Properties of leukotriene B4 20-hydroxylase from polymorphonuclear leukocytes

Human polymorphonuclear leukocytes (PMNL) convert arachidonic acid (20:4) to a number of dihydroxy metabolites, including leukotriene B4 (LTB4) 5S,12R-dihydroxy-6,8,10,14-EEEZ-icosatetraenoic acid (isomer-1), 5S,12S-dihydroxy-6,8,10,14-EEEZ-icosatetraenoic acid, 5S,12S-dihydroxy-6,8,10,14-EZEZ-icosatetraenoic acid (5S,12S-dh-20:4), 5,6-dihydroxy-7,9,11,14-icosatetraenoic acid, and 5,15-dihydroxy-6,8,11,13-icosatetraenoic acid. LTB4 was synthesized rapidly after stimulation of PMNL with the divalent cation ionophore, A23187, but its concentration rapidly declined after about 4 min, in contrast to the other dihydroxy metabolites of 20:4 whose concentrations remained stable for at least 20 min. The amounts of polar metabolites (identified primarily as 20-hydroxy-LTB4) increased steadily with time up to 20 min. These results suggest that LTB4 may be specifically converted to its 20-hydroxy metabolite by PMNL. We prepared 3H- and 14C-labeled analogs of the dihydroxyicosatetraenoic acid metabolites described above by incubation of labeled 20:4 with PMNL. Although all of these substances were metabolized to some extent by human PMNL, LTB4 (apparent Km, 1.0 microM) was metabolized the most rapidly, followed by 5S,12S-dh-20:4 (apparent Km, 2.4 microM) and isomer-1 (apparent Km, 4.8 microM). All three substrates were shown by mass spectrometry to be converted to their 20-hydroxy metabolites. LTB4 was also metabolized to its omega-carboxy derivative. Human mononuclear leukocytes and rabbit PMNL metabolized LTB4 very slowly, whereas rat PMNL metabolized this substrate at about one-sixth the rate of human PMNL. These results demonstrate that human PMNL contain an omega-hydroxylase that specifically converts LTB4 to its 20-hydroxy metabolite. This enzyme may be important for the regulation of LTB4 levels in vivo.     

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.     

Contrasting effects of Mycoplasma fermentans and M. felis on the viability and chemiluminescence response of human polymorphonuclear leukocytes

Abstract Trypan blue exclusion was used to estimate the viability of human polymorphonuclear leukocytes (PMNL) in the presence of Mycoplasma felis and two strains of M. fermentans (PG18 and incognitus). The competence of PMNL to mount a respiratory burst when challenged with the mycoplasmas was also monitored by luminol-dependent chemiluminescence (CL). Both un-opsonised and non-immune human serum opsonised M. felis cells had little effect on PMNL viability. In contrast, PMNL viability was reduced markedly by un-opsonised cells of M. fermentans strain incognitus and, to a lesser extent, strain PG18, and opsonisation of these mycoplasmas further enhanced killing. Death of PMNL in the presence of M. fermentans was not associated with the autonomous production of active oxygen species during the respiratory burst as M. felis induced a high CL response from PMNL, whereas that induced by M. fermentans strain incognitus was significantly lower. M. fermentans may invade mammalian cells and it is suggested that the mechanism of PMNL death could be related to the ability of M. fermentans to penetrate host cell membranes.     

Regulation of the lateral diffusion of WGA-labeled glycoconjugates in human leukocytes

Cell Biochemistry and Biophysics - The regulation of the membrane mobility of glycoconjugates in human polymorphonuclear leukocytes (PMNL) was studied by comparing adult PMNL with promyelocytic...     

