C3a is a chemotaxin for human eosinophils but not for neutrophils. I. C3a stimulation of neutrophils is secondary to eosinophil activation

Inflammatory action of the potent chemotaxin C5a has been well characterized on a variety of human cell types, including neutrophils, monocytes, basophils, and eosinophils. The cellular effects of C3a are less well defined. Contradictory reports have been published for C3a activation of neutrophils. Recent reports that C3a activates both basophils and eosinophils prompted us to reinvestigate the effects of C3a stimulation on eosinophils. We hypothesized that C3a activation of eosinophils, cells that are present in most neutrophil preparations, might lead to neutrophil activation. Using neutrophils of 98% purity, we observed no evidence of cellular activation after stimulation with either C3a, recombinant human C3a (rhC3a), or the synthetic C3a analogue C3a 57-77, Y57. Eosinophils purified to > 98% purity displayed concentration-dependent polarization, chemotaxis, and enzyme release by stimulation with C3a, rhC3a, and the synthetic C3a analogue. An inactive form of C3a, C3adesArg, failed to stimulate either eosinophils or neutrophils. Using neutrophil preparations containing 5-9% eosinophils, up to 20% of neutrophils became polarized after exposure to C3a. Likewise, we demonstrated that supernatant from C3a-stimulated eosinophils promotes neutrophil chemotaxis. Eosinophil polarization experiments were repeated in the presence of antibody to the C5a receptor (C5aR) to show that C3a and C5a interact with different receptors. C3a activates eosinophils in the presence of anti-C5aR antibody at concentrations that fully block C5a activation. We conclude that eosinophils are directly activated by either C3a or C5a, whereas C3a failed to activate neutrophils. C3a acts on eosinophils via a receptor that is distinct from C5aR. Since neutrophils are indirectly stimulated by C3a, eosinophils contaminating neutrophil preparations may explain earlier reports that C3a activates human neutrophils.     

Differentiation induction in non-lymphocytic leukemia cells upon treatment with mizoribine

The effectiveness of an ideal antimicrobial agent depends on its ability to kill microbes with minimal toxicity to host cells. Depending on the treatment regimen, antimicrobial agents come into contact with host cells for various intervals of time. Sanguinarium (SANG), chlorhexidine (CHX) and tetracycline (TET) are 3 antimicrobial agents frequently used in the management of periodontal infections. However, their effects on host immune cells during different treatment regimens are not known. Due to their ability to serve as the first line of host defense against microbial infections, we have compared the effects of these antimicrobial agents on human neutrophil functions and viability. The results show that SANG is not lytic to neutrophils from peripheral blood or crevicular fluid, at all concentrations tested. However, exposures of neutrophils to very low concentrations of SANG (0.001%) inhibits neutrophil chemotaxis, oxidative metabolism and degranulation within 5 min. Increasing the exposure time results in a similar inhibition of neutrophil functions, albeit at 50-100 fold lower concentrations of SANG. CHX rapidly disrupts the cell membrane of both crevicular and peripheral blood neutrophils at concentrations above 0.005% within 5 min, and inhibition of all neutrophil functions is due to its lytic properties. While TET is least toxic to neutrophils, a dose dependent inhibition of neutrophil functions is dependent on the calcium concentrations of the cellular environment, and is observed only above 0.04% or higher concentrations in the absence of calcium. The data suggest that a critical cumulative concentration of these drugs is essential for their toxicity and inhibition of neutrophil functions. Therefore, both the length of exposure and the dose of the drug both are critical while considering the effectiveness of SANG, CHX or TET in the treatment of infections. Furthermore, due to differences in their mechanisms of action, the consequences of their effects on neutrophils may have significant bearing on tissue pathology as well as on their therapeutic efficacy.     

Priming induces functional coupling of N-formyl-methionyl-leucyl-phenylalanine receptors in equine neutrophils

The synthetic formylpeptide fMLP is widely used as a model chemoattractant and secretagogue for mammalian neutrophils. Despite possessing fMLP receptors, equine neutrophils do not produce superoxide anions in response to fMLP and there is no inflammatory reaction in the horse when fMLP is injected intradermally. The functional capability of these receptors was investigated after pretreatment with recognized priming agents. Purified neutrophils were pretreated with lipopolysaccharide (LPS), platelet-activating factor (PAF), or tumor necrosis factor alpha (TNF-alpha) and superoxide anion generation and shape change quantified by lucigenin-dependent chemiluminescence (LDCL) and flow cytometry, respectively. LPS, TNF-alpha, and PAF pretreatment induced significant LDCL in response to fMLP; similarly LPS pretreatment was a prerequisite for fMLP-stimulated neutrophil polarization in response to fMLP. However, LPS failed to induce fMLP-mediated chemotaxis of equine neutrophils. These data indicate that equine neutrophil fMLP receptors are not vestigial as previously thought but can trigger both respiratory burst activity and cell polarization responses after priming.     

