Polymorphonuclear Leukocyte Phagocytosis Research Articles

TLR4 Recognizes Pseudallescheria boydii Conidia and Purified Rhamnomannans

Pseudallescheria boydii (Scedosporium apiospermum) is a saprophytic fungus widespread in the environment, and has recently emerged as an agent of localized as well as disseminated infections, particularly mycetoma, in immunocompromised and immunocompetent hosts. We have previously shown that highly purified α-glucan from P. boydii activates macrophages through Toll-like receptor TLR2, however, the mechanism of P. boydii recognition by macrophage is largely unknown. In this work, we investigated the role of innate immune receptors in the recognition of P. boydii. Macrophages responded to P. boydii conidia and hyphae with secretion of proinflammatory cytokines. The activation of macrophages by P. boydii conidia required functional MyD88, TLR4, and CD14, whereas stimulation by hyphae was independent of TLR4 and TLR2 signaling. Removal of peptidorhamnomannans from P. boydii conidia abolished induction of cytokines by macrophages. A fraction highly enriched in rhamnomannans was obtained and characterized by NMR, high performance TLC, and GC-MS. Preparation of rhamnomannans derived from P. boydii triggered cytokine release by macrophages, as well as MAPKs phosphorylation and IκBα degradation. Cytokine release induced by P. boydii-derived rhamnomannans was dependent on TLR4 recognition and required the presence of non-reducing end units of rhamnose of the rhamnomannan, but not O-linked oligosaccharides from the peptidorhamnomannan. These results imply that TLR4 recognizes P. boydii conidia and this recognition is at least in part due to rhamnomannans expressed on the surface of P. boydii.

Synergy of Caspofungin with Human Polymorphonuclear Granulocytes for Killing Candida albicans

The influence of caspofungin on polymorphonuclear leukocyte (PMN) phagocytosis and intracellular killing of Candida albicans was investigated. Caspofungin, at all of the concentrations tested (2, 3.2, and 8 microg/ml), significantly increased intracellular killing by PMNs through its direct action on both yeast cells and PMNs, indicating the potential ability of caspofungin to synergize with phagocytes for candidal killing. Caspofungin may therefore constitute an effective therapeutic option for the treatment of invasive fungal infections, including those refractory to conventional treatment with azole agents.

Effects of antibiotics on phagocytosis of polymorphonuclear leukocytes in experimental rabbit infection models

Effects of antibiotics on phagocytosis of polymorphonuclear leukocytes in experimental rabbit infection models

Synthesis and Biological Evaluation of Pyridine‐Containing Lipoxin A4 Analogues

A short and efficient synthesis of new pyridine-containing lipoxin A4 analogues was developed. These analogues induce phagocytosis of apoptotic polymorphonuclear leukocytes and display anti- inflammatory characteristics by suppressing pro-inflammatory cytokine production by macrophages.

Resistin Inhibits Essential Functions of Polymorphonuclear Leukocytes

The serum levels of resistin, a 12-kDa protein primarily expressed in inflammatory cells in humans, are increased in patients with chronic kidney disease and in those with diabetes mellitus. Both groups of patients have an increased risk of infections mainly as a result of disturbed polymorphonuclear leukocyte (PMNL) functions. Therefore, we investigated the influence of resistin on human PMNLs. Serum resistin concentrations were determined with a sandwich enzyme immunoassay. Using PMNLs from healthy subjects, chemotaxis was tested by the under-agarose method. Flow cytometric assays to measure oxidative burst and phagocytosis were conducted in whole blood. The uptake of deoxyglucose was determined as measure of the PMNL activation state. The activity of intracellular kinases was assessed by Western blotting and by in vitro kinase assays. Resistin inhibited PMNL chemotaxis and decreased the oxidative burst stimulated by Escherichia coli and by PMA, but did not influence PMNL phagocytosis of opsonized E. coli and PMNL glucose uptake. The inhibition of PMNLs by resistin was observed at concentrations found in serum samples of uremic patients, but not in concentrations measured in healthy subjects. Experiments with specific signal transduction inhibitors and measurements of intracellular kinases suggest that PI3K is a major target of resistin. In conclusion, resistin interferes with the chemotactic movement and the stimulation of the oxidative burst of PMNL, and therefore may contribute to the disturbed immune response in patients with increased resistin serum levels such as uremic and diabetic subjects.

