the TLR4 agonist LPS, and the synthetic TLR2 agonist Pam3CSK4, resulted in no platelet aggregation, no increase in CD62P surface expression and no increase in the cytosolic concentration of Ca2+. The TLR agonists were also unable to directly activate platelets primed with epinephrine, or pretreated with a low concentration of ADP or PAF. Pretreatment of platelets with LPS or Pam3CSK4 also failed to modulate the platelet response to submaximal concentrations of the classical platelet agonists ADP and PAF. We conclude that the TLR agonists LPS and Pam3CSK4 have no direct effect on platelet activation and that platelet TLRs may be a remnant from megakaryocytes. TLR2 and TLR4 agonists are thought to have a significant role in diseases such as atherosclerosis and DIC, but our research suggests that this is through a mechanism other than direct platelet activation or by modification of platelet responses to other agonists.

A role for potassium permeability in the recognition, clearance, and anti-inflammatory effects of apoptotic cells.

The benefits of programmed cell death by apoptosis are the safe and efficient clearance of damaged, infected, or surplus cells, primarily mediated by tissue-resident macrophages or tissue-infiltrating blood monocytes that differentiate into macrophages. Microglial cells are macrophages of the brain parenchyma, important immune surveillance cells that respond to various injuries and diseases of the brain. It is often stated that how a macrophage interacts with an apoptotic cell defines subsequent inflammatory responses, i.e., will engulfment be beneficial or detrimental for tissue repair, regeneration, and immunity. Our focus has been to better understand how macrophages discriminate between living and dying cells. Following our initial findings with platelet endothelial cell adhesion molecule (PECAM)-1, our studies have revealed a key role for potassium ion permeability in regulating integrin-dependent binding of apoptotic cells by macrophages and their subsequent response to proinflammatory stimuli. Specifically, apoptotic cells represent a depolarizing stimulus for macrophages where PECAM-1-mediated cell-cell interactions delay subsequent membrane repolarization. It is salient that potassium leak represents an early feature of cells destined to die by apoptosis that could trigger depolarization of macrophages that lie in close apposition. We speculate that how a tissue-resident macrophage responds to strong depolarizing stimuli has wider implications for inflammation and autoimmunity.

Skin exposure to micro- and nano-particles can cause haemostasis in zebrafish larvae.

Low mass ambient exposure to airborne particles is associated with atherothrombotic events that may be a consequence of the combustion-derived nanoparticle content. There is concern also over the potential cardiovascular impact of manufactured nanoparticles. To better understand the mechanism by which toxic airborne particles can affect cardiovascular function we utilised zebrafish as a genetically tractable model. Using light and confocal fluorescence video-microscopy, we measured heart-rate and blood flow in the dorsal aorta and caudal artery of zebrafish larvae that had been exposed to a number of toxic and non-toxic microparticles and nanoparticles. Diesel exhaust particles (DEP), carboxy-charged Latex beads (carboxy-beads) and toxic alumina (Taimicron TM300), but not non-toxic alumina (Baikalox A125), were found to promote both skin and gut cell damage, increased leukocyte invasion into the epidermis, tail muscle ischaemia and haemostasis within the caudal artery of free swimming zebrafish larvae. The presence of sodium sulfite, a reducing agent, or warfarin, an anticoagulant, within the system water abrogated the effects of both toxic alumina and carboxy-beads but not DEP. Genetic manipulation of skin barrier function augmented skin damage and haemostasis, even for the non-toxic alumina. The toxic effects of carboxy-beads were still apparent after leukocyte numbers were depleted with anti-Pu.1 morpholino. We conclude that particle uptake across skin epithelium and gut mucosal barriers, or the presence of leukocytes, is not required for particle-induced haemostasis while a compromised skin barrier function accentuated tissue injury and haemostasis.

Hypoglycaemia predisposes platelets to death by affecting calcium homeostasis and mitochondrial integrity.

Factors affecting platelet survival are poorly understood. To explore the hypothesis that platelet lifespan correlates with the lifespan of a key housekeeping process we subjected human platelets to in vitro incubation at 37 degrees C for 24 h to several days under hypoglycaemic conditions. Viability was assessed both by microscopy and flow cytometry using calcein-AM and/or FM4-64. In keeping with previous data we found that, under control conditions platelets died at a linear rate during 120 h of incubation. Hypoglycaemia did not affect the death rate but did lead to an increase in the frequency of platelets unable to accumulate the mitochondrial potentiometric dye 10-Nonyl Acridine Orange (NAO) and promoted platelet death in response to the pro-apoptotic molecule BH3I-2'. Hypoglycaemia led to an increase in intraplatelet calcium that could be prevented 2-aminoethoxydiphenylborate (2-APB), a store operated calcium channel (SOCC) blocker. However, this agent was unable to rescue the platelets' ability to accumulate NAO. These data suggest that extracellular glucose is utilised by platelets for calcium homeostasis and maintenance of mitochondrial integrity and that hypoglycaemia primes platelets for death.

Phagocytosis of apoptotic cells by human macrophages: analysis by multiparameter flow cytometry.

BACKGROUND: Phagocytic removal of apoptotic cells is an important regulatory event in development, tissue homoeostasis, and inflammation. There are several methodologic problems with most in vitro studies of the molecular mechanisms of apoptotic cell phagocytosis. First, cell loss occurs during rigorous washing of adherent macrophages required to ensure removal of noningested particles. Second, discrimination of adherent or internalised apoptotic cells is difficult. Third, microscopic quantification is time consuming and has the potential for significant interobserver error. Fourth, subsequent analysis of phagocyte populations is difficult. METHODS: We used a flow cytometric method that allows quantification of phagocytosis of fluorescently labelled apoptotic cells with the use of multiparameter flow cytometric analysis. RESULTS: Phagocytosis of apoptotic cells was validated by use of inhibitors (cytochalasins) or low temperature and counterstaining with cell surface markers for the phagocytic targets to exclude binding to the phagocytic surface. Populations of phagocytic macrophages were sorted, and the presence of internalized apoptotic material was validated by microscopy. CONCLUSIONS: The technique we used in this study allows observer-independent analysis of phagocytosis of apoptotic cells by macrophages. Importantly, phagocytic or nonphagocytic populations could be subjected to further characterization with the use of flow cytometry with additional fluorochrome reagents and can be re-cultured to study underlying regulatory mechanisms.