Immediate-early antigen expression and modulation of apoptosis after in vitro infection of polymorphonuclear leukocytes by human cytomegalovirus

Polymorphonuclear leukocytes (PMNL) are a major carrier of human cytomegalovirus (CMV) in viremic immunodepressed patients. We transmitted infectious virions and viral components to PMNL by coculturing these cells with infected human embryonic lung fibroblasts (HELF) or human umbilical vein endothelial cells (HUVEC). Quantitative time-course analysis of viral DNA and protein expression in PMNL, after functional separation from infected donor cells, indicated the initiation of viral cycling, with immediate-early protein expression. No viral replication or early or late gene expression was observed, but infected PMNL were able to infect naive fibroblasts more than 48 h after the end of co-culture. PMNL apoptosis was significantly delayed during co-culture with infected or uninfected HUVEC, and this phenomenon did not require contact between the two cell populations. The increased production of IL-8 in the same culture conditions that protect PMNL from apoptosis, associated with the reversion of this protection by inhibiting or depleting this factor in the culture media, targets this cytokine as a likely candidate for this protective effect. These data suggest that PMNL play a key role in virus dissemination in vivo, through their interactions with infected endothelial cells.     

The correlation between the number of polymorphonuclear leukocytes in human oral cavity and the counts of some bacteria

The aim of the study was to evaluate the relationship between the number of polymorphonuclear leukocytes (PMNL) and the number of selected bacteria in human oral cavity. Sixty one healthy people (periodontal index of Russel's did not exceed 0.2), aged 22-26 were investigated. PMNL were isolated by rinsing oral cavity with isotonic, buffered NaCl solution. Nonstimulated whole saliva was bacteriologically examined. The number of PMNL, which was obtained from mouth of examined people was between 100,000 and 4,200,000 in 100 mL of rinsings. Usually, i.e. in 34% of cases, the number was between 500,000-1,000,000. Streptococci were isolated from all tested people and their number was the biggest. Almost in all cases there were isolated cariogenic bacteria--Streptococcus mutans (95%) and lactobacilli (93%). Haemophili, staphylococci, including Staphylococcus aureus, were found relatively often (in 86%, 86% and 52% respectively). Gram-negative enteric rods were rarely isolated (33%) and were the least numerous group of all. It has been stated, that there is a comparatively strong negative correlation between the number of PMNL in oral cavity and the number of streptococci (r=-0.55; p<0.0001), haemophili (r=-0.564; p<0.0001), staphylococci (r=-0.538; p<0.0001), lactobacilli (r=-0.407; p=0.0017) and Streptococcus mutans (r=-0.483; p=0.0002). The results suggest that PMNL are one of the factors controlling the number of some bacteria in human oral cavity.     

Priming of polymorphonuclear leukocytes: a culprit in the initiation of endothelial cell injury

Peripheral polymorphonuclear leukocytes (PMNL) in hemodialysis (HD) patients are primed, continually releasing and exposing the vascular endothelium to soluble factors such as reactive oxygen species and inflammatory mediators. To mimic the close proximity between PMNL and the endothelial monolayer and to monitor and characterize the influence of soluble mediators released from PMNL, we developed a novel cocultivation system using primary human umbilical vein endothelial cell (HUVEC) cultures and PMNL, with a sieve separating the two cell types to prevent direct adhesive effects. PMNL (10(6)) from HD patients or from healthy normal controls were cocultivated with HUVEC (10(5)) for 15 min, and endothelial cell injury was assessed by HUVEC morphology, cell detachment, and apoptosis. Proinflammatory changes were estimated by expression of HUVEC adhesion molecule P-selectin and by endothelial IL-8 and endothelial nitric oxide synthase mRNA. The levels of intracellular tissue factor reflected the procoagulant state, whereas NADPH oxidase activity served as an indicator for prooxidative changes in HUVEC. Mediators released from the primed PMNL triggered activation/dysfunction of endothelial cells, causing 1) an increase in endothelial cell detachment and apoptosis, 2) a proinflammatory state manifested by increased IL-8 mRNA expression and P-selectin on the endothelial surface, 3) activation of endothelial NADPH oxidase, 4) an increase in endothelial cell tissue factor that directly correlated with PMNL priming index, and 5) a decrease in endothelial nitric oxide synthase mRNA. Our data support a pathogenic link between PMNL priming and endothelial dysfunction, suggesting that PMNL priming is a potential new nontraditional risk factor for the development of atherosclerosis.     

