Plasma Membrane Of Leukocytes Research Articles

Semi-Refined Carrageenan Attenuates Lipopolysaccharide-Induced Reactive Oxygen Species Production in Peripheral Blood Mononuclear Cells and Cell Membrane Alterations in Leukocytes

Aim: To assess the effects of semi-refined carrageenan (E407a) on lipopolysaccharide (LPS)-induced reactive oxygen species (ROS) generation in peripheral blood mononuclear cells (PBMCs) and LPS-mediated cell membrane alterations in leukocytes. Methods: Blood samples collected from 8 intact rats were incubated with E407a (10 mg/ml), E407a (50 mg/ml), LPS (1 µg/ml), E407a (10 mg/ml) + LPS (1 µg/ml), E407a (50 mg/ml) + LPS (1 µg/ml) and without those compounds (controls) for 2 h in RPMI 1640 medium enriched with 5% fetal bovine serum. ROS generation in PBMCs obtained from the incubated samples was estimated by flow cytometry using 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) staining. The impact of E407a, LPS and their mixture on leukocyte cell membranes was evaluated spectrofluorimetrically using the fluorescent probe 2-(2¢-hydroxy-phenyl)-5-phenyl-1,3-oxazole. Results: Expectedly, incubation with LPS induced ROS generation in PBMCs and decreased the lipid order of cell membranes in leukocytes. E407a alone was found to alter neither ROS production in PBMCs, nor membrane lipid order in leukocytes. Semi-refined carrageenan partially reduced LPS-mediated ROS overproduction in PBMCs and cell membrane alterations in leukocytes. Conclusion: E407a attenuates LPS-induced alterations of redox homeostasis in rat PBMCs and LPS-mediated modifications of cell membrane lipid order in leukocytes.

Experimental Evaluation of Food-Grade Semi-Refined Carrageenan Toxicity.

The safety of food additives E407 and E407a has raised concerns in the scientific community. Thus, this study aims to assess the local and systemic toxic effects of the common food additive E407a in rats orally exposed to it for two weeks. Complex evaluations of the effects of semi-refined carrageenan (E407a) on rats upon oral exposure were performed. Local effects of E407a on the intestine were analyzed using routine histological stains and CD68 immunostaining. Furthermore, circulating levels of inflammatory markers were assessed. A fluorescent probe O1O (2- (2′-OH-phenyl)-5-phenyl-1,3-oxazole) was used for evaluating the state of leukocyte cell membranes. Cell death modes of leukocytes were analyzed by flow cytometry using Annexin V and 7-aminoactinomycin D staining. Oral administration of the common food additive E407a was found to be associated with altered small and large intestinal morphology, infiltration of the lamina propria in the small intestine with macrophages (CD68+ cells), high systemic levels of inflammation markers, and changes in the lipid order of the phospholipid bilayer in the cell membranes of leukocytes, alongside the activation of their apoptosis. Our findings suggest that oral exposure to E407a through rats results in the development of intestinal inflammation.

Allylic Selenocyanates as Antifungal Agents Against Pathogenic Candida Species

AbstractCandida is a genus that causes the highest number of fungal infections on people around the world, responsible for high mortality rates in critical and immunocompromised patients. Even though several antifungals are commercially available, most of these have side effects or are only available for intravenous administration. In view of this aspect, the susceptibility of twelve allylic selenocyanate (AS) compounds, five of them novel, were tested against 36 Candida strains. The AS were active for all Candida strains reaching minimal inhibitory concentrations (MIC) in the order of ng mL−1 (0.39–50 μg mL−1). The sorbitol and ergosterol assay show that these compounds do not act on the cell wall and fungal membrane, suggesting a different mechanism of action. Cytotoxicity and irritability tests showed that only three molecules already studied (2 b, 2 j and 2 i) for this series manifested damage to the leukocyte cell and chorioallantoic membrane, but at higher concentrations than the MICs. Considering the susceptibility results and parameters of Lipinski, molecules 2 c (0.39–12.5 μg mL−1) and 2 d (0.78–6.2 μg mL−1) look promising for the development of a new antifungal agent against Candida infections.

