Phagocytosis of Trypanosoma cruzi by Human Polymorphonuclear Leukocytes1

Phagocytosis of culture forms of Trypanosoma cruzi was assayed by a radioisotopic method. Purified polymorphonuclear leukocytes (PMN) were mixed with ³H-uridine-labeled T. cruzi epimastigotes in the presence or absence of anti-T. cruzi antibodies. The reaction was stopped by adding N-ethyl-maleimide, and noningested parasites were lysed by complement. The percentage of radioactivity incorporated into the PMN pellet was recorded. The phagocytosis reaction was rapid, yielding maximum incorporation at 30 min at which point the radioactivity associated with the PMN cells decreased through release of the isotope to the supernatant. The degree of incorporation of radio-labeled parasites was a function of the effector/target cell ratio and the antibody concentration. The method is suitable for the quantitative determination of phagocytosis of T. cruzi by normal PMN.     

Hypochlorite-Modified Adenine Nucleotides: Structure,Spin-Trapping,and Formation by Activated Guinea Pig Polymorphonuclear Leukocytes

Adenosine and its nucleotides react with hypochlorite to form unstable products that have been identified as the N⁶ chloramine derivatives. These chloramines spontaneously oligomerize. form stable adducts with proteins and nucleic acids, and are converted with loss of chlorine to the original nucleoside or nucleotide by reducing agents. The chloramines are associated with a free radical. and the spin-trapping of adenosine chloramine with 5,5-dimethyl-l-pyrroline-N-oxide (DMPO) yielded a mixture of unstable nitroxyl adducts that corresponded to nitrogen-centered radicals from the parent nucleoside. When activated guinea pig polymorphonuclear leukocytes were stimulated with phorbol myristate acetate to produce hypochlorite, they actively incorporated [¹⁴C] adenosine into acid-insoluble products by a process that was dependent on oxygen and inhibited by azide and thiols. These findings suggest that adenine nucleotide chloramines are generated by activated phagocytic cells and form ligands with proteins and nucleic acids as observed in model systems. The results imply that nucleotide chloramines are among the cytotoxic and possibly mutagenic factors that are associated with the icflammatory process.     

Hypochlorite-Modified Adenine Nucleotides: Structure, Spin-Trapping, and Formation by Activated Guinea Pig Polymorphonuclear Leukocytes

Adenosine and its nucleotides react with hypochlorite to form unstable products that have been identified as the N⁶ chloramine derivatives. These chloramines spontaneously oligomerize. form stable adducts with proteins and nucleic acids, and are converted with loss of chlorine to the original nucleoside or nucleotide by reducing agents. The chloramines are associated with a free radical. and the spin-trapping of adenosine chloramine with 5,5-dimethyl-l-pyrroline-N-oxide (DMPO) yielded a mixture of unstable nitroxyl adducts that corresponded to nitrogen-centered radicals from the parent nucleoside. When activated guinea pig polymorphonuclear leukocytes were stimulated with phorbol myristate acetate to produce hypochlorite, they actively incorporated [¹⁴C] adenosine into acid-insoluble products by a process that was dependent on oxygen and inhibited by azide and thiols. These findings suggest that adenine nucleotide chloramines are generated by activated phagocytic cells and form ligands with proteins and nucleic acids as observed in model systems. The results imply that nucleotide chloramines are among the cytotoxic and possibly mutagenic factors that are associated with the icflammatory process.     

多形核白细胞上嘌呤受体的初步研究

ATP和ADP能激活多型核白细胞引起细胞内［Ca²⁺］_i的明显升高,AMP则无此作用.多型核白细胞对ATP和ADP具有不同的浓度依赖性.当细胞外的钙离子被螯合后,ATP和ADP仍能引起细胞内游离钙浓度的升高.结果表明多形核白细胞存在着对ATP和ADP敏感的P2型嘌呤受体,并且属于P2型受体中的P2Y亚类.     