Evidence for the denaturation of recombinant hepatitis B surface antigen on aluminium hydroxide gel

Eosinophils, prominent cells in asthmatic inflammation, undergo apoptosis or programmed cell death following deprivation of contact with survival-promoting cytokines such as IL-5 and GM-CSF. The aim of this study was to assess a number of techniques for the quantification of apoptosis in human eosinophils cultured with or without IL-5 or GM-CSF and following staurosporine treatment. The relationship between apoptosis and necrosis in eosinophils was also determined. Eosinophils 'aged' in vitro for 48 h exhibited endonuclease DNA degradation, apoptotic morphology, increased red autofluorescence and externalisation of phosphatidylserine (PS) as assessed by binding of FITC-labelled annexin V. Annexin V-FITC binding was first detectable in eosinophils maintained at 37 degrees C for 5 h post-purification. This method proved to be the most sensitive marker of apoptosis. Morphological assessment of wet preparations of eosinophils by Kimura staining was found to be the next most-sensitive marker followed by increased red autofluorescence. The latter was a relatively insensitive method for the detection of apoptosis. At 5, 20 and 24 h of culture trypan blue exclusion indicated that eosinophil viability was high (85-90% viable cells). However, propidium iodide (PI) staining and flow cytometry revealed that, by 24 h, approximately 75% of cells had compromised membrane integrity. Eosinophils maintained in IL-5 or GM-CSF exhibited a non-apoptotic morphology and levels of annexin V-FITC binding and PI uptake similar to that of freshly isolated cells. Staurosporine (10(-5) M) treatment of eosinophils maintained in IL-5 or GM-CSF resulted in significant levels of apoptotic morphology at 2 h (23.8% +/- 6.9, p < 0.025) which was associated with negligible annexin binding. At 6 h post-staurosporine treatment significant annexin-FITC binding (38% +/- 1.5, p < 0.025) was observed compared with 93% +/- 1.2 of eosinophils displaying apoptotic morphology. Exclusion of PI demonstrated membrane integrity at all time points up to 6 h. Thus, eosinophils aged in vitro in the absence of viability-promoting cytokines exhibit evidence of both apoptosis and necrosis simultaneously. In contrast, staurosporine-treated eosinophils exhibited both membrane integrity and rapid apoptosis-associated morphological changes detected by single step Kimura staining which preceded externalisation of PS.     

Identification of eosinophils in lysed whole blood using side scatter and CD16 negativity

The identification of eosinophils in lysed whole blood by flow cytometry can be problematic, since these cells overlap significantly with the neutrophil cluster on forward scatter versus side scatter plots of whole blood samples. Current methods can be time-consuming when running multiple samples or may compromise yield in the interests of greater accuracy. The use of eosinophil purification techniques prior to FACS analysis or sorting as a way of ensuring purity may have unpredictable effects on eosinophil activation, leading to questionable data interpretation. Here we describe a simple, single-step method for definition of eosinophils utilizing their high side scatter and CD16 fluorescence negativity to differentiate them from neutrophils. The purity of the neutrophil and eosinophil populations sorted with this gate is close to 100% regardless of the peripheral blood eosinophil count, while the population obtained by sorting on a plot of FSC/SSC was a mixture of eosinophils and neutrophils. We suggest this method as a simple, reproducible, and accurate way of defining eosinophils by flow cytometry for analysis or sorting.     