Membrane Compartmentalization and Scaffold Proteins in Leukocyte Migration.

Leukocyte migration across vessels into and within peripheral and lymphoid tissues is essential for host defense against invading pathogens. Leukocytes are specialized in sensing a variety of guidance cues and to integrate environmental stimuli to navigate in a timely and spatially controlled manner. These extracellular signals must be transmitted across the leukocyte’s plasma membrane in a way that intracellular signaling cascades enable directional cell movement. Therefore, the composition of the membrane in concert with proteins that influence the compartmentalization of the plasma membrane or contribute to delineate intracellular signaling molecules are key in controlling leukocyte navigation. This becomes evident by the fact that mislocalization of membrane proteins is known to deleteriously affect cellular functions that may cause diseases. In this review we summarize recent advances made in the understanding of how membrane cholesterol levels modulate chemokine receptor signaling and hence leukocyte trafficking. Moreover, we provide an overview on the role of membrane scaffold proteins, particularly tetraspanins, flotillins/reggies, and caveolins in controlling leukocyte migration both in vitro and in vivo.

Profiling the expression and function of oestrogen receptor isoform ER46 in human endometrial tissues and uterine natural killer cells.

STUDY QUESTIONDoes the oestrogen receptor isoform, ER46, contribute to regulation of endometrial function?SUMMARY ANSWERER46 is expressed in endometrial tissues, is the predominant ER isoform in first trimester decidua and is localised to the cell membrane of uterine natural killer (uNK) cells where activation of ER46 increases cell motility.WHAT IS KNOWN ALREADYOestrogens acting via their cognate receptors are essential regulators of endometrial function and play key roles in establishment of pregnancy. ER46 is a 46-kDa truncated isoform of full length ERα (ER66, encoded by ESR1) that contains both ligand- and DNA-binding domains. Expression of ER46 in the human endometrium has not been investigated previously. ER46 is located at the cell membrane of peripheral blood leukocytes and mediates rapid responses to oestrogens. uNK cells are a phenotypically distinct (CD56brightCD16−) population of tissue-resident immune cells that regulate vascular remodelling within the endometrium and decidua. We have shown that oestrogens stimulate rapid increases in uNK cell motility. Previous characterisation of uNK cells suggests they are ER66-negative, but expression of ER46 has not been characterised. We hypothesise that uNK cells express ER46 and that rapid responses to oestrogens are mediated via this receptor.STUDY DESIGN, SIZE, DURATIONThis laboratory-based study used primary human endometrial (n = 24) and decidual tissue biopsies (n = 30) as well as uNK cells which were freshly isolated from first trimester human decidua (n = 18).PARTICIPANTS/MATERIALS, SETTING, METHODSPrimary human endometrial and first trimester decidual tissue biopsies were collected using methods approved by the local institutional ethics committee (LREC/05/51104/12 and LREC/10/51402/59). The expression of ERs (ER66, ER46 and ERβ) was assessed by quantitative PCR, western blot and immunohistochemistry. uNK cells were isolated from first-trimester human decidua by magnetic bead sorting. Cell motility of uNK cells was measured by live cell imaging: cells were treated with 17β-oestradiol conjugated to bovine serum albumin (E2-BSA, 10 nM equivalent), the ERβ-selective agonist 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN; 10 nM) or dimethylsulphoxide vehicle control.MAIN RESULTS AND THE ROLE OF CHANCEER46 was detected in proliferative and secretory phase tissues by western blot and was the predominant ER isoform in first-trimester decidua samples. Immunohistochemistry revealed that ER46 was co-localised with ER66 in cell nuclei during the proliferative phase but detected in both the cytoplasm and cell membrane of stromal cells in the secretory phase and in decidua. Triple immunofluorescence staining of decidua tissues identified expression of ER46 in the cell membrane of CD56-positive uNK cells which were otherwise ER66-negative. Profiling of isolated uNK cells confirmed expression of ER46 by quantitative PCR and western blot and localised ER46 protein to the cell membrane by immunocytochemistry. Functional analysis of isolated uNK cells using live cell imaging demonstrated that activation of ER46 with E2-BSA significantly increased uNK cell motility.LARGE SCALE DATAN/A.LIMITATIONS, REASONS FOR CAUTIONExpression pattern in endometrial tissue was only determined using samples from proliferative and secretory phases. Assessment of first trimester decidua samples was from a range of gestational ages, which may have precluded insights into gestation-specific changes in these tissues. Our results are based on in vitro responses of primary human cells and we cannot be certain that similar mechanisms occur in situ.WIDER IMPLICATIONS OF THE FINDINGSE2 is an essential regulator of reproductive competence. This study provides the first evidence for expression of ER46 in the human endometrium and decidua of early pregnancy. We describe a mechanism for regulating the function of human uNK cells via expression of ER46 and demonstrate that selective targeting with E2-BSA regulates uNK cell motility. These novel findings identify a role for ER46 in the human endometrium and provide unique insight into the importance of membrane-initiated signalling in modulating the impact of E2 on uNK cell function in women. Given the importance of uNK cells to regulating vascular remodelling in early pregnancy and the potential for selective targeting of ER46, this may be an attractive future therapeutic target in the treatment of reproductive disorders.STUDY FUNDING/COMPETING INTEREST(S)These studies were supported by Medical Research Council (MRC) Programme Grants G1100356/1 and MR/N024524/1 to PTKS. H.O.D.C. was supported by MRC grant G1002033. The authors declare no competing interests related to the published work.