Inhibitory effect of stobadine on FMLP-induced chemiluminescence in human whole blood and isolated polymorphonuclear leukocytes.

The chemiluminescence (CL) technique with luminol and isoluminol was used to characterize the effect of stobadine on reactive oxygen metabolites (ROM) generation in human whole blood and in isolated polymorphonuclear leukocytes (PMNL) stimulated with N-formyl-methionyl-leucyl-phenyl-alanine (FMLP). In whole blood and in isolated PMNL, stobadine in the concentrations of 1, 10 and 100 micromol/L significantly inhibited the CL signal after FMLP, which activated predominantly extracellular generation of ROM. The same concentrations of stobadine were effective on CL in a cell-free system. On the other hand, myeloperoxidase (MPO) liberation was decreased by stobadine only in the concentration of 100 micromol/L. The results showed stobadine to act as a potent inhibitor/scavenger of extracellularly produced ROM in human PMNL and indicated interference of stobadine with ROM as well as with signalling events resulting in NADPH-oxidase activation and MPO liberation.     

Human polymorphonuclear leukocytes are sensitive in vitro to Helicobacter pylori vaca toxin

BACKGROUND: Interactions between bacterial components and polymorphonuclear leukocytes (PMNL) play a major pathogenic role in Helicobacter pylori-associated diseases. Activation of PMNL can be induced by contact with whole bacteria or by different H. pylori products released in the extracellular space either by active secretion or by bacterial autolysis. Among these products, H. pylori VacA is a secreted toxin inducing vacuolation and apoptosis of epithelial cells. METHODS AND RESULTS: We found that non-opsonic human PMNL were sensitive to the vacuolating effect of VacA+ broth culture filtrate (BCF) and of purified VacA toxin. PMNL incubated with VacA+ BCF showed Rab7-positive large intracytoplasmic vacuoles. PMNL preincubation with H. pylori BCF of different phenotypes dramatically potentialized the oxidative burst induced by zymosan, increased phagocytosis of opsonized fluorescent beads, and up-regulated CD11b cell surface expression, but independently of the BCF VacA phenotype. Moreover, by using purified VacA toxin we showed that vacuolation induced in PMNL did not modify the rate of spontaneous PMNL apoptosis measured by caspase 3 activity. CONCLUSIONS: Taken together, these data showed that human PMNL is a sensitive cell population to H. pylori VacA toxin. However, activation of PMNL (i.e., oxidative burst, phagocytosis, CD11b up-regulation) and PMNL apoptosis are not affected by VacA, raising question about the role of VacA toxin on PMNL in vivo.     

Affinity labeling of the membrane protein-binding component of human polymorphonuclear leukocyte receptors for leukotriene B4.

A radiolabeled N-(3-aminopropyl)-leukotriene B4 amide ([3H]LTB4-APA) analog of the potent leukocyte chemotactic factor leukotriene B4 (LTB4) binds to receptors for LTB4 in plasma membrane-enriched preparations from human blood polymorphonuclear leukocytes (PMNL) and intact PMNL with respective mean dissociation constants of 2.3 nM and 69 nM at 4 degrees C. The [3H]LTB4-APA bound to plasma membrane-enriched preparations from PMNL was covalently cross-linked to membrane proteins with disuccinimidyl suberate. Solubilization and resolution by SDS-PAGE of proteins from [3H]LTB4-APA-labeled PMNL membranes revealed predominant labeling of a 60-kDa protein. Labeling of the PMNL membrane protein was inhibited by LTB4 and its analogs at concentrations similar to those inhibiting the binding of [3H]LTB4 to its receptor, with an identical rank order of potency of LTB4 greater than 20-hydroxy-LTB4 greater than LTB4-APA = 5(S),12(R)-dihydroxy-eicosa-14-cis-6,8,10-trans-tetraenoic acid much greater than LTD4 = LTC4. GTP suppressed the labeling of the 60-kDa PMNL membrane protein to an extent consistent with the decrease in receptor affinity for LTB4 induced by GTP. The stereospecificity of the affinity cross-linking reaction and the regulation by GTP support the identification of an approximately 60-kDa protein as the binding component of the PMNL receptor for LTB4.     

