Transendothelial chemotaxis in vitro of human monocytes

Porcine aortic vascular endothelial cells were grown to confluence on microporous PTFE membranes and incorporated into a two-compartment chemotaxis assembly. Human peripheral blood mononuclear cells (20-25% monocytes) were placed in the upper compartment and zymosan-activated human serum (ZAHS) as chemoattractant in the lower compartment. Over a 3 h period monocytes migrated across the endothelialized membrane and adhered to a collecting filter sited in the lower compartment. Addition of ZAHS to the cell suspension in the upper compartment virtually abolished the migration response whilst only minimal leucocyte migration was supported by the bare unendothelialized PTFE membrane. The extent of transendothelial monocyte chemotaxis was dependent upon the concentration of chemoattractant in the lower compartment and upon the cell density of the suspension containing monocytes. A confluency test of fluid flow across the endothelialized filter showed that monocyte migration could take place without disturbing endothelial barrier function. The culture system is easy to assemble and the method provides experimental versatility.     

Anti-inflammatory effects of onions: inhibition of chemotaxis of human polymorphonuclear leukocytes by thiosulfinates and cepaenes

Seven different synthetic thiosulfinates, and cepaene- and/or thiosulfinate-rich onion extracts were found to inhibit in vitro the chemotaxis of human granulocytes induced by formyl-methionine-leucine-phenylalanine in a dose-dependent manner and at a concentration range of 0.1-100 microM. Diphenylthiosulfinate showed the highest activity and was found to be more active than prednisolone. The anti-inflammatory properties of onion extracts are related, at least in part, to the inhibition of inflammatory cell influx by thiosulfinates and cepaenes.     

Tolfenamic acid inhibits leukotriene B4-induced chemotaxis of polymorphonuclear leukocytes in vitro

Inhibition of prostanoid synthesis is usually regarded as the mode of action of nonsteroidal antiinflammatory drugs (NSAIDs). In addition, some NSAIDs have been reported to have prostanoid-independent inhibitory effects on neutrophil functions. In the present study, we examined the effects of acetylsalicylic acid, diclofenac, indomethacin, ketoprofen, piroxicam and tolfenamic acid on leukotriene B₄ (LTB₄)-induced chemotaxis of human polymorphonuclear leukocytes (PMNs) in vitro. Tolfenamic acid inhibited LTB₄-induced chemotaxis (IC₅₀ 59M), whereas the other compounds were ineffective. Tolfenamic acid inhibited also FMLP-induced chemotaxis at the same concentration range (IC₅₀ 46M). About 25% reduction in the chemotactic response was achieved with therapeutic concentrations of tolfenamic acid. We suggest that the inhibition of PMN chemotaxis is an additional mechanism in the antiinflammatory action of tolfenamic acid and that this action is not ligand specific.     

Leukotriene B4 modulates phospholipid methylation and chemotaxis in human polymorphonuclear leukocytes

Formation of phosphatidylcholine from phosphatidylethanolamine via the S-adenosylmethionine (AdoMet) pathway has been shown to be required for signal transduction of receptor-ligand interactions in a variety of cells. These interactions result in the remodeling of phospholipid pools and phospholipase activation. To extend these observations and to explore the role of the phosphatidylcholine synthesis pathway in transduction of the leukotriene B4 (LTB4) receptor-ligand response, we examined phospholipid methylation in human polymorphonuclear leukocytes (PMN) following stimulation by LTB4, a potent chemotactic agent that is a metabolite of arachidonic acid. At early time points (approximately 3-10 min), formation of methylated phospholipids was enhanced following LTB4 stimulation. The LTB4 analogs 6-trans LTB4 as well as LTB4 epimers induced less methylation compared with LTB4, and the potencies of these analogs in inducing methylation correlated with their diminished ability to induce chemotaxis. Furthermore, the ability of these agonists to induce methylation also correlated with the binding affinity of these agents to the LTB4 receptors on these cells. Synthesis of phosphatidylcholine by the choline transferase pathway was not affected by LTB4. Inhibition of the AdoMet reaction with 3- deazaadenosine, L-homocysteine homolactone, or erythro-9-[2-hydroxy-3-nonyl] adenine (EHNA) abrogated LTB4-induced phospholipid methylation and the chemotactic response. The potencies of these inhibitors in blocking phospholipid methylation also correlated with their ability to abrogate the LTB4-induced chemotactic response. These data suggest that phospholipid methylation and phospholipase activation play an important role in transduction of the LTB4 receptor-ligand interaction in PMN, which results in chemotaxis.     

Effects of nicotine on the functions of human polymorphonuclear leukocytes in vitro

Effects of nicotine on migration, extracellular release of lysosomal enzymes, and superoxide anion (O-2) production of human polymorphonuclear leukocytes (PMN) were studied. Nicotine (5 X 10(-6) to 5 X 10(-4) M) had no effect on random migration, chemotaxis to fMet-Leu-Phe, nor on chemokinesis induced by fMet-Leu-Phe. Nicotine, however, inhibited both extracellular release of lysosomal enzymes from PMN and O-2 production of PMN, both of which were induced by fMet-Leu-Phe and cytochalasin B. The inhibition of enzyme release and O-2 production by nicotine was not affected by atropine, hexamethonium, or acetyl beta-methylcholine, suggesting a direct action of nicotine on PMN functions. It is presumed that nicotine does not affect PMN migration to inflammatory sites, but inhibits the microbicidal functions of PMN. Exposure to PMN to nicotine introduced into the body by smoking could suppress their functions. This might result in harmful influences on the host defense mechanism, including antitumor function.     