Role of neuroendocrine modulation and biochemistry in the sepsis in Piaractus mesopotamicus

Sepsis is a systemic process with multifactorial pathophysiology that affects most animal species. It is responsible for high rates of morbidity and mortality. This work aimed to study the biochemical and neuroendocrine changes of the sepsis process in Piaractus mesopotamicus after Aeromonas hydrophila inoculation analyzing changes in blood leukocyte and differences in neuroendocrine-biochemical modulation using RNA-seq. Fish showed hypercortisolemia, inhibition of glucose absorption, followed by hypocortisolemia and then hyperglycemia. Thyroid hormones (T3 and T4) showed immediate decrease in serum and T4 increased 6 h post-inoculation (HPI). Sepsis-induced hormonal alterations triggered changes in the metabolic pathways increasing protein and lipid catabolism, use of transient anaerobic glycolysis and liver injury. A reference transcriptome was constructed based on blood leukocytes from P. mesopotamicus. The assembly resulted in total 266,272 contigs with a N50 of 2786 bp. There was a reorganization of plasma membrane of leukocytes at the beginning of the septic process with increased expression of neuroendocrine receptors and with continuous flow of neurotransmitters, hormones and solutes with compensatory regulation at 6 HPI. Three and nine HPI seemed to be critical, the expression of a number of transcription factors was increased, including the modulatory DEGs related to glucocorticoid and thyroid hormones induced and suppressed (FDR < 0.05). Neuroendocrine modulation can regulate leukocytes and biochemical parameters of peripheral blood, being important sources for the study of the pathophysiology of sepsis. These finding highlights the importance of further studies focusing on biochemical-neuroendocrine changes in blood leukocytes and systemic sepsis.

Influence of Chemokine N-Terminal Modification on Biased Agonism at the Chemokine Receptor CCR1.

