Lipids of alveolar macrophages, polymorphonuclear leukocytes, and their phagocytic vesicles

Phagocytic vesicles were isolated from rabbit alveolar macrophages and guinea pig polymorphonuclear leukocytes that had ingested emulsified paraffin oil. Phospholipids and their fatty acids were determined in whole cells and in the phagocytic vesicle and pellet fractions separated from them. The cholesterol-to-phospholipid ratios in the vesicle fractions were distinctly higher than those of the respective whole cells or pellet fractions. The vesicle fractions also had higher phospholipid-to-protein ratios than did the whole cells. The phospholipids of the phagocytic vesicle fraction from macrophages contained relatively more sphingomyelin, lyso-(bis)phosphatidic acid, and phosphatidylserine and less lecithin, phosphatidylethanolamine, and phosphatidylinositol than did the whole cells or pellet fractions. The phospholipids of phagocytic vesicles from polymorphonuclear leukocytes contained significantly more phosphatidylinositol than did the pellet fractions. Lyso(bis)phosphatidic acid, which constituted 15% of the phospholipid in rabbit alveolar macrophages and 25% of that in their phagocytic vesicles, contained almost 60% oleic acid and 20% linoleic acid. This lipid was not detected in rabbit peritoneal macrophages or in rat alveolar macrophages.The polyunsaturated fatty acids of leukocyte phospholipids were chiefly linoleic, whereas in macrophages arachidonic accounted for almost 20% of the total fatty acids. The macrophages produced malondialdehyde when ingesting polystyrene beads or emulsified paraffin oil, from which it was inferred that peroxidation of endogenous lipid can occur during phagocytosis. Polymorphonuclear leukocytes in which less than 3% of phospholipid fatty acids were arachidonic did not produce malondialdehyde during phagocytosis of these inert particles, but did when ingesting an emulsion containing linolenate, thus providing evidence for peroxidation of ingested lipid. Isolated phagocytic vesicles from alveolar macrophages contained lipid peroxides and generated malondialdehyde when incubated with ADP, FeCl(3), and NADH.     

The alteration of superoxide dismutase, catalase, glutathione peroxidase, and NAD(P)H cytochrome c reductase in guinea pig polymorphonuclear leukocytes and alveolar macrophages during hyperoxia.

Superoxide dismutase, catalase, glutathione peroxidase and NAD(P)H cytochrome c reductase were quantitated in polymorphonuclear leukocytes (PMN) and alveolar macrophages (AM) obtained from guinea pigs exposed up to 90 h to 85% oxygen. PMN and AM were sonicated and separated into a 16,000-g pellet, a 100,000-g pellet, and a 100,00-g supernate. Superoxide dismutase activity increased in both cells within 18 h, persisted for 66 h and decreased by 90 h. The highest rate of increase was in the 100,000-g pellet containing 3.4% of total enzyme activity in PMN but 28% in AM. The enzyme induction in PMN and AM was partially inhibited by daily intracardiac injections of 50 mg/kg actinomycin D. During oxygen exposure, catalase activity in PMN and AM decreased to 60% of its original activity, and gluthathione peroxidase was reduced in PMN to 60% and in AM to 20% of control values. Although NAD(P)H cytochrome c reductase decreased to 50% in PMN, no change was noted in AM. Upon exposure to superoxide anion, purified catalase, the glutathione peroxidase of the 100,000-g supernate, NADH, and NADPH cytochrome c reductases of the 16,000-g pellet decreased to 66+/-5%, 72+/-4%, 52+/-8%, and 40+/-9%, respectively, of their original activity. This inactivation was prevented by 0.1 mg superoxide dismutase. These in vitro observations could explain the decreased catalase and glutathione peroxidase activity demonstrated in vivo that may lead to an intracellular accumulation of hydrogen peroxide. Increased hydrogen peroxide concentrations have been found to inactivate superoxide dismutase thus impairing the first defense mechanism against superoxide anion.     