Priming Effect of Pseudomonal Leukocidin on Chemiluminescence Response of Rabbit Polymorphonuclear Leukocytes

To clarify effects of pseudomonal leukocidin (42.5 kd) on chemiluminescence (CL) production of polymorphonuclear leukocytes (PMNs), rabbit PMNs were stimulated by zymosan or phorbol myristate acetate (PMA) after pretreatment with the leukocidin, which by itself stimulated little chemiluminescence response. The extent of CL responses stimulated by zymosan or PMA was respectively 5.3- or 3.5-fold greater in leukocidin (1.5 μg/ml)-pretreated PMNs than in non-pretreated ones. The priming effect of the leukocidin was greater than that of G-CSF and related to some steps before NADPH oxidase activation. The increased CL productions might be related to tissue damages caused by pseudomonal infections in vivo.     

Myeloperoxidase-Based Chemiluminescence of Polymorphonuclear Leukocytes and Monocytes

Luminol and lucigenin chemiluminescence (CL) responses produced by separated human blood polymorphonuclear leukocytes (pmn) and monocytes (mono) have been studied following stimulation with the surface-receptor agonist fMLP (a synthetic chemotactic peptide) and the protein kinase C activator phorbol myristate acetate (PMA). Pmn produced two- to threefold the luminol CL and superoxide anion (O₂⁻) levels of mono; lucigenin CL was similar for both cell-types. The myeloperoxidase (MPO) inhibitor salicylhydroxamic acid (SHA) abrogated luminol but not lucigenin CL in both cell types, but did not further inhibit the already grossly subnormal luminol CL responses seen with MPO-deficient cells which produced normal lucigenin CL. SHA also profoundly inhibited the luminol CL response in a cell-free MPO–H₂O₂ system. Mono lucigenin CL does not appear to specifically measure O₂⁻ production. These data show that luminol CL provides a useful measure of pmn and also mono MPO activity. However, analysis of the effects of various reactive oxygen species (ROS) scavengers, assessed on phagocyte and cell-free CL systems (both MPO–H₂O₂ and superoxide generating) suggest that the luminol CL signal is not entirely dependent on MPO activity.     

Effects of Antioxidants on Surfactant Peroxidation by Stimulated Human Polymorphonuclear Leukocytes

Production of oxygen radicals by stimulated phagocytes followed by surfactant lipid peroxidation (LPO) and loss of surfactant function have all been implicated in the pathogenesis of acute lung injury. We studied the interactions between natural lung surfactant (Curosurf) and neutrophils in vitro, and compared various antioxidants; (superoxide dismutase (SOD), vitamin E, vitamin C, ebselen and melatonin), or combinations of them in duplicate and triplicate regarding their ability to decrease superoxide production and the peroxidation level of surfactant caused by activated phagocytes. The superoxide production of neutrophils activated by Candida albicans was measured with the nitroblue tetrazolium (NBT) test. The subsequent LPO was estimated as the content of malondialdehyde (MDA) and 4-hydroxyalkenals (4-HNE). We found that lung surfactant decreased the superoxide production by activated neutrophils (29.7%) and that Curosurf was peroxidized with elevated MDA/4-HNE values. With supplements of antioxidants (except vitamin C), superoxide radical production and the surfactant LPO level fell in a dose-dependent manner. The protective effect of the antioxidants differed in each test. SOD had a slight effect in both tests. The findings with vitamin E, melatonin and ebselen were similar. The best combination was that of a natural and a synthetic antioxidant (melatonin-ebselen) with a 60% decrease in comparison to the corresponding control. These findings suggest that antioxidants, particularly in combination, prevent LPO of lung surfactant.     

Cryptococcus neoformans II. Phagocytosis by Human Leukocytes

Twenty-four per cent of the leukocytes from healthy human subjects phagocytized an encapsulated strain of Cryptococcus neoformans. Phagocytosis was approximately three times more effective with nonencapsulated mutants of C. neoformans. When the mutants reverted to the encapsulated state, the percentages of phagocytosis decreased. These data indicate that cryptococcal polysaccharide inhibits the phagocytosis of C. neoformans by human leukocytes.     