Leukocyte migration, a hallmark of the inflammatory response, is stimulated by the interactions between chemokines, which are expressed in injured or infected tissues, and chemokine receptors, which are G protein-coupled receptors (GPCRs) expressed in the leukocyte plasma membrane. One mechanism for the regulation of chemokine receptor signaling is biased agonism, the ability of different chemokine ligands to preferentially activate different intracellular signaling pathways via the same receptor. To identify features of chemokines that give rise to biased agonism, we studied the activation of the receptor CCR1 by the chemokines CCL7, CCL8, and CCL15(Δ26). We found that, compared to CCL15(Δ26), CCL7 and CCL8 exhibited biased agonism towards cAMP inhibition and away from β-Arrestin 2 recruitment. Moreover, N-terminal substitution of the CCL15(Δ26) N-terminus with that of CCL7 resulted in a chimera with similar biased agonism to CCL7. Similarly, N-terminal truncation of CCL15(Δ26) also resulted in signaling bias between cAMP inhibition and β-Arrestin 2 recruitment signals. These results show that the interactions of the chemokine N-terminal region with the receptor transmembrane region play a key role in selecting receptor conformations coupled to specific signaling pathways.

Evaluation and extension of the two-site, two-step model for binding and activation of the chemokine receptor CCR1

Interactions between secreted immune proteins called chemokines and their cognate G protein-coupled receptors regulate the trafficking of leukocytes in inflammatory responses. The two-site, two-step model describes these interactions. It involves initial binding of the chemokine N-loop/β3 region to the receptor's N-terminal region and subsequent insertion of the chemokine N-terminal region into the transmembrane helical bundle of the receptor concurrent with receptor activation. Here, we test aspects of this model with C-C motif chemokine receptor 1 (CCR1) and several chemokine ligands. First, we compared the chemokine-binding affinities of CCR1 with those of peptides corresponding to the CCR1 N-terminal region. Relatively low affinities of the peptides and poor correlations between CCR1 and peptide affinities indicated that other regions of the receptor may contribute to binding affinity. Second, we evaluated the contributions of the two CCR1-interacting regions of the cognate chemokine ligand CCL7 (formerly monocyte chemoattractant protein-3 (MCP-3)) using chimeras between CCL7 and the non-cognate ligand CCL2 (formerly MCP-1). The results revealed that the chemokine N-terminal region contributes significantly to binding affinity but that differences in binding affinity do not completely account for differences in receptor activation. On the basis of these observations, we propose an elaboration of the two-site, two-step model-the "three-step" model-in which initial interactions of the first site result in low-affinity, nonspecific binding; rate-limiting engagement of the second site enables high-affinity, specific binding; and subsequent conformational rearrangement gives rise to receptor activation.

Biomimetic carriers mimicking leukocyte plasma membrane to increase tumor vasculature permeability.

Recent advances in the field of nanomedicine have demonstrated that biomimicry can further improve targeting properties of current nanotechnologies while simultaneously enable carriers with a biological identity to better interact with the biological environment. Immune cells for example employ membrane proteins to target inflamed vasculature, locally increase vascular permeability, and extravasate across inflamed endothelium. Inspired by the physiology of immune cells, we recently developed a procedure to transfer leukocyte membranes onto nanoporous silicon particles (NPS), yielding Leukolike Vectors (LLV). LLV are composed of a surface coating containing multiple receptors that are critical in the cross-talk with the endothelium, mediating cellular accumulation in the tumor microenvironment while decreasing vascular barrier function. We previously demonstrated that lymphocyte function-associated antigen (LFA-1) transferred onto LLV was able to trigger the clustering of intercellular adhesion molecule 1 (ICAM-1) on endothelial cells. Herein, we provide a more comprehensive analysis of the working mechanism of LLV in vitro in activating this pathway and in vivo in enhancing vascular permeability. Our results suggest the biological activity of the leukocyte membrane can be retained upon transplant onto NPS and is critical in providing the particles with complex biological functions towards tumor vasculature.

Biomimetic proteolipid vesicles for targeting inflamed tissues.