Galectin-3 inhibits the chemotaxis of human polymorphonuclear neutrophils in vitro

In the recent years, the participation of the animal lectin galectin (gal)-3 in inflammation and in host defence mechanisms was extensively studied. In vivo studies implied - among others - a role of gal-3 in the recruitment of polymorphonuclear neutrophils (PMN) to sites of bacterial infection. In that context, we asked the question whether gal-3 was chemotactic for PMN. Functional assays revealed that gal-3 was not chemotactic for PMN, but that it inhibited the spontaneous migration and the chemotaxis of PMN towards complement C5a, interleukin (IL)-8, or ATP. Moreover, gal-3 inhibited the shape change and the actin polymerisation of PMN that occurs in response to C5a or IL-8. By use of FITC-labelled gal-3, we found that it attached rapidly to the PMN membrane in a lactose-sensitive manner. In response to gal-3 the MAP kinase p38 was phosphorylated. This kinase is crucial for the migration of PMN towards end-target chemokines, such as C5a, and is activated in response to C5a or IL-8. When PMN were preincubated with gal-3, the C5a-induced p38 phosphorylation was transiently enhanced, but eventually down-modulated. We conclude that by interfering with the chemokine-induced p38 phosphorylation gal-3 inhibits chemotaxis of PMN.     

Studies on gonococcus infection. VII. In vitro killing of gonococci by human leukocytes.

The comparative killing of pilated and nonpilated forms of Neisseria gonorrhoeae by human peripheral blood leukocytes was studied in vitro. Some nonpilated gonococci (T2) were killed to a lesser extent than were pilated, T2 organisms, which were killed less readily than another nonpilated (T4*) form of gonococcus. Thus, the relative order of killing of gonococci by human peripheral blood leukocytes appears to be: T4 less than T2 less than T4*. These data suggest that pilation, though correlated with virulence of gonococci, has little influence on the survival or killing of these organisms by human leukocytes.     

Adverse effects of acetylcysteine on human and guinea pig bronchial asthma in vivo and on human fibroblasts and leukocytes in vitro

The term 'allergen tachyphylaxis' denotes decreasing bronchial reactivity to allergen after repeated allergen inhalation challenges. In guinea pig bronchial asthma this self-protecting mechanism depends on endogenous prostaglandin E biosynthesis and can be inhibited by certain thiols. Therefore, we tested the effect of acetylcysteine (AC), a secretolytic thiol, on allergen tachyphylaxis in 25 guinea pigs. We observed inhibition of allergen tachyphylaxis and prolongation of each single asthmatic reaction. A possible clinical relevance of this observation was tested by the following experiments: Human lung fibroblasts (Wi-38) were stimulated with arachidonic acid and calcium ionophore and exposed to increasing amounts of AC. PgE biosynthesis was reduced from 2,408 pg/ml (control) to 84.2 pg/ml (0.6% AC) and 18.6 pg/ml (6% AC). Histamine release (HR) from human peripheral leukocytes was induced by anti-IgE. AC (0.016, 0.16, 1.6%) augmented both spontaneous HR (0-51.8%) and anti IgE induced HR (23.5-57.9%, p less than 0.001). Patients with isolated immediate bronchial reactions after allergen challenge inhaled 3 times a constant allergen dose. In few cases the reaction decreased from one test to another. This 'allergen tachyphylaxis' was inhibited by AC. We conclude that AC should be used with caution in patients suffering from bronchial asthma.     

缺氧对大鼠中性粒细胞趋化能力的影响

目的：研究急、慢性缺氧对大鼠中性粒细胞（PMNs）趋化能力的影响。方法:应用细胞微管吸吮实验技术，从单细胞角度研究在血清趋化作用下不同缺氧状态PMNs伪足生长的变化。结果:自身血清作趋化剂，急性、慢性缺氧组PMNs伪足长度分别为(1.200±0.178)μm和(1.094±0.164)μm，显著大于常氧对照组(0.914±0.156)μm（P＜0.05）；常氧、急性缺氧和慢性缺氧血清作用于常氧PMNs时，急、慢性缺氧血清作用下PMNs伪足长度分别为(1.059±0.179)μm和(1.041±0.175)μm，显著大于常氧血清作用（P＜0.05）；常氧血清作用于常氧、急性和慢性缺氧PMNs时，急性、慢性缺氧PMNs伪足长度分别为(1.058±0.163)μm和(1.118±0.126)μm，显著大于常氧对照组（P＜0.05）。结论:急、慢性缺氧促进了大鼠PMNs趋化能力，缺氧后血液微环境的变化和PMNs响应能力的变化是其重要原因。     

