Polymorphonuclear Leukocytes Viability Research Articles

Blood polymorphonuclear leukocyte responses against Staphylococcus aureus in primiparous and pluriparous Lacaune and Santa Inês ewes

• This study provides novel insights into PMNLs’ functionality in primiparous and pluriparous ewes. • A difference was observed in the intensity of the phagocytic response to S. aureus in pluriparous ewes. • Antibody-mediated opsonization of S. aureus was associated with a lower intensity of phagocytosis. In this study, we evaluated the viability, intracellular production of reactive oxygen species (ROS) and phagocytosis of Staphylococcus aureus by blood polymorphonuclear leukocytes (PMNLs) in primiparous and pluriparous Santa Inês and Lacaune ewes. Blood was collected from 14 Santa Inês ewes (seven primiparous and seven pluriparous) and 14 Lacaune ewes (seven primiparous and seven pluriparous). The viability of PMNLs, intracellular ROS production, and S. aureus phagocytosis were evaluated using flow cytometry. No parity effect on blood’s PMNLs functionality was found. However, comparing primiparous and pluriparous ewes, we noticed a difference in the opsonized phagocytic response to S. aureus , suggesting that pluriparous ewes could be better protected against S. aureus , although further field studies are needed. Antibody-mediated opsonization of S. aureus was associated with a lower intensity of S. aureus phagocytosis in both primiparous and pluriparous ewes, while it had no effect on the percentage of S. aureus phagocytosis. The percentage and geometric mean fluorescence intensity (GMFI) of S. aureus phagocytosis behaved differently in Lacaune and Santa Inês ewes, which could be explained by breed and farm effects. The intensity of intracellular ROS production by PMNLs when stimulated with S. aureus was higher than that of unstimulated basal PMNLs in both primiparous and pluriparous ewes, while no effect on the percentage of PMNLs that produced ROS was observed. Thus, our study provides novel insights into PMNLs’ functionality in primiparous and pluriparous ewes, which could impact their health, although further research is needed to extrapolate conclusions to the field.

Blood and milk polymorphonuclear leukocyte and monocyte/macrophage functions in naturally caprine arthritis encephalitis virus infection in dairy goats

The exact influence of caprine arthritis encephalitis virus (CAEV) infection on blood and milk polymorphonuclear leukocytes (PMNLs) and monocyte/macrophages of goats remains unclear. Thus, the present study sought to explore the blood and milk PMNL and monocyte/macrophage functions in naturally CAEV-infected goats. The present study used 18 healthy Saanen goats that were segregated according to sera test outcomes into serologically CAEV negative (n=8; 14 halves) and positive (n=10; 14 halves) groups. All milk samples from mammary halves with milk bacteriologically positive outcomes, somatic cell count ≥2×106cellsmL−1, and abnormal secretions in the strip cup test were excluded. We evaluated the percentage of blood and milk PMNLs and monocyte/macrophages, the viability of PMNLs and monocyte/macrophages, the levels of intracellular reactive oxygen species (ROS) and the nonopsonized phagocytosis of Staphylococcus aureus and Escherichia coli by flow cytometry. In the present study, a higher percentage of milk macrophages (CD14+) and milk polymorphonuclear leukocytes undergoing late apoptosis or necrosis (Annexin-V+/Propidium iodide+) was observed in CAEV-infected goats; we did not find any further alterations in blood and milk PMNL and monocyte/macrophage functions. Thus, regarding our results, the goats naturally infected with CAEV did not reveal pronounced dysfunctions in blood and milk polymorphonuclear leukocytes and monocytes/macrophages.

Assessment and comparison of phagocytic function and viability of polymorphonuclear leukocytes in saliva of smokers and non-smokers

OBJECTIVESTobacco use is one of the most important public health problems worldwide. It is also linked to impairment of normal immunologic surveillance and defence mechanism of polymorphonuclear leukocytes. Tobacco smoke and its components have been seen to affect the phagocytic ability and viability of polymorphonuclear leukocytes suggesting the pathogenesis of tobacco induced oral diseases. Aim of this study was to assess and compare the phagocytic function and viability of polymorphonuclear leukocytes in saliva of smokers and non smokers. DESIGNThe study comprised of 35 smokers and 35 non-smokers, age matched. Saliva was collected by rinsing method and the polymorphonuclear leukocytes were separated. Phagocytic activity was determined by using latex spheres as targets. Cell viability was measured using trypan blue stain. RESULTSSalivary polymorphonuclear leukocytes in smokers showed significant reduction in the phagocytic activity by ingesting few latex spheres when compared to the non-smokers. The viability of these cells in saliva of smokers was significantly reduced. CONCLUSIONSThe present study revealed reduced phagocytic activity and viability of salivary polymorphonuclear leukocytes in smokers compared to non-smokers. These findings indicate that smokers are more prone to gingival, periodontal and other oral diseases. Thus indicating that the health care professionals should encourage smoking cessation as an aid in preventing oral diseases.