A multitude of micro- and nanoparticles have been developed to improve the delivery of systemically administered pharmaceuticals, which are subject to a number of biological barriers that limit their optimal biodistribution. Bioinspired drug-delivery carriers formulated by bottom-up or top-down strategies have emerged as an alternative approach to evade the mononuclear phagocytic system and facilitate the transport across the endothelial vessel wall. Here, we describe a method that leverages the advantages of bottom-up and top-down strategies to incorporate proteins derived from the leukocyte plasma membrane into lipid nanoparticles. The resulting proteolipid vesicles - which we refer to as leukosomes - retained the versatility and physicochemical properties typical of liposomal formulations, preferentially targeted inflamed vasculature, enabled the selective and effective delivery of dexamethasone to inflamed tissues, and reduced phlogosis in a localized model of inflammation.

Targeting ADAM17 in leukocytes increases neutrophil recruitment and reduces bacterial spread during polymicrobial sepsis.

A rapid and robust recruitment of circulating neutrophils at sites of infection is critical for preventing bacterial spread. The efficiency of this process, however, is greatly diminished during sepsis, a severe systemic inflammatory response to infection. The proteolytic activity of a disintegrin and metalloprotease-17 is induced in the cell membrane of leukocytes upon their activation, resulting in the conversion of membrane to soluble TNF-α and the release of assorted receptors from the surface of neutrophils important for their effector functions. We show that conditional knockout mice lacking a disintegrin and metalloprotease-17 in all leukocytes had a survival advantage when subjected to polymicrobial sepsis. Bacteremia and the levels of circulating proinflammatory cytokines, key determinants of sepsis severity, were significantly reduced in conditional a disintegrin and metalloprotease-17 knockout mice during sepsis. Although cecal bacterial microbiota and load were similar in unmanipulated conditional a disintegrin and metalloprotease-17 knockout and control mice, peritoneal spread of bacteria was significantly reduced in conditional a disintegrin and metalloprotease-17 knockout mice following sepsis induction, which was associated with an amplified recruitment of neutrophils. Taken together, our findings suggest that extensive a disintegrin and metalloprotease-17 induction during sepsis may tip the balance between efficient and impaired neutrophil recruitment.

Polymeric capsule-cushioned leukocyte cell membrane vesicles as a biomimetic delivery platform.

We report a biomimetic delivery of microsized capsule-cushioned leukocyte membrane vesicles (CLMVs) through the conversion of freshly reassembled leukocyte membrane vesicles (LMVs), including membrane lipids and membrane-bound proteins onto the surface of layer-by-layer assembled polymeric multilayer microcapsules. The leukocyte membrane coating was verified by using electron microscopy, a quartz crystal microbalance, dynamic light scattering, and confocal laser scanning microscopy. The resulting CLMVs have the ability to effectively evade clearance by the immune system and thus prolong the circulation time in mice. Moreover, we also show that the right-side-out leukocyte membrane coating can distinctly improve the accumulation of capsules in tumor sites through the molecular recognition of membrane-bound proteins of CLMVs with those of tumor cells in vitro and in vivo. The natural cell membrane camouflaged polymeric multilayer capsules with the immunosuppressive and tumor-recognition functionalities of natural leukocytes provide a new biomimetic delivery platform for disease therapy.

More than just a fruit: Grapefruit-derived nanovectors deliver cancer drugs

Grapefruit-derived nanoparticles coated with active leukocyte plasma membrane can target tumors and effectively deliver anticancer therapeutics.

Multiple low-affinity interactions support binding of human osteopontin to integrin αXβ2

Integrin αXβ2 (also known as complement receptor 4, p150,95, or CD11c/CD18) is expressed in the cell membrane of myeloid leukocytes. αXβ2 has been reported to bind a large number of structurally unrelated ligands, often with a shared molecular character in the presence of polyanionic stretches in poorly folded proteins or glucosaminoglycans. Nevertheless, it is unclear what chemical sources of polyanionicity enable the binding by αXβ2. Osteopontin (OPN) is an intrinsically disordered protein, which facilitates phagocytosis via the integrin αXβ2. Unlike for other integrins, neither the RGD nor the SVVYGLR motifs account for this binding, and the molecular basis of OPN binding by αXβ2 remains uncharacterized. Here, we show that the monovalent interactions between the ligand-binding domain of αXβ2 and OPN, its fragments, or caseins are weak, with dissociation constants higher than 10−5M but with high apparent stoichiometries. From comparison with cell adhesion studies, the discrimination between αXβ2 ligands and non-ligands appears to rely on these apparent stoichiometries in a way, which involves glutamate rather than aspartate side chains. Surprisingly, the extensive, negatively charged phosphorylation of OPN is not contributing to αXβ2 binding. Furthermore, synchrotron radiation circular spectroscopy excludes that the phosphorylation affects the general folding of OPN. Taken together, our quantitative analyses reveal a mode of ligand recognition by integrin αXβ2, which seem to differ in principles considerably from other OPN receptors.