Biochemical Properties of Human Oral Polymorphonuclear Leukocytes

Polymorphonuclear leukocytes (PMN) isolated from the oral cavity of healthy human volunteers, spontaneously generated superoxide, nitric oxide (NO) and other reactive oxygen species (ROS) which exhibited strong luminol chemiluminescence (LCL). To understand the physiological roles of oral PMN (OPMN), biochemical properties of the cells were analyzed. Biochemical analysis revealed that OPMN were already primed under physiological conditions. Western blot analysis revealed that they strongly expressed the inducible type of NO synthase (NOS II) and exhibited the activity to catalyze tyrosine phosphoryla-tion of various proteins including a 115 kDa protein (cbl product). OPMN also generated H₂O₂ and OH by some superoxide dismutase (SOD)-sensitive mechanism and released myeloperoxidase (MPO). Kinetic analysis using specific inhibitors revealed that OCI generated by OPMN was predominantly responsible for the enhanced LCL. During the incubation under standard culture conditions, OPMN underwent apoptosis which proceeded more rapidly than that of the circulating PMN (CPMN). Immunochemical analysis revealed that expression of apoptosis-related gene products, such as Bcl-2, Bcl-xL and Bax, was below detectable levels with both cell types. However, caspase-3 but not caspase-1 was markedly activated in OPMN. These results indicate that the primed OPMN spontaneously generate ROS and play an important role in the defense mechanism in the oral cavity and that the generated ROS activate caspase-3 thereby inducing apoptosis of the cells.     

三个品种豚鼠血液蛋白多态性的比较分析

目的比较分析白毛黑眼（WHBE）豚鼠和DHP豚鼠、花色豚鼠三个品种豚鼠在13个血液蛋白位点上的多态性。方法采用垂直板浓度和pH均不连续的聚丙烯酰胺凝胶电泳法对WHBE豚鼠、DHP豚鼠和花色豚鼠的66只个体的后白蛋白（Po）、前转铁蛋白1（Prt1）、前转铁蛋白2（Prt2）、转铁蛋白1（Tf1）、转铁蛋白2（Tf2）、后转铁蛋白（Ptf）、慢α球蛋白（Sag）、红细胞酯酶（Es）、血清酯酶1（Est1）、血清酯酶3（Est3）、血红蛋白α（Hbα）、血红蛋白β（Hbβ）和白蛋白（Alb）共13个蛋白位点进行了电泳及染色,再利用电泳图谱对各蛋白位点基因频率、平均杂合度和遗传距离进行计算,然后结合聚类分析。结果 Tf1、Tf2、Ptf、Est1和Es在三个豚鼠品种中表现为多态,其中Tf1可作为识别WHBE豚鼠的遗传标记。Po、Prt1、Prt2、Sag、Est3、Hbα、Hbβ和Alb等位点在三个豚鼠品种中的表型一致。Hardy-Weinberg平衡状态分析表明,Es为DHP豚鼠的高度不平衡位点。Ptf为花色豚鼠的高度不平衡位点。在WHBE豚鼠中,Tf1为高度不平衡位点,Est1为不平衡位点。在三个豚鼠品种中,所检测的13个蛋白位点的平均杂合度的排列顺序为：花色豚鼠（0.350 1）〉WHBE豚鼠（0.339 0）〉DHP豚鼠（0.313 5）。聚类分析结果表明,花色豚鼠和WHBE豚鼠的遗传遗传距离最近（0.064 3）,DHP豚鼠与花色豚鼠的遗传距离最远（0.179 2）。结论利用这些蛋白位点可以有效鉴别WHBE豚鼠、DHP豚鼠和花色豚鼠血液蛋白的遗传多态性。     

Augmentation and Suppression of TNF Release from Macrophages by Inflammatory Polymorphonuclear Leukocytes

It is known that polymorphonuclear leukocytes (PMNs) emerge first in local inflammatory sites, and then they are followed and scavenged by macrophages. We focused on the effect of PMN on tumor necrosis factor (TNF) release activity of macrophages, which is viewed as a possible indicator of the status of macrophage activation. One day after macrophages were cultured with fresh, intact murine PMNs which were induced with sodium casein, the release of TNF triggered by lipopolysaccharide (LPS) was augmented by low concentrations of PMNs, but suppressed by their high concentrations. When the PMN samples were fractionated into soluble and insoluble fractions, the augmenting and suppressing activity was partitioned; the relatively high concentrations of soluble fraction showed the suppressive effect whereas the insoluble fraction in lower concentrations showed augmentation. The suppressive activity was stable at 100 C, but the filtrates of the soluble fraction with membranes having cut-offs of 5,000 or 10,000 were not suppressive at all, suggesting the suppression is not due to low molecular compounds. It was also suggested that the suppressive effect for TNF release was not due to contaminating LPS or transforming growth factor-β. Inflammatory processes may thus be positively and negatively controlled by a quantitative factor of initial PMN populations by regulating the TNF release activity of the subsequent macrophages.     