Efeito da nicotina sobre fagócitos ativados

Smokers show increased rates of polymorphonuclear leukocytes (PMNL), including in pulmonary tissue, resulting increased levels of proteolytic enzymes and reactive oxygen species, which have a destructive effect on the cellular matrix and contribute to the progression of pulmonary disease, in addition to promoting the onset of microbial infections which cause phagocytic cells to be in a state of frequent activation (phagocytosis). This work evaluated the influence of nicotine (NIC) on the viability of PMNL and macrophages (activated or not), by stimuli zymosan and phorbol myristate acetate. The results indicated that NIC led to increased viability of PMNL and activated macrophages compared to these cells activated without NIC, measured ex vivo by trypan blue exclusion test. This effect was significantly higher on PMNL than on macrophages. This reduction in cytotoxicity favors cell survival, and may exacerbate its deleterious effect, especially in the active state, due to increased production of reactive oxygen species.

Apoptosis and necrosis of polymorphonuclear leukocytes in goat milk with high and low somatic cell counts

The purpose of the present trial was to compare the percentages of necrotic and apoptotic polymorphonuclear leukocytes (PMNL) in goat milk with low and high somatic cell count (SCC). Twenty eight milk samples were collected from 20 lactating goats, determined to be negative in bacteriological examination, and divided in three groups, according to their SCC: samples with SCC lower than 500×103cells/mL; between 500 and 1500×103cells/mL; and higher than 1500×103cells/mL. SCC was performed in an automatic somatic cell counter. Apoptosis and necrosis were quantified using dual-color flow cytometry with fluorescein labeled annexin-V and propidium iodide (PI). Results of the present study showed a significant positive correlation between the percentage of the viable PMNL and milk SCC (r=0.495, P=0.008), as well as a significant negative correlation between apoptotic PMNL and milk SCC (r=−0.486, P=0.009). Results also pointed out lower PMNL viability rates due to higher apoptosis rates in milk samples with SCC lower than 5×105cells/mL.

The Effects of Peritoneal Dialysis Solutions on Peritoneal Host Defense

Conventional peritoneal dialysis fluid (PDF) is a bioincompatible solution owing to the acidic pH, the high glucose concentrations and the associated hyperosmolarity, the high lactate concentrations, and the presence of glucose degradation products (GDPs). This unphysiologic composition adversely affects peritoneal host defense and may thus contribute to the development of PD-related peritonitis. The viability of polymorphonuclear leukocytes, monocytes, peritoneal macrophages, and mesothelial cells is severely depressed in the presence of conventional PDF. In addition, the production of inflammatory cytokines and chemoattractants by these cells is markedly affected by conventional PDF. Further, conventional PDF hampers the recruitment of circulating leukocytes in response to an infectious stimulus. Finally, phagocytosis, respiratory burst, and bacterial killing are markedly lower when polymorphonuclear leukocytes, monocytes, and peritoneal macrophages are exposed to conventional PDF. Although there are a few discrepant results, all major PDF components have been implicated as causative factors. Generally, novel PDF with alternative osmotic agents or with alternative buffers, neutral pH, and low GDP content have much milder inhibitory effects on peritoneal host defense. Clinical studies, however, still need to demonstrate their superiority with respect to the incidence of PD-related peritonitis.

Leukotriene B 4 generation by blood polymorphonuclear leukocytes after incubation in urine

To investigate the capacity of leukocytes in urine to produce leukotriene B 4 (LTB 4), we evaluated calcium ionophore A23187-induced LTB 4 generation by human blood polymorphonuclear leukocytes (PMNL) after these cells were incubated in urine under various conditions. LTB 4 production was markedly reduced after incubation of PMNL in urine of high or low pH and high osmolarity. The production was also inhibited by a prolonged incubation period. There was a significant correlation (r = 0.82, p < 0.01) between LTB 4 production and PMNL viability. Our results indicate that it is possible to evaluate the LTB 4 production capacity of urinary PMNL by determining PMNL viability only. Furthermore acidification of urine hamper the LTB 4 generation which might be unfavorable in vivo.

Attenuation of ecto-5'-nucleotidase activity and adenosine release in activated human polymorphonuclear leukocytes.

To examine whether activation of polymorphonuclear leukocytes attenuates release of adenosine through attenuation of their own ecto-5'-nucleotidase activity, human polymorphonuclear leukocytes were incubated with and without exposure to either N-formyl-methionyl-leucyl-phenylalanine (FMLP) or complement C5a. Ecto-5'-nucleotidase activity of polymorphonuclear leukocytes was attenuated by both FMLP and complement C5a (22.7 +/- 3.6 vs 9.7 +/- 2.6 nmol/min per 10(7) cells at 10(-6) M FMLP, P < .05; 21.5 +/- 2.2 vs 10.2 +/- 1.2 nmol/min per 10(7) cells at 5 x 10(-7) g/mL complement C5a, P < .001), whereas cytosolic 5'-nucleotidase activity was not affected by either FMLP or complement C5a. These reductions of ecto-5'-nucleotidase activity that were caused by both FMLP and complement C5a were dose and time dependent and were inhibited by superoxide dismutase. Desferrioxamine did not inhibit the decreases in ecto-5'-nucleotidase. In accordance with the decreases in ecto-5'-nucleotidase activity, release of adenosine was attenuated in the FMLP-pretreated and complement C5a-pretreated polymorphonuclear leukocytes, which were restored by concomitant administration of superoxide dismutase. The viability of FMLP-pretreated and complement C5a-pretreated polymorphonuclear leukocytes was markedly decreased compared with the untreated group after 60 minutes of hypoxia followed by 60 minutes of reoxygenation. Thus, we conclude that: (1) activation of polymorphonuclear leukocytes attenuates their own ecto-5'-nucleotidase activity and thereby reduces adenosine release, (2) reduction of ecto-5'-nucleotidase activity is attributable to generated superoxide anion in polymorphonuclear leukocytes, and (3) viability of polymorphonuclear leukocytes after hypoxia and reoxygenation largely depends on the extents of decreases in ecto-5'-nucleotidase activity.