Structure and Dynamics of the Integrin LFA-1 I-Domain in the Inactive State Underlie its Inside-Out/Outside-In Signaling and Allosteric Mechanisms

SummaryLymphocyte function-associated antigen 1 (LFA-1) is an integrin that transmits information in two directions across the plasma membrane of leukocytes, in so-called outside-in and inside-out signaling mechanisms. To investigate the structural basis of these mechanisms, we studied the conformational space of the apo I-domain using replica-averaged metadynamics simulations in combination with nuclear magnetic resonance chemical shifts. We thus obtained a free energy landscape that reveals the existence of three conformational substates of this domain. The three substates include conformations similar to existing crystallographic structures of the low-affinity I-domain, the inactive I-domain with an allosteric antagonist inhibitor bound underneath α helix 7, and an intermediate affinity state of the I-domain. The multiple substates were validated with residual dipolar coupling measurements. These results suggest that the presence of three substates in the apo I-domain enables the precise regulation of the binding process that is essential for the physiological function of LFA-1.

Immunological Markers for PML Prediction in MS Patients Treated with Natalizumab.

Natalizumab (NTZ), a monoclonal antibody recognizing the alpha4 integrin chain, has been approved for the treatment of active multiple sclerosis, but expose to the onset of a rare side effect, progressive multifocal leukoencephalopathy (PML). Estimating the individual risk of PML in NTZ-treated patients is a major challenge, and therapeutic strategies are mainly guided by the overall PML risk assessed by identified risk factors: JC virus (JCV) seropositivity, treatment duration (with peak incidence after 24 months), and the previous use of immunosuppressive therapies. Given that this stratification does not yet allow a precise individual prediction of PML, other predictive markers are needed, and several immunological biomarkers have been described. Quantification of anti-JCV antibody levels may improve individual predictive value, with higher baseline titers indicating increased risk. Other immunological biomarkers such as leukocyte cell membrane markers (CD49d, CD11a, and CD62L), detection of circulating JCV-specific activated T effector memory cells (TEM) or genetic screening have been proposed. In this review, we discuss how recent progress in immunology has paved the way for «new combined monitoring», which will include immunological screening, in NTZ-treated patients.

Detection of CFTR protein in human leukocytes by flow cytometry

Leukocytes have previously been shown to express detectable levels of the protein cystic fibrosis transmembrane conductance regulator (CFTR). This study aims to evaluate the application of flow cytometric (FC) analysis to detect CFTR expression, and changes thereof, in these cells. Aliquots (200 μL) of peripheral whole blood from 12 healthy control volunteers (CTRLs), 12 carriers of a CFTR mutation (CFC), and 40 patients with cystic fibrosis (CF) carrying various combinations of CFTR mutations were incubated with specific fluorescent probes recognizing CFTR protein expressed on the plasma membrane of leukocytes. FC was applied to analyze CFTR expression in monocytes, lymphocytes, and polymorphonuclear (PMN) cells. CFTR protein was detected in monocytes and lymphocytes, whereas inconclusive results were obtained from the analysis of PMN cells. Mean fluorescence intensity (MFI) ratio value and %CFTR-positive cells above a selected threshold were the two parameters selected to quantify CFTR expression in cells. Lowest variability and the highest reproducibility were obtained when analyzing monocytes. ANOVA results indicated that both parameters were able to discriminate monocytes of healthy controls and CF individuals according to CFTR mutation classes with high accuracy. Significantly increased MFI ratio values were recorded in CFTR-defective cells that were also able to improve CFTR function after ex vivo treatment with PTC124 (Ataluren), an investigative drug designed to permit the ribosome to read through nonsense CFTR mutations. The method described is minimally invasive and may be used in the monitoring of responses to drugs whose efficacy can depend on increased CFTR protein expression levels. © 2014 International Society for Advancement of Cytometry.