Influence of Diesel Soot Particles and Sulfite on Functions of Polymorphonuclear Leukocytes

Aqueous suspensions of diesel soot particles in combination with sulfite influence certain functions of human polymorphonuclear neutrophils in vitro. Chemiluminescence, generated after activation by opsonized zymosan as well as oxygen uptake were decreased, whereas phagocytosis was increased. An enhancement of degranulation could not be observed. The single substances show little or no effects on the above properties. The results indicate that combinations of air pollutants such as diesel soot and sulfite may modulate vital functions of activated leukocytes in vivo.     

Oscillations in Superoxide Anion Release by Polymorphonuclear Leukocytes and its Inhibition by Human Saliva

The release of superoxide anion (O 2 -) by inflammatory polymorphonuclear leukocytes (PMN), obtained from the intraperitoneal cavity of mice, was investigated during two different times of the day. NaF-stimulated PMN at nigth-phase showed greater release of O 2 - than at day. Addition of human saliva in the culture medium dramatically inhibits O 2 - generation by both day- and night-phase cells. Also, the inhibitory effect of diurnal saliva on the cell activation is higher than the nocturnal saliva.     

Influence of Diesel Soot Particles and Sulfite on Functions of Polymorphonuclear Leukocytes

Aqueous suspensions of diesel soot particles in combination with sulfite influence certain functions of human polymorphonuclear neutrophils in vitro. Chemiluminescence, generated after activation by opsonized zymosan as well as oxygen uptake were decreased, whereas phagocytosis was increased. An enhancement of degranulation could not be observed. The single substances show little or no effects on the above properties. The results indicate that combinations of air pollutants such as diesel soot and sulfite may modulate vital functions of activated leukocytes in vivo.     

Oscillations in Superoxide Anion Release by Polymorphonuclear Leukocytes and its Inhibition by Human Saliva

The release of superoxide anion (O 2 - ) by inflammatory polymorphonuclear leukocytes (PMN), obtained from the intraperitoneal cavity of mice, was investigated during two different times of the day. NaF-stimulated PMN at nigth-phase showed greater release of O 2 - than at day. Addition of human saliva in the culture medium dramatically inhibits O 2 - generation by both day- and night-phase cells. Also, the inhibitory effect of diurnal saliva on the cell activation is higher than the nocturnal saliva.     

Inactivation of alpha(2)-Macroglobulin by Activated Human Polymorphonuclear Leukocytes

The proteolytic activity of trypsin releases the dye Remazol Brilliant Blue from its high molecular weight substrate, the skin powder (Hide Powder Azure, Sigma), with an increase in absorbance at 595 nm. Active alpha(2)- macroglobulin (80 mug/ml) totally inhibits the proteolytic activity of trypsin (14 mug/ml) by trapping this protease. But after a 20 min incubation of alpha(2)-macroglobulin at 37 degrees C with 2 x 10(6) human polymorphonuclear leukocytes activated by N-formyl-L-methionyl-L-leucyl-L-phenylalanine (10(-7) M) and cytochalasin B (10(-8) M), 100% of trypsin activity was recovered, indicating a total inactivation of alpha(2)-macroglobuHn. Incubation with granulocyte myeloperoxidase also inactivates alpha(2)-macroglobulin. Hypochlorous acid, a by-product of myeloperoxidase activity, at a concentration of 10(-7) M also inactivates alpha(2)-macroglobulin, which indicates that an important cause of alpha(2)-macroglobulin inactivation by activated polymorphonuclear leukocytes could be the activity of myeloperoxidase.     