The intravenous administration of tumor necrosis factor alpha, interleukin 8 and macrophage-derived neutrophil chemotactic factor inhibits neutrophil migration by stimulating nitric oxide production

1. The i.v. administration of tumor necrosis factor-alpha (TNF-alpha), interleukin-8 (IL-8) and the recently described macrophage-derived neutrophil chemotactic factor (MNCF) inhibits the recruitment of neutrophils to the inflammatory site. 2. Pretreatment of mice with the NO synthase antagonist, NG-monomethyl-L-arginine (L-NMMA, 15-60 mg kg(-1)), but not the inactive enantiomer D-NMMA (30 mg kg(-1)), prevented in a dose-dependent manner the TNF-alpha, IL-8 and MNCF-mediated inhibition of neutrophil migration into thioglycollate-challenged peritoneal cavities. 3. Treatment of the neutrophils with TNFalpha (10(-7) M), IL-8 (10(-7) M) or MNCF blocked their migration towards FMLP in the chemotaxis assay. The pretreatment of the neutrophils with L-NMMA (50-200 microM) prevented in a dose-dependent manner the inhibition of FMLP-induced chemotaxis by IL-8, but did not alter the inhibition caused by TNF-alpha or MNCF. Different concentrations of the NO donors, S-nitroso-N-acetylpenicillamine (SNAP) or 3-morpholino-sydnonimine (SIN-1), did not alter this chemotaxis. 4. Preincubating the neutrophils with L-NMMA (200 microM) significantly increased the TNF-alpha (10(-7) M) and MNCF-mediated neutrophil adhesion to unstimulated endothelial cells, but had no effect on IL-8 (10(-7) M)-mediated adhesion. 5. Although NO donors did not directly affect the mechanisms of neutrophil motility, NO is involved in the in vitro inhibitory action of IL-8 on chemotaxis. The TNF-alpha and MNCF-mediated inhibition of neutrophil migration seems to be indirect, by affecting the mechanisms of adhesion. It was concluded that TNF-alpha-, IL-8- and MNCF-mediated inhibition of neutrophil migration is associated with the stimulation of NO production.     

Characterization of antichemotactic factor extracted from the gastric mucosa of rats

The gastric mucosa of normal rats exhibits no detectable inflammation or visible damage. We examined the effect of the gastric mucosal extract of rats on neutrophil chemotaxis and tried to purify antichemotactic factor. The chemotaxis of neutrophils was examined by the modified Boyden's method. After mucosal layer was scraped and then homogenized and centrifuged at 20,000 x g for 30 min, the supernatant was used as rat gastric mucosal extract (RGME). Prior exposure of neutrophils to the gastric mucosal extract caused a dose-dependent reduction in the neutrophil migration induced by formyl-methionyl-leucyl-phenylalanine (FMLP), leukotriene B4 (LTB4) and interleukin 8 (IL-8) without affecting the cell viability. The antichemotactic factor was partially purified by lectin affinity chromatography on wheat germ lectin (WGL)-Sepharose, anion exchange chromatography on Mono-Q and gel filtration on Superose 12. The molecular weight of the antichemotactic factor was estimated to be around 60 k by gel filtration. The activity was markedly abolished by boiling for 5 min, heating at 60 degrees C for 30 min, and treatment with 1% acetic acid, 0.1 M Na2CO3 or trypsin. Furthermore, the FMLP-induced migration of neutrophils pretreated with the antichemotactic factor for 5 min followed by washing with fresh medium was inhibited, although the factor was not added to the chamber. These results suggest that the gastric mucosa of rats intrinsically generates an antichemotactic factor which might play a crucial role in maintenance of the integrity of the gastric mucosa.     

Isolation of a novel mouse gene MA-3 that is induced upon programmed cell death

Haematopoietic progenitor cells were isolated from human fetal liver, obtained between 6 and 15 weeks gestation. After preparation of a single cell suspension, the cells were stored using a stepwise freezing protocol; taking the cells from room temperature through -70 degrees C to liquid nitrogen. Viability (trypan blue exclusion), morphology (Leishman stain), identification of cell type (flow cytometry) and growth characteristics in semi-solid culture medium were assessed using the fresh cell suspension. We were able to confirm that the predominant cells in human fetal liver up to about 15 weeks gestation are those of the erythroid lineage. It was established that viability in excess of 75% was required to ensure adequate growth in culture after frozen storage and it was deemed important to ensure morphological integrity of the cell preparations. The colonies formed in culture were observed to be producing haemoglobin between 7 and 9 days after initial seeding. We have determined that cells can be stored in liquid nitrogen for up to 2 years without loss of (1) viability, (2) morphological features and (3) ability to form colonies and produce haemoglobin in culture. These findings offer encouragement for the implementation of a cell bank to support an in utero transplantation programme.     

