Conversion of arachidonic acid to two novel products by a cytochrome P450-dependent mixed-function oxidase in polymorphonuclear leukocytes

Canine polymorphonuclear leukocytes metabolize [14C] arachidonic acid into 2 unidentified products, separated by thin-layer chromatography and high performance liquid chromatography, and called peak 1 and peak 2. The formation of peak 1 is maximal at 5 minutes and then declines, while the synthesis of peak 2 increases throughout the 30 minute incubation period. The formation of peak 1 and, to a lesser extent, peak 2, was enhanced after dual inhibition of lipoxygenase and cyclo-oxygenase enzymes with BW755C (94 microns) or nafazatrom (37 microns), or after incubation in a calcium-free buffer. In contrast, the formation of these products was inhibited by SKF-525A (50 microns), suggesting a cytochrome P450-dependent mechanism. The presence of cytochrome P450 in neutrophil microsomes was confirmed by measuring aryl hydrocarbon hydroxylase activity and cytochrome P450 content.     

Concanavalin a capping of rhesus monkey polymorphonuclear leukocytes

The technique of capping, a probe designed to evaluate the fluidity and functional competence of human polymorphonuclear leukocytes (PMNs), has been successfully adapted for rhesus monkey PMNs. The capping characteristics of rhesus monkey PMNs are very similar to those of human PMNs. Utilizing this capping technique as an evaluation tool and with fetal/neonatal rhesus monkey as a functional animal model, the ontogeny of movement and chemotactic characteristics of PMNs can now be studied.     

Isolation of functional polymorphonuclear leukocytes from rhesus monkeys using discontinuous Ficoll-Hypaque density gradients

A simple, rapid method for separating polymorphonuclear leukocytes (PMNs) from adult rhesus monkey blood, based on the use of a discontinuous gradient of Ficoll-Hypaque (densities, 1.100, 1.077) has been developed. Using this method, 71.1% of the PMNS were recovered in a layer with a purity of 91% PMNs with very few contaminating erythrocytes. The added advantage of the method described here is that a higher chemotactic activity was retained.     

TNF-alpha induced PMN apoptosis in whole human blood: Protective effect of SSR180575, a potent and selective peripheral benzodiazepine ligand

The peripheral benzodiazepine receptor (PBR) has been shown to play a key role in the regulation of the mitochondrial process leading to apoptosis. Despite much controversy in the literature on this subject, PBR synthetic ligands (and specifically agonists such as Ro5-4864 and SSR180575) are described as presenting potent anti-apoptotic effect against oxidative stress, TNFα- and tamoxifen-induced apoptosis when the PBR ligand is administrated at a low dose, close to the affinity range of the ligand to its receptor. Such anti-apoptotic activity has already been correlated with a protective effect of PBR ligands against ischemia-reperfusion induced tissue dysfunction.Previously, we had shown that SSR180575 is a specific and high affinity PBR ligand of potential interest in pathological cardiovascular, renal and neurodegenerative indications. Beyond its expression in steroid-producing tissues, heart, liver and kidney, the PBR is also known to be highly expressed in blood cells. In this work, we demonstrate by flow cytometry experiments, that SSR180575, at low concentrations, is able to protect polymorphonuclear leukocytes (PMNs) against TNFα-induced apoptosis in whole blood. Thus, in a new context, SSR180575 again shows potent anti-apoptotic properties. Moreover, TNFα- induced PMN apoptosis appears to be a good surrogate marker for determining SSR180575 blood availability and activity in treated patients.     