Effects of recombinant human gamma interferon on intracellular survival of Bordetella pertussis in human phagocytic cells

Abstract Several studies have demonstrated that Bordetella pertussis has the ability to enter and survive intracellularly within human polymorphonuclear leukocytes (PMNL) and human monocytes/macrophages. The effects of human recombinant gamma interferon (IFN-γ) on the survival of B. pertussis in PMNL and human monocytes, and on the oxidative burst activity of PMNL and human monocytes in response to B. pertussis were assessed in this study. IFN-γ partially increased intracellular killing of phagocytosed B. pertussis in human monocytes, as determined by an orange acridine-crystal violet assay. In contrast, IFN-γ did not enhance intracellular killing of B. pertussis in PMNL. No significant increase of superoxide production was noted in human monocytes in response to B. pertussis when stimulated with various concentrations of IFN-γ. The partial increase of B. pertussis killing by IFN-γ within monocytes, together with poor production of superoxide may explain how B. pertussis can survive within human phagocytic cells, and thus cause a more prolonged course of the disease.     

Modulation of polymorphonuclear leukocyte locomotion by synthetic amphiphiles

The capacity of synthetic amphiphiles, poly(ethyleneglycol) 6000 (PEG) esterified with saturated fatty acids (C₂–C₁₈), to modify polymorphonuclear leukocyte (PMNL) locomotion has been investigated. It was noticed that PEG-myristate (M-PEG; C₁₄) stimulated the random locomotion of PMNL populations in concentrations up to about 1 g/L. The esters with shorter aliphatic chains had negligible effects, whereas those with longer chains, PEG-palmitate (P-PEG; C₁₆) and PEG-stearate (S-PEG; C₁₈) reduced the locomotion, irrespectively of concentration. The ability of the PMNL to be stimulated by an attractant liberated from normal human serum was slightly impaired by M-PEG, but not by P-PEG. The response to M-PEG of individual PMNL was heterogeneous in that some cells were stimulated and others were inhibited. However, the average result was a reduction of the motility. This indicates that methods used for the study of the locomotion of cell populations may not always reflect the average behavior of the whole population. It was also concluded that the different effects of M-PEG and P-PEG owed to dissimilar effects on the membrane structure of the PMNL since (1) M-PEG perturbated the PMNL membrane more than P-PEG, as assayed by the release of superoxide anion (0 ₂ ⁻ , although the binding was smaller, and (2) M-PEG and P-PEG increased and decreased the membrane fluidity, respectively, as measured with fluorescent bleaching and recovery after bleaching of labeled PMNL. The results indicate a subtle coupling between membrane structure and PMNL locomotion.     

Disruption of POF1B binding to nonmuscle actin filaments is associated with premature ovarian failure

Premature ovarian failure (POF) is characterized by elevated gonadotropins and amenorrhea in women aged <40 years. In a Lebanese family with five sisters who received the diagnosis of POF, we established linkage to the long arm of the X chromosome (between Xq21.1 and Xq21.3.3), using whole-genome SNP typing and homozygosity-by-descent mapping. By sequencing one candidate gene within that region, POF1B, we identified a point mutation localized in exon 10. This substitution of a nucleotide (G-->A), at position 1123, results in an arginine-->glutamine mutation of the protein sequence at position 329 (mutation R329Q). All the affected family members were homozygous for the mutation, whereas the unaffected members were heterozygous. Because POF1B shares high homology with the tail portion of the human myosin, we assessed the ability of both wild-type and mutant POF1B proteins to bind nonmuscle actin filaments in vitro. We found that the capacity of the mutant protein to bind nonmuscle actin filaments was diminished fourfold compared with the wild type, suggesting a function of POF1B in germ-cell division. Our study suggests that a homozygous point mutation in POF1B influences the pathogenesis of POF by altering POF1B binding to nonmuscle actin filaments.     