Symptoms of depression in residents: a South Carolina Family Practice Research Consortium study

OBJECTIVES: To test the hypothesis that loss of polymorphonuclear neutrophil tumor necrosis factor alpha (TNF-alpha) receptors during transmigration renders the exudate neutrophil refractory to TNF-alpha-mediated stimulation of apoptosis; and to investigate the surface expression of Fas on both circulating and exudate neutrophils. DESIGN: A prospective cohort study. SETTING: Surgical laboratory of a tertiary care hospital. PARTICIPANTS: Twenty-one healthy human volunteers. INTERVENTIONS: All subjects had circulating neutrophils and exudate neutrophils collected by venipuncture and skin window methods, respectively. MAIN OUTCOME MEASURES: Circulating and exudate neutrophils were incubated in culture medium (1.0x10(6) neutrophils per milliliter) alone or with TNF-alpha (100 ng/mL). Apoptosis was evaluated by flow cytometry (annexin V-fluorescein isothiocyanate and propidium iodide). Tumor necrosis factor alpha-phycoerythrin and anti-human Fas-fluorescein isothiocyanate were used to evaluate neutrophil TNF-alpha receptors and surface expression of Fas. RESULTS: Exudate neutrophils had a significant delay in apoptosis rates when compared with circulating neutrophils. The percentage of neutrophils expressing TNF-alpha receptors was significantly diminished after exudation (80%+/-15% vs 33%+/-9%; P<.001), as was the median channel number of TNF-alpha phycoerythrin fluorescence (8.1+/-1.6 vs 5.2+/-0.5; P=.001). However, the expression of Fas was unchanged after transmigration (percentage positive for Fas: 98.7%+/-0.7% vs 92.8%+/-3.4%, P=.89; Fas antibody-fluorescein isothiocyanate median channel fluorescence: 12.2+/-1.1 vs 13.1+/-1.2; P=.80). Exposure of exudate neutrophils to TNF-alpha failed to increase their rate of apoptosis. CONCLUSIONS: Exudate polymorphonuclear neutrophils are confirmed to have delayed apoptosis. Loss of TNF-alpha receptors during transmigration is necessary for neutrophil survival in the extravascular inflammatory milieu.     

The colorectal adenoma-carcinoma sequence: the limits between polypectomy and intestinal resection

A hemocytometer-based trypan blue dye exclusion cell quantitation and viability assay was compared with a similar assay using simultaneous fluorometric staining with fluorescein diacetate and propidium iodide. Viable and nonviable cell densities were measured, and culture viability was calculated both during the normal growth cycle of a murine hybridoma and in response to the application of millimolar concentrations of either tert-butyl hydroperoxide or ferrous iron. During the early phase of rapid hybridoma cell growth, assay-based differences in viable cell density were not significant. As the culture aged, the trypan blue dye exclusion assay significantly overestimated cell viability, thereby underestimating nonviable cell density and yielding an erroneous estimation of the overall viability of the culture. Because of its lack of ambiguity in the identification of stained, nonviable cells and its resulting increased accuracy in the estimation of culture viability, the fluorometric assay was considered a better choice for the evaluation of cell viability.     

The isoflavone genistein inhibits copper and peroxyl radical mediated low density lipoprotein oxidation in vitro

OBJECTIVES: Hormone-independent and cytotoxic drug-resistant tumor growth in osteoblastic metastases defines poor survival in patients with advanced prostate cancer. Therefore, we analyzed the ability of human osteoblast-like cells (MG-63 cells) and MG-63 conditioned media (MG-63 CM) to protect PC-3 human prostate cancer cells from adriamycin cytotoxicity in vitro. METHODS: Adriamycin cytotoxicity was assessed in MG-63 osteoblast-like and PC-3 prostate cancer monolayer and three-dimensional collagen coculture systems using the DNA content and trypan blue exclusion assays, analysis of indexes of cell cycle by flow cytometry, determination of DNA fragmentation on simple agarose gel and terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay, and immunocytochemistry. RESULTS: Adriamycin (100 nM) arrested both the PC-3 and MG-63 cells at the G2/M phase in the cell cycle but induced apoptosis only in PC-3 cells, as assessed by flow cytometry, trypan blue exclusion, and agarose gel. Optimal doses of MG-63 CM (50 microg/mL), insulin-like growth factor I (50 ng/mL), and transforming growth factor-beta-1 (25 ng/mL), as determined by DNA content assay, partially neutralized the adriamycin cytotoxicity of PC-3 cells detected by flow cytometry and trypan blue exclusion. In addition, MG-63 cells rescued PC-3 cells from adriamycin apoptosis in the three-dimensional type I collagen gel coculture system, as analyzed by TUNEL assay. CONCLUSIONS: These data suggest that osteoblast-like cells and osteoblast-derived growth factors can optimize survival of metastatic prostate cancer cells, thereby helping to develop cytotoxic drug-resistant growth in vitro.     