Leukotriene B4-loaded microspheres: a new therapeutic strategy to modulate cell activation

BackgroundLeukotriene B4 (LTB4) is a potent inflammatory mediator that also stimulates the immune response. In addition, it promotes polymorphonuclear leukocyte phagocytosis, chemotaxis, chemokinesis and modulates cytokines release. Regarding chemical instability of the leukotriene molecule, in the present study we assessed the immunomodulatory activities conferred by LTB4 released from microspheres (MS). A previous oil-in-water emulsion solvent extraction-evaporation method was chosen to prepare LTB4-loaded MS.ResultsIn the mice cremasteric microcirculation, intraescrotal injection of 0.1 ml of LTB4-loaded MS provoked significant increases in leukocyte rolling flux, adhesion and emigration besides significant decreases in the leukocyte rolling velocity. LTB4-loaded MS also increase peroxisome proliferator-activated receptor-α (PPARα) expression by murine peritoneal macrophages and stimulate them to generate nitrite levels. Monocyte chemoattractant protein-1 (MCP-1) and nitric oxide (NO) productions were also increased when human umbilical vein and artery endothelial cells (HUVECs and HUAECs, respectively) were stimulated with LTB4-loaded MS.ConclusionLTB4-loaded MS preserve the biological activity of the encapsulated mediator indicating their use as a new strategy to modulate cell activation, especially in the innate immune response.

Homeostatic Cytokines and Expansion of Regulatory T Cells Accompany Thymic Impairment in Children with Down Syndrome

Down syndrome (DS), the most common chromosomal abnormality in humans, is characterized by precocious immunologic aging that results, among other things, in alterations of B and T lymphocyte subsets and natural killer cells, defective phagocytosis, and chemotaxis of polymorphonuclear leukocytes. We studied 30 children affected by DS, compared them to 29 healthy controls, and evaluated the functionality of the thymus (by measuring the amount of lymphocytes that express the signal-joint T cell receptor rearrangement excision circles [sj-TREC+]), the plasma levels of interleukin (IL)-7 and IL-15, the proliferative T cell response to these cytokines, the expression of the alpha chain of the IL-7 receptor (CD127), the extrathymic differentiation of T lymphocytes, and the presence of natural regulatory T cells (Tregs) in peripheral blood. We found that DS children had a significantly lower number of sj-TREC+ lymphocytes, the levels of which were strongly correlated with age. We found higher plasma levels of IL-7 and IL-15 than in healthy controls, and a higher proliferative T cell response to IL-15. DS children also showed a lower percentage of CD4(+) cells and profound alterations of T cell differentiation, along with increased amount of Tregs and of cells expressing markers of apoptosis. We can thus hypothesize that the precocious thymic involution occurring in DS is mirrored by a high production of IL-7 and IL-15, which is crucial for cell survival and proliferation. The complex alterations present in the periphery are likely the result of a compensatory mechanism: the overproduction of homeostatic cytokines could be a reaction to the impaired intrathymic production of T lymphocytes and/or to the expansion of Treg in the periphery, and could be required to allow the survival of T cells.

Identification of squalamine in the plasma membrane of white blood cells in the sea lamprey, Petromyzon marinus

It is well established that innate mechanisms play an important role in the immunity of fish. Antimicrobial peptides have been isolated and characterized from several species of teleosts. Here, we report the isolation of an antimicrobial compound from the blood of bacterially challenged sea lamprey, Petromyzon marinus. An acetic acid extract from the blood cells of challenged fish was subjected to solid-phase extraction, cation-exchange chromatography, gel-filtration chromatography, and reverse-phase high-performance liquid chromatography, with the purified fractions assayed for antimicrobial activity. Surprisingly, antimicrobial activity in these fractions originated from squalamine, an aminosterol previously identified in the dogfish shark, Squalus acanthias. Further chromatographic and mass spectrometric analyses confirmed the identity of squalamine, an antimicrobial and antiangiogenic agent, in the active fraction from the sea lamprey blood cells. Immunocytochemical analysis localized squalamine to the plasma membrane of white blood cells. Therefore, we postulate that squalamine has an important role in the innate immunity that defends the lamprey against microbial invasion. The full biochemical and immunological roles of squalamine in the white blood cell membrane remain to be investigated.