Periodontal therapy increases neutrophil extracellular trap degradation

In periodontitis, polymorphonuclear leucocytes (PMNs) are activated. They entrap and eliminate pathogens by releasing neutrophil extracellular traps (NETs). Abnormal NET degradation is part of a pro-inflammatory status, affecting co-morbidities such as cardiovascular disease. We aimed to investigate the ex vivo NET degradation capacity of plasma from periodontitis patients compared to controls (part 1) and to quantify NET degradation before and after periodontal therapy (part 2). Fresh NETs were obtained by stimulating blood-derived PMNs with phorbol 12-myristate 13-acetate. Plasma samples from untreated periodontitis patients and controls were incubated for 3 h onto freshly generated NETs (part 1). Similarly, for part 2, NET degradation was studied for 91 patients before and 3, 6 and 12 mo after non-surgical periodontal therapy with and without adjunctive systemic antibiotics. Finally, NET degradation was fluorospectrometrically quantified. NET degradation levels did not differ between periodontitis patients and controls, irrespective of subject-related background characteristics. NET degradation significantly increased from 65.6 ± 1.7% before periodontal treatment to 75.7 ± 1.2% at 3 mo post periodontal therapy, and this improvement was maintained at 6 and 12 mo, irrespective of systemic usage of antibiotics. Improved NET degradation after periodontitis treatment is another systemic biomarker reflecting a decreased pro-inflammatory status, which also contributes to an improved cardiovascular condition.     

Impact of angiogenic and innate immune systems on the corpus luteum function during its formation and maintenance in ruminants

The corpus luteum (CL) is formed from an ovulated follicle, and grows rapidly to secrete progesterone (P4) thereby supporting implantation and maintenance of pregnancy. It is now evident that angiogenesis is necessary to form the structure of the developing CL as well as to acquire the steroidogenic capacity to secrete large amounts of P4. It is of interest that the increases in CL size, plasma P4 concentration and luteal blood flow are occurring in parallel during the first seven days after ovulation. Angiogenic factors, such as vascular endothelial growth factor-A (VEFGA) and basic fibroblast growth factor (FGF2), play a central role in promoting cell proliferation and angiogenesis in the developing CL. Angiopoietins regulate the stability of blood vessels, which directly affects angiogenesis or angiolysis via angiogenic factors. Vasohibin-1 is a novel negative feedback regulator, which inhibits VEGF-based vasculogenesis. It became evident that the immune cells, i.e., macrophages, eosinophils and neutrophils are recruited into the CL – using the innate immune system – just after ovulation which is accompanied by bleeding. The immune cells support active angiogenesis and thus the growth of the CL. In cows, the lymphatic system, but not blood vascular system, is reconstituted during early pregnancy, and embryonic trophoblast-derived interferon tau could play a crucial role in inducing lymphangiogenesis. This novel phenomenon may support a maternal recognition of pregnancy in shifting the local systems in such a way that they ensure a long-term supply of P4 over the period of pregnancy. Overall, the current findings support the concept that several major components involved in the regulation of the CL development and maintenance overlap in stimulating steroidogenesis, angiogenesis, vascular function and the innate immune system.     

Glycerol monomycolate, a latent tuberculosis-associated mycobacterial lipid, induces eosinophilic hypersensitivity responses in guinea pigs

Dynamic changes in the lipid composition of the cell wall occur in pathogenic mycobacteria that are often intended for adaptation to the host environment. Dormant mycobacteria should have evolved efficient maneuvers for cohabitation, allowing the microbes to persist for years within the host. Glycerol monomycolate (GroMM) has been implicated as a specific immune target in human individuals with latent, but not active, tuberculosis, but the in vivo response to GroMM and the relevance of it to latent infection remain poorly understood. Here, we immunized guinea pigs with bacillus Calmette–Guerin (BCG) expressing high levels of GroMM and then, monitored skin reactions at the site of challenge with GroMM-containing liposome. We found that BCG-immunized guinea pigs mounted enhanced skin reactions to GroMM with prominent local infiltration by eosinophils. Consistent with this, GroMM-stimulated lymph node cells upregulated the expression of T helper (Th)2-type cytokines, such as interleukin (IL)-5 and IL-10, that could potentially counteract the microbe-eliminating Th1-type cytokine response. On the basis of these observations, we predict that the host response to GroMM produced by dormant mycobacteria would contribute to their long-term survival in the host.     