Aortoiliac stent placement in patients treated for intermittent claudication

Inhaled nitric oxide (NO) is an important new therapeutic agent used to treat pulmonary arterial hypertension in a variety of disease states. However, the effects of NO on cells in the lung are uncertain. Previously, we have shown that NO gas depresses neutrophil oxidative cell function and increases neutrophil cell death. The purpose of this in vitro study was to determine the mechanism of neutrophil death. We hypothesized that NO hastened cell death by inducing apoptosis. To mimic the clinical environment of patients with respiratory failure, we also studied the effects of hyperoxia on neutrophil cell viability and apoptosis. Isolated human neutrophils were exposed to 80% O2 (O2), NO at 20 ppm in room air (NO/RA), 20 ppm NO blended with 80% O2 (NO/O2), or RA alone (control) for 2 to 24 h. Experiments were repeated with NO concentrations of 5 and 50 ppm and with 20 ppm in the presence of superoxide dismutase (SOD). Neutrophils were also incubated in the absence or presence of neutrophil stimulant fMLP (10 nM). Neutrophil cell viability was measured by fluorescence viability/cytotoxicity assay. Neutrophil apoptosis was assessed by cell death detection ELISA for histone-associated DNA fragments, TdT transferase-mediated fluorescence-labeled dUTP nick end labeling (TUNEL) assay, and DNA fragmentation gel electrophoresis. NO/O2-exposed neutrophils showed decreased viability at 2 h (31.7 +/- 3.7%, mean % viability +/- SD) compared with control (94.7 +/- 4.7%), O2 (75.6 +/- 9.3%), and NO/RA (62.8 +/- 14.9%; P < 0.05 by ANOVA; n = 9). Although control neutrophils demonstrated marked apoptosis at 24 h, there was no significant apoptosis at 2, 4, or 6 h (P < 0.001 by Kruskal-Wallis, n = 20) as assessed by ELISA and TUNEL assays. When compared with RA controls at 2 h, neutrophils exposed to NO/O2 showed significantly more apoptosis (292% of control, range: 106 to 2,488%, P < 0.001 by ANOVA and Kruskal-Wallis) but not with exposure to NO/RA or O2 alone. These findings were confirmed by TUNEL assay (n = 4, P < 0.05). NO/ RA and NO/O2-exposed neutrophils demonstrated both evidence of necrosis and enhanced DNA fragmentation at 2 h by gel electrophoresis (n = 2). Fifty parts per million NO produced similar findings, but exposure to 5 ppm NO did not induce significant DNA fragmentation. Coincubation with SOD inhibited NO/ O2-associated apoptosis, suggesting peroxynitrite contributed to cell death. Stimulation with fMLP did not alter apoptosis induced in neutrophils exposed to NO/RA or NO/O2. We conclude that exogenous NO gas, at clinically relevant concentrations under hyperoxic conditions, induces cell death in neutrophils in part by enhancing DNA fragmentation.     

Endotoxin-stimulated alveolar macrophage recruitment of neutrophils and modulation with exogenous surfactant

OBJECTIVE: To determine whether endotoxin-stimulated alveolar macrophages would attract neutrophils and whether exogenous surfactant treatment would modulate this chemoattraction. DESIGN: Alveolar macrophages were harvested from bronchoalveolar lavage fluid and neutrophils from the blood of anesthetized guinea pigs. SUBJECTS: Hartley guinea pigs. INTERVENTIONS: Alveolar macrophages were suspended in RPMI 1640 and stimulated with 1 microg/mL of lipopolysaccharide (LPS), the supernatant removed and the alveolar macrophages were incubated in either RPMI or RPMI with surfactant at two different doses (292 microg/mL or 875 microg/mL) for 16 hrs. MEASUREMENTS AND MAIN RESULTS: The supernatant was extracted from the alveolar macrophages and placed in a chemotaxis plate and the migration of neutrophils was measured. Chemotaxis of all cell types to be tested was measured by a change of absorbance on a microplate reader set at 492 nm. Results were compared with alveolar macrophages not stimulated with LPS, RPMI alone, and N formyl-methionyl-leucyl-phenylalanine (FMLP). The supernatant of the stimulated alveolar macrophages increased neutrophil chemotaxis as compared with unstimulated alveolar macrophages, and RPMI (p < .05). Surfactant treatment with 292 microg/mL significantly decreased LPS-stimulated alveolar macrophages induced neutrophil chemotaxis. Treatment with 875 microg/mL of surfactant did not alter neutrophil chemotaxis. CONCLUSIONS: Alveolar macrophages stimulation with LPS increased the chemotaxis of neutrophils. Treatment with surfactant at a concentration of 875 microg/mL did not alter neutrophil migration; however, treatment with 292 microg/mL significantly decreased neutrophil chemotaxis suggesting that at low concentrations, surfactant inhibits chemokine release and may reduce pulmonary neutrophil sequestration in vivo.     