Aromatic Lipoxin A4and Lipoxin B4Analogues Display Potent Biological Activities

Lipoxins are a group of biologically active eicosanoids typically formed by transcellular lipoxygenase activity. Lipoxin A4 (LXA4) and Lipoxin B4 (LXB4) biosynthesis has been detected in a variety of inflammatory conditions. The native lipoxins LXA4 and LXB4 demonstrate potent antiinflammatory and proresolution bioactions. However, their therapeutic potential is compromised by rapid metabolic inactivation by PG dehydrogenase-mediated oxidation and reduction. Here we report on the stereoselective synthesis of aromatic LXA4 and LXB4 analogues by employing Sharpless epoxidation, Pd-mediated Heck coupling, and diastereoselective reduction as the key transformations. Subsequent biological testing has shown that these analogues display potent biological activities. Phagocytic clearance of apoptotic leukocytes plays a critical role in the resolution of inflammation. Both LXA4 analogues (1R)-3a and (1S)-3a were found to stimulate a significant increase in phagocytosis of apoptotic polymorphonuclear leukocytes (PMN) by macrophages, with comparable efficacy to the effect of native LXA4, albeit greater potency, while the LXB4 analogue also stimulated phagocytosis with a maximum effect observed at 10-11 M. LX-stimulated phagocytosis was associated with rearrangement of the actin cytoskeleton consistent with that reported for native lipoxins. Using zymosan-induced peritonitis as a murine model of acute inflammation (1R)-3a significantly reduced PMN accumulation.

Ceramide kinase expression is altered during macrophage-like cell differentiation of the leukemia cell line HL-60

Ceramide kinase (CerK) has important roles in leukocyte functions, including the role in degranulation of mast cells and the phagocytosis of polymorphonuclear leukocytes, so its expression levels should be strictly regulated. Here, we report that the mRNA expression and enzyme activity of CerK were decreased during macrophage-like cell differentiation of the leukemia cell line HL-60, yet neither was altered during granulocytic differentiation of the same cells. Our findings demonstrate that HL-60 cells are useful for studying CerK functions in leukocyte differentiation, and they also suggest that CerK might have an important role in such differentiation.

Inhibition of apoptosis induced by heat shock preconditioning is associated with decreased phagocytosis in human polymorphonuclear leukocytes through inhibition of Rac and Cdc42

The functionality of polymorphonuclear leukocytes (PMNL) and the exact process of the protective program employed by these cells in response to the heat shock (HS) remain ill-defined and debated. Particularly, the mechanism of phagocytic impairment induced by the HS and the molecular events associated with the delay of apoptosis used by these cells in such condition have given conflictual data. The aim of the present work is to study the consequences of the HS in different pathways involved in human PMNL apoptosis and subsequently in human PMNL phagocytic function. We demonstrated that HS (41 degrees C, 1 h) preconditioning induced inhibition of spontaneous PMNL apoptosis observed at 18 h in control cells incubated at 37 degrees C. This inhibition was characterized by absence of morphological nuclear changes, decrease of DNA fragmentation, low level of annexin V expression and decrease of caspase-3 activity. In parallel, HS increased both Hsp70 and Mcl-1 protein levels in PMNL. Phagocytosis of latex beads by PMNL was inhibited by HS (41 degrees C, 1 h) preconditioning despite an upregulation of CD11b, CD16 and CD47. Moreover, HS induced prolonged F actin depolymerization and inhibited both Rac and Cdc42 activation in PMNL. Finally, our results identify a new function of Mcl-1 in HS protection against apoptosis.