Monoclonal antibody to human IgG Fc receptors. Cross-linking of receptors induces lysosomal enzyme release and superoxide generation by neutrophils

The monoclonal antibody KuFc79 binds to a determinant on the Fc receptors (Fc gamma R) of human leukocytes. We examined the biologic effects of the interaction of this antibody with Fc gamma R on human neutrophils (PMNL). The univalent Fab fragment of KuFc79 inhibits the formation of rosettes with IgG-sensitized sheep erythrocytes by as much as 91.7%. In other experiments in which PMNL were washed after exposure to Fab of KuFc79, phagocytosis of IgG-sensitized sheep erythrocytes was inhibited by 36%. Fab fragments of other mouse IgG2b monoclonal proteins did not have these effects. When PMNL are exposed to coverslips coated with univalent Fab fragments of this antibody, the Fc gamma R are removed from the surface of the PMNL. Under these conditions, rosetting could be inhibited by 85.4%. We examined cross-linking of receptor bound monoclonal antibody or its Fab fragment by either Protein A or F(ab')2 of an anti-mouse Ig. As much as 31.7% of beta-glucuronidase, a marker for lysosomal enzymes, is specifically released by cross-linking the Fc gamma R on PMNL. The generation of O2- is also induced by specifically cross-linking Fc gamma R with Fab and anti-Fab. The data constitute the first formal demonstration that cross-linking of Fc gamma R on PMNL leads to enzyme release and superoxide generation.     

Different influences of cytochalasin B on the activation of human neurophils settled onto petri dishes displayed by simultaneously detected native and luminol-dependent luminescence

Cytochalasin B (CB) is known to interfere reversibly with the cytoplasmic contractile filamental network of mammalian cells. The role of the microfilament system in the mechanism of the reactive oxygen intermediates release of polymorphonuclear leukocytes (PMNL) was studied for different kinds of stimuli. PMNL from fresh human blood were treated with CB and stimulated by adherence on plastic surfaces, by opsonized zymosan, by phorbol myristate acetate and by N-formylmethionyl-phenylalaline. The production of reactive oxygen species were monitored by simultaneous detection of native, luminol-independent, luminescence (NL) and luminol-dependent luminescence (LDL) using a method of spectral discrimination. Different influences of CB on NL with respect to LDL as well stimuli-dependent influences of CB on the luminescence response of PMNL were observed. Especially phagocytosis-associated activation of PMNL was strongly inhibited by CB, whereas LDL was reduced to a much greater extent in comparison with NL. A firm involvement of the microfilament system is indicated, but it depends on the kind of stimulus engaged.     

Autoimmune targeted disruption of the pituitary-ovarian axis causes premature ovarian failure