Comparative study of classical, colorimetric and immunologic staining methods for the assessment of tumor cell viability

The trypan blue exclusion test, the MTT test and an immunostaining test for apoptosis were performed before and after incubation of SW620 human colonic carcinoma cells with different cytotoxic agents (CTAs) in order to assess tumor cell viability and CTA efficacy in vitro. A modified MTT test using light microscopy was also performed. A good correlation was found between the trypan blue assay and the MTT test, as determined by spectrophotometry. There was no 'overestimation' of cell viability as measured by the trypan blue test. The monitoring of formazan formation by light microscopy was feasible, but not very reliable since it did not show a good correlation with findings determined by spectrophotometry. The apoptosis test failed to show good correlation with other tests. Distilled water had no relevant cytotoxic effect, while chlorhexidine cetrimide (HAC 3.5%), chloramine 0.5% and polyvinylpyrrolidone iodine (PVP-I) > or = 0.05% damaged a large majority of cells. PVP-I at a concentration of > or = 5% was found to be the most effective CTA.     

Propofol inhibits human neutrophil functions

Neutrophils play important roles in the antibacterial host defense mechanism and in the pathogenesis of tissue injury. Propofol has been reported to impair the production of reactive oxygen species from neutrophils. We examined the effect of propofol (2,6-diisopropylphenol), at clinically relevant concentrations and at 10 and 100 times this concentration, on several aspects of human neutrophil functions using an in vitro system. Propofol significantly inhibited chemotaxis, phagocytosis, and reactive oxygen species (ROS) (O2-, H2O2, OH) production of neutrophils in a dose-dependent manner. At clinically relevant concentrations, propofol suppressed these neutrophil functions, but it did not decrease ROS generation by the cell-free (xanthine-xanthine oxidase) system. Increase in intracellular calcium concentrations in neutrophils stimulated by N-formyl-L-methionyl-L-leucyl-L-phenylalanine was dose-dependently attenuated by propofol. This decreasing effect on [Ca2+]i in neutrophils may represent one of the mechanisms responsible for the inhibition of neutrophil functions by propofol. IMPLICATIONS: Neutrophils play a pivotal role in the antibacterial host defense system and tissue injury. We found that at clinically relevant concentrations, propofol impaired neutrophil functions. Further studies may determine whether this impairment, observed in vitro, leads to clinical immunological suppression.     

Hemodynamic effects of transdermal estradiol alone and combined with norethisterone acetate

Of various vital dyes used to assess schistosomula viability, toluidine blue enabled differential counting of the schistosomula on microscope slides but not in culture wells, whereas methylene blue could be added directly to the schistosomula suspension in culture wells of microtiter plates. Toluidine blue uptake by dead parasites was very fast. It mostly also partially stained damaged but not dead organisms. Its main disadvantage was rapid, nonspecific staining of live schistosomula, requiring prompt counting of a preparation and additional reliance on motility for assessment of viability. Methylene blue staining of dead worms was slower, but it did not stain the live worms until about 1 h after dye application, enabling its addition to a series of preparations for consecutive counting. It did not always stain flattened, dead schistosomula or it stained them an uncontrasting pale blue. This dye remarkably induced movement in seemingly inert and probably damaged worms, thus enabling determination of viability even following poor staining or a lack of staining.     