Toll-Like Receptors’ Pathway Disturbances are Associated with Increased Susceptibility to Infections in Humans

Toll-like receptors (TLRs) sense microbial products and play an important role in innate immunity. Currently, 11 members of TLRs have been identified in humans, with important function in host defense in early steps of the inflammatory response. TLRs are present in the plasma membrane (TLR1, TLR2, TLR4, TLR5, TLR6) and endosome (TLR3, TLR7, TLR8, TLR9) of leukocytes. TLRs and IL-1R are a family of receptors related to the innate immune response that contain an intracellular domain known as the Toll-IL-1R (TIR) domain that recruits the TIR-containing cytosolic adapters MyD88, TRIF, TIRAP and TRAM. The classical pathway results in the activation of both nuclear factor κB and MAPKs via the IRAK complex, with two active kinases (IRAK-1 and IRAK-4) and two non-catalytic subunits (IRAK-2 and IRAK-3/M). The classical pro-inflammatory TLR signaling pathway leads to the synthesis of inflammatory cytokines and chemokines, such as IL-1β, IL-6, IL-8, IL-12 and TNF-α. In humans, genetic defects have been identified that impair signaling of the TLR pathway and this may result in recurrent pyogenic infections, as well as virus and fungi infections. In this review, we discuss the main mechanisms of microbial recognition and the defects involving TLRs.

Activation of Neutrophils by the Two-Component Leukotoxin LukE/D from Staphylococcus aureus: Proteomic Analysis of the Secretions

Staphylococcus aureus is responsible for severe bacterial infections in hospitals and healthcare facilities. It produces single and bicomponent toxins (leukotoxins and hemolysins) that hinder innate immune function. Leukotoxin subunits bind to leukocyte cell membrane thus inducing transmembrane pores and subsequently, cell lysis. Leukotoxin LukE/D is a member of the bicomponent toxin family, but to date, no study concerning its involvement in host-pathogen interactions has been reported. In the present study, we performed the proteomic analysis of the secretions recovered after activation of human neutrophils by leukotoxin LukE/D. The neutrophil secretions were purified by RP-HPLC and different fractions were analyzed by Edman sequencing, LC-MS/MS, immunoblotted for chromogranin-derived peptides and further analyzed for antimicrobial properties. Proteomic analysis revealed that neutrophil secretions constitute a large number of proteins related with immune boosting mechanisms, proteolytic degradation, inflammatory process and antioxidant reactions.

L‐selectin shedding by NSAIDs: Old friends in new dresses

The recruitment of leukocytes to sites of inflammation requires the highly organized interplay of cell adhesion molecules on both leukocytes and inflamed endothelial cells, and disrupting the interaction of these molecules may compromise efficient recruitment of immune cells. Non-steroidal anti-inflammatory drugs inhibit inflammatory responses by several mechanisms including inhibition of prostaglandin synthesis and decreasing the expression of cell surface adhesion molecules. A report by Herrera-Garcia et al. [Eur. J. Immunol. 2013. 43: 55-64] in this issue of the European Journal of Immunology shows that the non-steroidal anti-inflammatory drug N-phenylanthranilic acid (N-Ph) causes L-selectin to be shed from the leukocyte plasma membrane and that this process in turn causes a decrease in leukocyte recruitment during inflammation in vivo. This finding may lead to novel approaches using N-Ph in the control of inflammatory processes as discussed in this Commentary.