Effects of cigarette smoke fractions on the chemotaxis of polymorphonuclear leukocytes

The effects of cigarette smoking fractions on polymorphonuclear leukocyte (PMN) chemotaxis were determined using the 51Cr-assay. Water-soluble fractions (WSF) of cigarette smoke produced from several tobacco types differed in inhibitory potencies (i.e., flue cured greater than or equal to Maryland greater than or equal to blended greater than Burley greater than or equal to Turkish) corresponding to the respective unsaturated aldehyde content of the smoke from these tobaccos. Fractionation of cigarette smoke condensate (CSC) demonstrated that the more polar fractions were potent inhibitors of chemotaxis whereas those containing nicotine and the polycyclic hydrocarbons were weak inhibitors of chemotaxis. Unlike the inhibitory effects of WSFs, CSC fractions did not inhibit random migration and their inhibition of chemotaxis could not be completely prevented by reduced glutathione. These data suggest that while the alpha, beta-unsaturated aldehydes present in the vapor phase of smoke are among the most potent inhibitors of in vitro PMN chemotaxis, other polar, nonvolatile constituents of cigarette smoke also inhibit chemotaxis and by a mechanism which differs from that of the unsaturated aldehydes.     

Computer-assisted image analysis assay of human neutrophil chemotaxis in vitro

We have developed a computer-based image analysis system to measure in-filter migration of human neutrophils in the Boyden chamber. This method is compared with the conventional manual counting techniques. Neutrophils from healthy individuals and from patients with reduced chemotactic activity were used. The cells migrating into the filter were counted automatically at depths 20 microns apart commencing from the upper surface on the filter. The major advantages of this method are reproducibility and the counting of many fields which provides better data for statistical analysis of the results. Another advantage of the assay is that it can be used to show the migration pattern of different populations of neutrophils from both healthy individuals and patients.     

In vitro and in vivo effects produced by the immunomodulating agent RU 41740 on human polymorphonuclear leukocytes in the elderly

The activity of RU 41740, a glycoprotein extract from Klebsiella pneumoniae has been investigated on some polymorphonuclear (PMN) functions. Chemotaxis, random migration and oxidative metabolism (assessed by chemiluminescence, O2 consumption and O2- generation) were studied in parallel. PMN were collected from adult and aged human volunteers. Experiments were performed either in vitro or in vivo in a double blind placebo assay. In both PMN populations RU 41740 enhanced oxidative metabolism either in in vivo or in vitro experiments. However, a higher and dose-related activity was observed on PMN collected from the younger subjects whereas maximal effective concentration was reached earlier with PMN collected from aged subjects. RU 41740 did not modify random migration but inhibited chemotaxis of PMN collected from the younger population in a dose-related manner. These data corroborated previous results observed on PMN collected from various animal species and suggested an interaction of RU 41740 on PMN membrane. Moreover drug-induced macrophage and lymphocyte stimulation might also explain, at least in part, the in vivo effects described in this study. Thus RU 41740 could partly account for the protective effects exerted against bacterial and fungal infections through its activity on PMN functions.     

Studies on the effects of disease-modifying antirheumatic drugs on lipoxygenase product synthesis in human neutrophils in vitro

The disease-modifying antirheumatic drugs sodium aurothiomalate, D-penicillamine and chloroquine phosphate were tested on leukotriene (LT) synthesis in human blood polymorphonuclear leukocytes stimulated with either the ionophore A23187, zymosan or f-Met-Leu-Phe. Lipoxygenase products were analyzed by reversed-phase high-performance liquid chromatography. The study demonstrated that the drugs can either inhibit or enhance 5-lipoxygenase product synthesis in human leukocytes, depending on the stimulus used to activate the cells and the drug concentration. The data also suggested that increased substrate availability accounted for the stimulatory effects of the drugs on leukotriene synthesis.     

Upregulatory effects of cefpimizole natrium on human leukocytes.

Cefpimizole natrium (CPIZ), an antibiotic belonging to the cephalosporins, was examined regarding its influence on neutrophil functions. Neutrophil superoxide (O2-) generation increased by intravenous CPIZ in patients with maxillofacial diseases. In vitro examination revealed that CPIZ directly stimulates neutrophils to generate O2- in a dose-dependent manner, though the induction ability is not as strong as phorbol myristate acetate (PMA). Protein kinase C (PKC) activity in the neutrophil plasma membrane increased after CPIZ treatment, while the activity in the cytosol fraction decreased. CPIZ cooperated with biological response modifiers (BRMs) such as sizofilan, lentinan, OK-432, rIL-2 and rIFN-gamma in neutrophil O2- generation. Non-specific cytotoxicity against K562 cells and candida cells was also enhanced by neutrophil pretreatment with both CPIZ and one of the BRMs except for sizofilan and rIL-2. From these results it can be concluded that CPIZ directly enhanced neutrophil O2- generation and that these CPIZ activations are further beneficial to protection against bacterial infections.     

Histamine,dithiaden and human polymorphonuclear leukocytes in vitro

Inflammation Research -