Glycerophospholipids and glycerophospholipid-derived lipid mediators: a complex meshwork in Alzheimer's disease pathology

An increasing body of evidence suggested that intracellular lipid metabolism is dramatically perturbed in various cardiovascular and neurodegenerative diseases with genetic and lifestyle components (e.g., dietary factors). Therefore, a lipidomic approach was also developed to suggest possible mechanisms underlying Alzheimer’s disease (AD). Neural membranes contain several classes of glycerophospholipids (GPs), that not only constitute their backbone but also provide the membrane with a suitable environment, fluidity, and ion permeability. In this review article, we focused our attention on GP and GP-derived lipid mediators suggested to be involved in AD pathology. Degradation of GPs by phospholipase A₂ can release two important brain polyunsaturated fatty acids (PUFAs), e.g., arachidonic acid and docosahexaenoic acid, linked together by a delicate equilibrium. Non-enzymatic and enzymatic oxidation of these PUFAs produces several lipid mediators, all closely associated with neuronal pathways involved in AD neurobiology, suggesting that an interplay among lipids occurs in brain tissue. In this complex GP meshwork, the search for a specific modulating enzyme able to shift the metabolic pathway towards a neuroprotective role as well as a better knowledge about how lipid dietary modulation may act to slow the neurodegenerative processes, represent an essential step to delay the onset of AD and its progression. Also, in this way it may be possible to suggest new preventive or therapeutic options that can beneficially modify the course of this devastating disease.     

Cellular crosstalk between TNF-α, NADPH oxidase, PKCβ2, and C2GNT in human leukocytes

Increasing evidence suggests that chronic, sub-clinical inflammation plays an important role in the pathogenesis of diabetic retinopathy. We have established the potential role of the inflammatory enzyme, core 2 β-1, 6-N-acetylglucosaminyltransferase (C2GNT) in diabetic retinopathy. The present study was designed to explore the NADPH oxidase signaling pathway in the tumor necrosis factor-alpha (TNF-α)-induced activity of C2GNT in leukocytes. Human leukocytes (U937 cells) and an Epstein-Barr-transformed lymphoblastoid cell line deficient in p47phox (F10007 cells) were used for the study. Cells were exposed to TNF-α for 24 h in the presence and absence of 1) NADPH oxidase inhibitors (apocynin and scrambled and unscrambled gp91ds-tat), 2) LY379196 (specific protein kinase C β1/2 (PKCβ1/2) inhibitor), and 3) the antioxidant tiron. Subsequent C2GNT and NADPH activity was measured and the adhesion of U937 and F10007 cells to endothelial cells was assessed. TNF-α-induced C2GNT activity (1813 ± 326 pmol/h/mg protein) (mean ± SEM) in human leukocytes was significantly reversed with apocynin (153 ± 82 pmol/h/mg protein), unscrambled gp91ds-tat (244 ± 122 pmol/h/mg protein) and tiron (756 ± 87 pmol/h/mg protein). We further supported this C2GNT-NADPH oxidase link using p47phox-deficient leukocytes. The deficiency in p47phox prevented TNF-α-induced NADPH oxidase and C2GNT activity and adherence to endothelial cells. The response to TNF-α was restored by transfection with an expression plasmid containing a p47phox cDNA inserted in the sense direction. Our results demonstrate for the first time a novel signaling crosstalk between TNF-α, NADPH oxidase, PKCβ1/2 and C2GNT in leukocytes.     

Characteristics of the epitope of leukotriene B4 recognized by a highly specific mouse monoclonal antibody

Mouse monoclonal IgG2b antibodies to leukotriene B4 bind [3H]leukotriene B4 with an affinity one-thirtieth to one-third that of different rabbit antibodies to leukotriene B4. The concentrations of related ligands required to inhibit by 50% the binding of [3H]leukotriene B4 define cross-reactivities of approximately 100% for carboxyl-derivatives of leukotriene B4, 10% for 12(S)-leukotriene B4 and 8 cis-leukotriene B4, which were not distinguished from leukotriene B4 by polyclonal antibodies, 3-5% for the two isomers of 6 trans-leukotriene B4, 5% for 20-OH-leukotriene B4 and 20-COOH-leukotriene B4, and less than 1% for other leukotrienes, mono-hydroxy-eicosatetraenoic acids, and the two leukotriene B4-like isomers of 8, 15-di-hydroxy-eicosatetraenoic acid. Thus the monoclonal combining site is highly specific for the di-hydroxy-triene portion of leukotriene B4.