Investigation of the role of nitric oxide and cyclic GMP in both the activation and inhibition of human neutrophils

1. The aim of this study was to establish the role of nitric oxide (NO) and cyclic GMP in chemotaxis and superoxide anion generation (SAG) by human neutrophils, by use of selective inhibitors of NO and cyclic GMP pathways. In addition, inhibition of neutrophil chemotaxis by NO releasing compounds and increases in neutrophil nitrate/nitrite and cyclic GMP levels were examined. The ultimate aim of this work was to resolve the paradox that NO both activates and inhibits human neutrophils. 2. A role for NO as a mediator of N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced chemotaxis was supported by the finding that the NO synthase (NOS) inhibitor L-NMMA (500 microM) inhibited chemotaxis; EC50 for fMLP 28.76 +/- 5.62 and 41.13 +/- 4.77 pmol/10(6) cells with and without L-NMMA, respectively. Similarly the NO scavenger carboxy-PTIO (100 microM) inhibited chemotaxis; EC50 for fMLP 19.71 +/- 4.23 and 31.68 +/- 8.50 pmol/10(6) cells with and without carboxy-PTIO, respectively. 3. A role for cyclic GMP as a mediator of chemotaxis was supported by the finding that the guanylyl cyclase inhibitor LY 83583 (100 microM) completely inhibited chemotaxis and suppressed the maximal response; EC50 for fMLP 32.53 +/- 11.18 and 85.21 +/- 15.14 pmol/10(6) cells with and without LY 83583, respectively. The same pattern of inhibition was observed with the G-kinase inhibitor KT 5823 (10 microM); EC50 for fMLP 32.16 +/- 11.35 and > 135 pmol/10(6) cells with and without KT 5823, respectively. 4. The phosphatase inhibitor, 2,3-diphosphoglyceric acid (DPG) (100 microM) which inhibits phospholipase D, attenuated fMLP-induced chemotaxis; EC50 for fMLP 19.15 +/- 4.36 and 61.52 +/- 16.2 pmol/10(6) cells with and without DPG, respectively. 5. Although the NOS inhibitors L-NMMA and L-canavanine (500 microM) failed to inhibit fMLP-induced SAG, carboxy-PTIO caused significant inhibition (EC50 for fMLP 36.15 +/- 7.43 and 86.31 +/- 14.06 nM and reduced the maximal response from 22.14 +/- 1.5 to 9.8 +/- 1.6 nmol O2-/10(6) cells/10 min with and without carboxy-PTIO, respectively). This suggests NO is a mediator of fMLP-induced SAG. 6. A role for cyclic GMP as a mediator of SAG was supported by the effects of G-kinase inhibitors KT 5823 (10 microM) and Rp-8-pCPT-cGMPS (100 microM) which inhibited SAG giving EC50 for fMLP of 36.26 +/- 8.77 and 200.01 +/- 43.26 nM with and without KT 5823, and 28.35 +/- 10.8 and 49.25 +/- 16.79 nM with and without Rp-8-pCTP-cGMPS. 7. The phosphatase inhibitor DPG (500 microM) inhibited SAG; EC50 for fMLP 33.93 +/- 4.23 and 61.12 +/- 14.43 nM with and without DPG, respectively. 8. The NO releasing compounds inhibited fMLP-induced chemotaxis with a rank order of potency of GEA 3162 (IC50 = 14.72 +/- 1.6 microM) > GEA 5024 (IC50 = 18.44 +/- 0.43 microM) > SIN-1 (IC50 > 1000 microM). This order of potency correlated with their ability to increase cyclic GMP levels rather than the release of NO, where SIN-1 was most effective (SIN-1 (EC50 = 37.62 +/- 0.9 microM) > GEA 3162 (EC50 = 39.7 +/- 0.53 microM) > GEA 5024 (EC50 = 89.86 +/- 1.62 microM)). 9. In conclusion, chemotaxis and SAG induced by fMLP can be attenuated by inhibitors of phospholipase D, NO and cyclic GMP, suggesting a role for these agents in neutrophil activation. However, the increases in cyclic GMP and NO induced by fMLP, which are associated with neutrophil activation, are very small. In contrast much larger increases in NO and cyclic GMP, as observed with NO releasing compounds, inhibit chemotaxis.     

Determination of Ag ions in water, urine and blood

Isolated human polymorphonuclear leukocytes were submitted to long wave ultraviolet light (UVA) with and without preincubation of the cells with 8-methoxypsoralen (8-MOP). Leukocyte chemotaxis was determined in modified Boyden chambers using caseine as the attractant. Combined treatment (8-MOP + UVA) significantly inhibited the chemotactic activity with 8-MOP concentrations above 0.1 mug/ml and 2 J/cm(2). However, with high dosage of UVA (5 J/cm(2)) with and without 8-MOP still 25-30% cells migrated through the filters. Also, cell viability as determined by trypan blue exclusion was only moderately affected by combined treatment. The results indicate that these nonreplicating cells are comperatively insensitive to UVA and 8-MOP.     