In vitro and in vivo activities of leukotriene B 4-loaded biodegradable microspheres

Leukotriene B 4 (LTB 4) is a potent inflammatory mediator and stimulates the immune response. In addition, LTB 4 promotes leukocyte functions such as phagocytosis, chemotaxis and chemokinesis of polymorphonuclear leukocytes, as well as modulates cytokine release. However, some physicochemical characteristics of leukotrienes, such as poor solubility in water and chemical instability, make them difficult to administer in vivo. The aim of this study was to develop LTB 4-loaded microspheres (MS) that prolong and sustain the in vivo release of this mediator. An oil-in-water emulsion solvent extraction–evaporation method was chosen to prepare the lipid-loaded MS. We determined their diameters, evaluated the in vitro release of LTB 4, using enzyme immunoassay and evaluated in vitro MS uptake by peritoneal macrophages. To assess the preservation of neutrophil chemoattractant activity, LTB 4-loaded MS were tested in vitro (in a modified Boyden microchamber) and in vivo, after intratracheal administration.

Abundance of the long pentraxin PTX3 at sites of leukocytoclastic lesions in patients with small‐vessel vasculitis

The prototypical tissue pentraxin PTX3 inhibits phagocytosis of late apoptotic polymorphonuclear leukocytes (PMNs) by macrophages. Levels of PTX3 parallel disease activity in small-vessel vasculitis. Small-vessel vasculitis is often characterized by leukocytoclasia, a phenomenon of accumulation of nuclear remnants from unscavenged PMNs in or near the vessel wall. We therefore hypothesized that PTX3 accumulates at sites of leukocytoclastic vasculitis and, as such, is a key factor for the induction of leukocytoclasis. We examined skin biopsy samples from 13 patients with small-vessel vasculitis and from 4 healthy and 3 inflammatory skin disease controls. Biopsy tissues, characterized histopathologically as leukocytoclastic vasculitis, were studied for the presence of PTX3 using rabbit anti-PTX3 polyclonal antibodies. Sections were scored morphometrically for leukocytoclastic infiltrates in conjunction with PTX3 staining. Morphometric scores were expressed as percentages of staining of the total tissue area. Biopsy specimens from patients with leukocytoclastic vasculitis revealed an abundant up-regulation of PTX3 at sites of leukocytoclastic infiltrates. Significantly more PTX3 was found in tissues from the 13 patients with vasculitis (mean +/- SEM 48.9 +/- 6.1%) than in tissues from the 7 controls (4.5 +/- 2.7%) (P = 0.0003). PTX3 was localized around vessels, as well as spread diffusely throughout the tissue. PTX3 is abundantly present at sites of leukocytoclastic infiltrates in patients with small-vessel vasculitis, but not in controls. Since PTX3 inhibits phagocytosis of late apoptotic PMNs by macrophages and is strongly up-regulated at sites of leukocytoclastic infiltration, PTX3 is a candidate factor in the phenomenon of leukocytoclasia in small-vessel vasculitis.

Modulation of Phagocytosis of Apoptotic Neutrophils by Supernatant from Dexamethasone-Treated Macrophages and Annexin-Derived Peptide Ac2–26

Phagocytic clearance of apoptotic leukocytes plays an important role in the resolution of inflammation. The glucocorticoid-inducible protein annexin 1 and annexin 1-derived peptides show potent anti-inflammatory responses in acute and chronic inflammation. In this study, we report that the annexin 1-derived peptide (Ac(2-26)) significantly stimulates nonphlogistic phagocytosis of apoptotic polymorphonuclear leukocytes (PMNs) by human monocyte-derived macrophages (Mphi). Peptide Ac(2-26)-stimulated phagocytosis is accompanied by rearrangement of the Mphi actin cytoskeleton. To investigate the potential role of endogenous annexin on clearance of apoptotic cells, Mphi were cultured for 5 days in the presence of dexamethasone. Supernatants collected from dexamethasone-treated Mphi significantly enhanced the ability of naive Mphi to engulf apoptotic PMNs. This effect was blocked by an annexin blocking Ab, by immunodepletion of the supernatants, and by the formyl peptide receptor/lipoxin receptor antagonist Boc1. In addition, we show that bone marrow-derived Mphi from annexin 1-null mice present a 40% decreased phagocytosis of apoptotic PMNs compared with cells taken from littermate controls. In conclusion, these results emphasize the pivotal role of annexin 1 as mediator for clearance of apoptotic cells and expand its potential therapeutic role in controlling inflammatory diseases.