Premature ovarian failure (POF) is characterized by amenorrhea and high serum levels of follicle-stimulating hormone (FSH). POF causes female infertility and represents a substantial women's health risk affecting 1% of women by age 40. Although ovarian autoimmunity has been associated with POF, the identity of ovarian Ags recognized is unknown. In this study, we show that autoimmune-targeted disruption of the pituitary-ovarian axis leads to POF. Immunization of SWXJ female mice with the p215-234 peptide derived from mouse inhibin-alpha activates CD4(+) T cells and induces experimental autoimmune oophoritis with a unique biphasic phenotype characterized by an early stage of enhanced fertility followed by a delayed stage of POF. Affected mice show high serum levels of inhibin-alpha-neutralizing Abs that prevent inhibin-mediated down-regulation of activin-induced pituitary FSH release. The loss of activin/FSH down-regulation leads to prolonged metestrus-diestrus, superovulation, increased numbers of mature follicles, increased offspring, accelerated depletion of primordial follicles, and ultimately premature infertility. Thus, inhibin-alpha-targeted experimental autoimmune oophoritis is initiated by CD4(+) Th1 T cells that stimulate B cells to produce inhibin-alpha-neutralizing Abs directly capable of mediating POF and transferring disease into naive recipients. Our inhibin-alpha autoimmune model of POF shows how premature infertility may develop in the context of elevated FSH levels thereby closely mimicking the hallmark features of human POF.     

Modulation of human polymorphonuclear leukocyte chemotaxis and superoxide anion production by Pseudomonas aeruginosa exoproducts, IL-1β and piroxicam

Abstract Whereas addition of 200 ng ml⁻¹ exotoxin A (exoA) did not modify PMNL chemotaxis, 20 U ml⁻¹ human recombinant interleukin-1β (hrIL-1β) primed polymorphonuclear leukocytes (PMNL) for migration towards Pseudomonas aeruginosa peptide chemotactins (PAPCs). Piroxicam (100 μg ml⁻¹), a non-steroidal anti-inflammatory agent (NSAIA), inhibited PMNL chemotaxis and abolished the priming effect of hrIL-1β. Both PAPCs and exoA induced PMNL superoxide anion production, but neither hrIL-1β nor piroxicam modified significantly PMNL superoxide anion production induced by PAPCs. The fact that hrIL-1β can prime PMNL for chemotaxis towards PAPCs and that piroxicam can abolish activation by primed PMNL are findings relevant to the pharmacological control of lung tissue damage during P. aeruginosa pneumonia.     

Role of interleukin-1 and tumour necrosis factor in leukocyte recruitment to acute dermal inflammation

The cytokines IL-1 and TNF-alpha are involved in inflammation and their production is stimulated by various agents, especially endotoxin (LPS). Here, using the human IL-1 receptor antagonist (IL-1RA) and a new monoclonal antibody (mAb 7F11) to rabbit TNF, the role of endogenous IL-l and TNF production in acute (3h) leukocyte (PMNL) recruitment to dermal inflammation in rabbits has been studied. IL-1RA inhibited by 27% the PMNL accumulation in reactions induced by killed Escherichia coli (p < 0.05) but not by LPS. The monoclonal antibody to TNF inhibited by 27% and 38% (p < 0.002) the PMNL accumulation in LPS and E. coli reactions respectively, but a combination of the mAb with IL-1RA was not more effective. Treatment of human umbilical vein endothelium with LPS for 3 h activated endothelium to induce PMNL transendothelial migration in vitro, which was not inhibited by IL-1RA, antibody to TNF-alpha, IL-1 or to IL-8. In conclusion, TNF and IL-1 may partially mediate acute PMNL infiltration in vivo to LPS and Gram negative bacteria, but there is a major IL-1/TNF independent mechanism, at least in dermal inflammation, which may be due to direct LPS activation of the microvasculature or perhaps the generation of cytokines other than IL-1 and TNF.     

The effect of antifungal agents on the chemoluminescence of polymorphonuclear leukocytes

The intensity of the formation of active forms of oxygen by Staphylococcus-activated polymorphonuclear leukocytes (PMNL) was registered according to the level of luminol-dependent chemiluminescence. Peritoneal PMNL of rats were shown to be already in an activated state. Activation probably occurred in the process of their penetration into the abdominal cavity. For this reason, further studies of three chloral and griseofulvin derivatives with respect to their influence on the metabolic activity of phagocytes were made on PMNL of human peripheral blood. The compounds under study were found to produce a suppressive effect, which was probably linked with their influence of the enzymatic cell systems taking part on the formation of active forms of oxygen.