Association between neutrophil functions and periparturient disorders in cows

Neutrophil functions were examined in healthy periparturient dairy cows (n = 46) and in cows with retained placenta and metritis complex (n = 20); metritis (n = 18); or mastitis (n = 13). Blood samples (50 ml) were collected from each cow via jugular vein twice weekly from 1.5 weeks before to 4 weeks after parturition. Neutrophil function was evaluated, using 6 tests: random migration, chemotaxis, ingestion, myeloperoxidase activity (iodination), superoxide production (cytochrome C reduction), and antibody-dependent cell-mediated cytotoxicity. Ability to ingest bacteria and random migration activity of neutrophils from clinically normal cows were high around parturition and increased immediately after parturition, whereas myeloperoxidase activity and antibody-dependent cell-mediated cytotoxicity ability of neutrophils from these cows decreased after parturition. Measurement of neutrophil function in 4 ovariectomized cows revealed significant (P < 0.0005) seasonal changes in results of all 6 functional assays. We observed various defects of neutrophil function in all cows with abnormal conditions after parturition. Before parturition, superoxide production activity by neutrophils from cows with metritis and chemotaxis by neutrophils from cows with mastitis were significantly (P < 0.001 and P < 0.05, respectively) lower, indicating that a defect of neutrophil function may be a predisposing factor in the development of these disorders. In conclusion, the host defense role of neutrophils in periparturient cows was impaired, principally because of a defect in killing capacity, which may increase susceptibility to infections. We also investigated the in vitro effects of arachidonic acid metabolites and recombinant human colony-stimulating factors (rhCSF) on functions of neutrophils from clinically normal and postparturient cows with abnormalities, including retained placenta, metritis, or mastitis (n = 5/group). Each abnormal cow was matched for postpartum period with a clinically normal cow. Neutrophils from individual cows were preincubated with arachidonic acid metabolites (prostaglandin F2 alpha, 10(-7) M; prostaglandin E2, 10(-6) M; leukotriene B4, 10(-8) M; and lipoxin B, 10(-8) M) and rhCSF (rh-granulocyte-CSF, 1,000 or 6,000 U/ml; rh-granulocyte-macrophage-CSF, 5 or 15 ng/ml) in a 37 C water bath for 30 minutes before submitting them to function assays. There was no response by neutrophils from either clinically normal or abnormal postparturient cows to treatment with either arachidonic acid metabolites or rhCSF in any of the 6 functional assays. However, preincubation of neutrophils alone in a 37 C water bath for 30 minutes resulted in some alteration of neutrophil function.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of thalidomide on neutrophil respiratory burst, chemotaxis, and transmigration of cytokine- and endotoxin-activated endothelium

Vascular endothelium activated by endotoxin and cytokines plays an important role in organ inflammation and blood leukocyte recruitment. Neutrophils, which are a homogeneous population of effector cells, are rapidly attracted in large numbers to sites of inflammation where they form an early response to infection or injury. Excessive production of various interleukins, TNF, arachidonic acid metabolites, and other substances by neutrophils and macrophages results in systemic endothelial cell injury, a fundamental problem. In the present study, we investigated in vitro the effects of thalidomide (THD) on activation of endothelial cells for enhanced transmigration of neutrophils by lipopolysaccharide (LPS), tumor necrosis factor-alpha (TNF), and interleukin-1 (IL-1). Modulation of endotoxin- and cytokine-induced neutrophil chemotaxis and respiratory burst by THD were also studied. Treatment of HUVEC with THD in combination with LPS, TNF, and IL-1, respectively, antagonized LPS-activated transmigration of neutrophils but stimulated the effects of TNF and IL-1. All of the agents used-THD, LPS, TNF, and IL-1-inhibited neutrophil chemotaxis. Addition of THD to the neutrophils had no effect on LPS-inhibited chemotaxis whereas the TNF- and IL-1-induced chemotaxis was modulated in a bimodal manner. However, THD failed to influence neutrophil respiratory burst activity. Results demonstrate that THD differentially affects mediator-induced activation of HUVEC and neutrophils.