Granulocyte function in patients with L-ferritin iron-responsive element (IRE) 39C-->T-positive hereditary hyperferritinaemia-cataract syndrome.

Hereditary hyperferritinaemia-cataract syndrome (HHCS) is an autosomal dominant trait associated with mutations in the iron responsive element (IRE) of the ferritin light-chain (L-ferritin) gene. Patients typically show elevated serum ferritin concentrations without iron overload and a bilateral cataract. Hyperferritinaemia can be associated with granulocyte dysfunction in patients with thalassemia beta and in haemodialysis patients. The effect of increased L-ferritin levels on granulocyte function in patients with HHCS is unknown. We examined glucose uptake, oxidative burst, chemotaxis, phagocytosis, apoptosis and intracellular calcium concentrations in polymorphonuclear leucocytes (PMNLs) of five affected members of a family with HHCS and in five healthy individuals matched for age and gender. Mutation testing revealed a 39C-->T transition in IRE in all five patients with HHCS. Serum ferritin levels of patients ranged between 907 and 2030 microg L(-1), respectively. In comparison with healthy individuals, PMNLs of patients with HHCS showed a significant increase in PMA-mediated stimulation of the oxidative burst, as well as a significantly higher stimulation of glucose uptake but no difference with respect to chemotaxis, phagocytosis, apoptosis and intracellular calcium concentrations. In summary, our study suggests that hyperferritinaemia in patients with IRE 39C-->T-positive HHCS is associated with activation of PMNLs but not with disturbance of fundamental PMNL function.

Effects of Tamm-Horsfall protein on polymorphonuclear leukocyte function.

Tamm-Horsfall protein (THP), a glycoprotein produced exclusively by renal tubular cells, is thought to be involved in several inflammatory disorders such as bacterial interstitial nephritis as well as in defence against uropathogenic microorganisms. The specific effects of THP on inflammatory cells, however, are not yet well known. Therefore, the present study investigates the effects of THP in its soluble form on distinct polymorphonuclear leukocyte (PMNL) functions. PMNL were isolated from the venous blood of healthy adult donors and incubated at low THP concentrations (70-350 ng/ml), resembling plasma concentrations, and at high THP concentrations (1.75-8.75 micro g/ml), resembling urinary concentrations. High (urinary) THP concentrations inhibited PMNL apoptosis and chemotaxis and stimulated PMNL phagocytosis, while low (plasma) THP concentrations increased PMNL chemotaxis. These data indicate that THP influences several PMNL functions, suggesting a crucial immunomodulatory role for this glycoprotein in host defence mechanisms of the kidney and genitourinary tract.

Nuclear Factor-κB Regulates Inflammatory Cell Apoptosis and Phagocytosis in Rat Carrageenin-Sponge Implant Model

In the present study we investigated whether apoptosis and phagocytosis are regulated by nuclear factor (NF)-kappaB in a model of chronic inflammation. The subcutaneous implant of lambda-carrageenin-soaked sponges elicited an inflammatory response, characterized by a time-related increase of leukocyte infiltration into the sponge and tissue formation, which was inhibited by simultaneous injection of wild-type oligodeoxynucleotide decoy to NF-kappaB. Molecular and morphological analysis performed on infiltrated cells demonstrated: 1) an inhibition of NF-kappaB/DNA binding activity; 2) an increase of polymorphonuclear leukocyte apoptosis correlated either to an increase of p53 or Bax and decrease of Bcl-2 protein expression; and 3) an increase of phagocytosis of apoptotic polymorphonuclear leukocytes by macrophages associated with an increase of transforming growth factor-beta1 and decrease of tumor necrosis factor-alpha as well as nitrite/nitrate production. Our results, showing that blockade of NF-kappaB by oligodeoxynucleotide decoy increases inflammatory cell apoptosis and phagocytosis, may contribute to lead to new insights into the mechanisms governing the inflammatory process.