Fungistatic Activity of Human Neutrophils against Histoplasma capsulatum: Correlation with Phagocytosis

The interaction of human polymorphonuclear leukocytes (PMNL) and Histoplasma capsulatum was studied. In limiting dilution assays and in a 2-h coculture system, PMNL were ineffective in killing H. capsulatum whereas Candida albicans was readily killed. Although PMNL could not reduce inoculum colony-forming units of H. capsulatum in short-term assays, they were highly fungistatic in 24- (47%-55%) and 72-h (65%-75%) cocultures. Phagocytosis of yeast cells by PMNL was 81.3% +/- 3.7%. Fungistasis persisted even though PMNL viability decreased with time. Fungistatic activity of PMNL was dose dependent. Fungistasis was verified by direct hemocytometer counts of fungal units. In transwell experiments, PMNL contact with H. capsulatum was required for fungistatic activity. Lymphocytes, PMNL lysates, or PMNL supernatants were not fungistatic in this system.

Trospectomycin Enhances Surface Phagocytosis of Bacteroides and Staphylococcus by Altering the Bacterial Glycocalyx

The influence of trospectomycin on phagocytosis of Bacteroides thetaiotaomicron, Bacteroides fragilis and Staphylococcus epidermidis was studied in the presence or absence of glycocalyx isolated from these microorganisms. Bacteria were grown with or without 0.25 or 0.5 of the minimal inhibitory concentration (MIC) of trospectomycin, a new chemically synthesized analog of spectinomycin. Surface phagocytosis by human polymorphonuclear leukocytes (PMNL) was determined using a modified fluorochrome assay. Subinhibitory concentrations of trospectomycin significantly enhanced surface phagocytosis of Bacteroides and Staphylococcus. When homologous or heterologous isolated glycocalyx was added to trospectomycin treated bacteria prior to incubation with PMNL, phagocytosis was reduced to levels observed in the untreated bacteria. Addition of glycocalyx to untreated strains produced no significant reduction of phagocytosis. The glycocalyx preparations were free of lipopolysaccharide and did not affect PMNL viability.

Effects of alpha and theta toxins from Clostridium perfringens on human polymorphonuclear leukocytes.

Two toxins, alpha (phospholipase C) and theta (oxygen-labile hemolysin), were purified from Clostridium perfringens type A and assayed for toxic effects on human polymorphonuclear leukocytes (PMNLs). Crude preparations containing both toxins totally inhibited chemotaxis and chemiluminescence responses of PMNLs and reduced PMNL viability. Purified alpha toxin did not alter PMNL viability, chemotactic responsiveness, or morphology but did enhance opsonized zymosan-induced PMNL chemiluminescence over a wide range of toxin concentrations. theta Toxin, at 12.5 hemolytic units (HU) per 10(5) PMNLs, reduced cell viability and induced marked PMNL morphological changes. Concentrations of theta toxin between 4 and 32 HU per 10(5) PMNLs inhibited PMNL chemiluminescence in a dose-dependent manner, whereas a lower concentration enhanced the PMNL chemiluminescent response to opsonized zymosan. Effects on chemotaxis were also dose dependent. Increased PMNL random migration was observed at a concentration of theta toxin of 0.06 HU per 2.5 X 10(5) PMNLs (P less than .05), whereas concentrations of greater than 0.08 HU per 2.5 X 10(5) PMNLs reduced both directed and random migration (P less than .05).

Inhibition of human polymorphonuclear leukocyte respiratory burst, bactericidal activity, and migration by pneumolysin.

The in vitro effects of pneumolysin, a sulfhydryl-activated toxin produced by Streptococcus pneumoniae, on various functions of human polymorphonuclear leukocytes (PMNLs) was investigated. Treatment of PMNLs with highly purified toxin significantly inhibited respiratory burst (in response to stimulation), ability to kill opsonized pneumococci, chemotaxis, and random migration. These inhibitions were observed at very low toxin doses (less than or equal to 1 hemolytic unit (2 ng) per 10(6) PMNLs), which had no effect on PMNL viability. These results suggest that pneumolysin could function in pathogenicity by interfering with the ability of PMNLs to migrate toward and kill pneumococci.