Spin Trapping of Superoxide from Glass Adherent Polymorphonuclear Leukocytes Induced by N-Formylmethionyl-Leucyl-Phenylalanine

Dahinden et al. reported that N-formylmethionyl-leucyl-phenylalanine (fMLP)-induced superoxide release from polymorphonuclear leukocytes (PMNs) lasted more than 60 min when the cells were allowed to attach to a petri dish before induction. In contrast, it lasted only for 2.5 min when cells were in suspension (J. Clin. Invest. 72: 113-121, 1983). In spite of this report, the effect of cell adhesion has been ignored in most spin trapping studies of superoxide release from PMNs. This study shows that most PMNs in a quartz flat electron paramagnetic resonance (EPR) cuvette which was placed horizontally adhered to the wall within 3 min. In contrast, if the cuvette was placed vertically, only 20-30% of the cells became adherent in 30 min. We performed spin trapping studies using 5,5-dimethylpyrroline-N-oxide (DMPO) as a spin trap, and monitored the effect of cell adhesion on superoxide generation. When spin trapping was conducted on PMNs in suspension, the EPR signal of superoxide adduct (DMPO-OOH) was undetectable after stimulation with fMLP. However, PMNs which were allowed to adhere to the cuvette after stimulation generated superoxide for hours. Moreover, when PMNs were allowed to adhere prior to the stimulation, the magnitude of superoxide release was augmented three-to fourfold. Unlike fMLP, phorbol myristate acetate (PMA), which has been most commonly used in spin trapping studies, induced superoxide release which was not influenced by cell adhesion. We emphasize the importance of specifying the cell-adhesion-state in spin trapping studies.     

High-Molecular-Weight Hyaluronic Acids Inhibit Chemotaxis and Phagocytosis but Not Lysosomal Enzyme Release Induced by Receptor-Mediated Stimulations in Guinea Pig Phagocytes

The effects of high-molecular-weight (HMW) hyaluronic acids (HAs) of 1.9 × 10⁶ Da, 8 × 10⁵ Da and 3 × 10⁵ Da on the receptor-mediated functions of guinea pig peritoneal phagocytes were studied. HMW-HAs of 1.9 × 10⁶ Da (HA190) and 8 × 10⁵ Da (HA80) effectively inhibited the chemotactic activity of polymorphonuclear leukocytes (PMNs) for formyl-Met-Leu-Phe (fMLP). The degree of inhibition was dose-dependent and the concentrations of HA190 and HA80 required for 50% inhibition were 0.5–1.5 mg/ml and 1.5–2.5 mg/ml, respectively. HMW-HA of 3 × 10⁵ Da (HA30) hardly affected the chemotaxis within a concentration range of 0.5–5.0 mg/ml. The phagocytic activities of PMNs and macrophages (Møs) for serum-opsonized zymosan (SOZ) and polystyrene latex particles were also inhibited by these HAs in a dose- and molecular-weight-dependent manner and HA190 was again the most inhibitory. By contrast, the release of lysosomal enzyme from Møs stimulated with SOZ was not significantly affected by HMW-HAs at any concentration used. Furthermore, the binding of [³H]fMLP with PMNs and the rosette formation of Møs with SOZ were not influenced by the presence of HMW-HAs. These findings suggested that the binding of HMW-HAs to the HA receptors on PMNs and Møs might produce certain intracellular signals which would be responsible for the suppression of the chemotaxis and the phagocytosis but not for the release of lysosomal enzyme. For the generation of such signals, higher-molecular-weight HMW-HAs would be more effective than lower one.     

体外刺激人外周血多形核白细胞诱发化学发光研究

分别测量酵母多糖、伴刀豆球蛋白及佛波醇酯刺激人外周血多形核白细胞诱发的化学发光,结果表明：只有ＰＭＡ刺激的效果最好,即能产生持续稳定高水平的ＣＬ;进一步探索ＰＭＡ刺激ＰＭＮ产生ＣＬ的规律发出：不仅ＰＭＡ的浓度对ＰＭＮ不生ＣＬ的时相、释放速度及效率有影响,测量体系中血含量也影响其ＰＭＮ受激诱发产生的ＣＬ（最适的血浓度在１０％左右）;此外,文中还对保存不同时间的血受ＰＭＡ刺激产生ＣＬ的情况进行了观察。