Sulfur mustard primes phagocytosis and degranulation in human polymorphonuclear leukocytes

Sulfur mustard (2,2′-bis-chloroethyl-sulfide; SM) is a chemical warfare vesicant that causes debilitating skin lesions. Although a great deal of work has focused on the direct effects of SM exposure on the epithelium, it is unclear how much the inflammatory response, induced by exposure, contributes to lesion pathogenesis. Keratinocytes exposed to SM express a number of inflammatory mediators and elicit a cellular infiltrate consisting largely of polymorphonuclear leukocytes (PMN). PMN infiltration into SM lesions occurs as early as 30 min and peaks after several hours postexposure, and, despite the relatively short half-life of SM, PMN infiltrating a lesion could be exposed to micromolar concentrations of the agent. Previously, we have shown that exposure to low doses of sulfur mustard prime oxidative function in human PMN. The current study was undertaken to evaluate the effects of low-dose SM exposure on PMN phagocytosis, degranualtion and chemotaxis. PMN exposed to low doses of SM (50–200 μM) showed a dose-dependent enhancement of phagocytic function. Exocytosis of PMN azurophilic and specific granules [determined by analysis of granule-specific intravesicular receptors, Interleukin 10 receptor (IL-10R) and CD63] was also enhanced by SM exposure. Finally, we examined the effect of SM as a chemoattractant for PMN and show that SM is not itself a chemotaxin. These results suggest that SM injury may, in part, be caused by normal inflammatory function, and that therapeutic strategies aimed at down-regulating PMN activation could lessen the severity of SM injury and the time required for its resolution.

Dialyzer membrane type and reuse practice influence polymorphonuclear leukocyte function in hemodialysis patients

Polymorphonuclear leukocyte (PMNL) production of reactive oxygen species (ROS) has been linked to hemodialysis (HD) associated morbidity. The effect of dialyzer membrane type and reuse on PMNL function has not been clearly defined. The present report is a cross-sectional study undertaken in a cohort of patients undergoing regular HD, at enrollment into the Hemodialysis (HEMO) Study, to study the association between patient and dialysis-related factors and PMNL function. PMNL function was assessed by measuring PMA- and N-formyl methionyl-leucyl-phenylalanine (fMLP) -induced respiratory burst, and phagocytic activity toward Staphylococcus aureus. PMNL from patients dialyzed with polysulphone (PS) or cuprophane (CU) membranes showed higher PMA-induced respiratory burst activity compared with those exposed to substituted cellulose (cellulose acetate, cellulose triacetate, CA/CT) membranes, regardless of dialyzer reuse. The use of bleach as a cleansing agent during reuse was associated with higher PMA-induced PMNL superoxide production, as was the use of renalin when compared to aldehydes. In a subgroup of patients using PS dialyzers, reuse itself was associated with higher fMLP-induced superoxide production. The type of bleach-germicide combination during reuse showed that use of renalin as a germicide was also associated with higher PMNL phagocytosis index. The number of years on HD correlated inversely with PMA-induced PMNL superoxide response. Weaker PMNL response to fMLP was associated with greater comorbidity and poor functional status as quantified by Index of Coexisting Diseases (ICED) and Karnofsky scores, respectively. Our results indicate that dialyzer membrane type and the reuse process influence the oxidative response of PMNL among HD patients. The implications of these observations on clinical morbidity need to be further evaluated in prospective studies.

Design of soymetide-4 derivatives to potentiate the anti-alopecia effect.

Previously, we found that soymetide-4 (MITL), an N-formyl-methionyl-leucyl-phenylalanine (fMLP) agonist peptide derived from soybean beta-conglycinin alpha' subunit, stimulated phagocytosis of human polymorphonuclear leukocytes, and inhibited alopecia induced by etoposide, an anticancer drug, in neonatal rats after oral administration. We found that the fMLP receptor affinity and phagocytosis-stimulating activity of soymetide-4 was potentiated by replacement of Thr(3) with hydrophobic residues. Among the derivatives synthesized, [Trp](3)-soymetide-4 (MIWL) was the most potent, stronger by 180 and 130 times than soymetide-4 in receptor affinity and phagocytosis-stimulating activity, respectively. The anti-alopecia effect of [Trp](3)-soymetide-4 was about 3 times larger than that of soymetide-4 after oral administration.