Amphotericin B formulations exert additive antifungal activity in combination with pulmonary alveolar macrophages and polymorphonuclear leukocytes against Aspergillus fumigatus

Deoxycholate amphotericin B (DAMB) and amphotericin B lipid complex (ABLC) additively augmented the fungicidal activity of pulmonary alveolar macrophages against the conidia of Aspergillus fumigatus. DAMB, ABLC, and liposomal amphotericin B similarly displayed additive effects with polymorphonuclear leukocytes in damaging the hyphal elements of A. fumigatus.     

Effect of linezolid on the phagocytic functions of human polymorphonuclear leukocytes

The effect of linezolid on the phagocytic and bactericidal functions of human polymorphonuclear neutrophils (PMN) against gram-positive cocci was evaluated. Preincubation (30 min; 37 degrees C) of PMN with different concentrations of linezolid (2, 10 and 20 mg/l) did not significantly affect the phagocytosis of either Staphylococcus aureus (methicillin susceptible and resistant) or Enterococcus faecalis (vancomycin susceptible and resistant). Overnight exposure of vancomycin-resistant E. faecalis to 1/4 minimum inhibitory concentrations (MIC) of linezolid slightly increased phagocytosis by PMN. Preincubation of the other three strains with 1/4 MIC of linezolid did not affect phagocytosis by these cells. Preincubation of PMN (30 min; 37 degrees C) using different extracellular concentrations of linezolid (2, 10 and 20 mg/l) did not affect their production of either superoxide or hydrogen peroxide radicals. In conclusion, linezolid did not affect the phagocytic and bactericidal functions of human PMN.     

Effect of ciprofloxacin on killing of Actinobacillus actinomycetemcomitans by polymorphonuclear leukocytes

Actinobacillus actinomycetemcomitans, a pathogen associated with aggressive periodontitis, resists phagocytic killing by polymorphonuclear leukocytes (PMNs). It is susceptible to ciprofloxacin, which PMNs actively accumulate. This study tested the hypothesis that ciprofloxacin-loaded PMNs are more effective at killing A. actinomycetemcomitans than control PMNs. Isolated human PMNs were loaded by brief incubation with 0.5 microg of ciprofloxacin/ml. Opsonized bacteria (ATCC 43718) were incubated at 37 degrees C with control and ciprofloxacin-loaded PMNs and in the presence and absence of 0.5 microg of ciprofloxacin/ml. When assayed at bacteria-to-PMN ratios of 30:1 and 90:1, ciprofloxacin-loaded PMNs killed significantly more bacteria and achieved significantly shorter half times for killing than control PMNs (P < 0.05; Tukey's test). At ratios of 3:1 and 10:1, these differences were not significant.     

Cytotoxic effects of polymorphonuclear leukocytes and macrophages in patients undergoing lymph depletion via thoracic duct drainage

It has been previously reported that both human peripheral blood monocyte derived macrophages and polymorphonuclear leukocytes acquire enhanced cytotoxicity for tumor cells. Lymphocyte depletion by thoracic duct cannulation prior to renal transplantation has been shown to suppress allograft rejection. However, the effects of thoracic duct drainage on macrophage and polymorphonuclear leukocyte function is not known. When the macrophages obtained from thoracic duct drainage patients were studied prior to cannulation, four of the five patients possessed cytotoxic macrophages. At 1 to 2 weeks post thoracic duct drainage, cytotoxicity was significantly depressed whereas by 3 weeks post thoracic duct cannulation most of the patients' macrophages exhibited maximal cytotoxicity. Approximately 3 1/2 weeks after cannulation these five patients received cadaveric renal transplants. The cytotoxic effects of the macrophages were tested again after transplantation and it was found that the macrophages became incapable of killing the tumor targets. In contrast to our findings with macrophage mediated cytotoxicity, the polymorphonuclear leukocytes generally retained their cytotoxic capabilities at all time points tested. However, it was noted that cytotoxic activity reached maximal levels within the first 3 weeks after cannulation but fell to low cytotoxic levels at approximately 4 to 5 weeks after cannulation. When tested several months post cannulation and transplantation, polymorphonuclear leukocyte mediated cytotoxicity increased dramatically in four of the five patients studied.     

Tumor necrosis factor-alpha-stimulated polymorphonuclear leukocytes suppress migration and bactericidal activity of polymorphonuclear leukocytes in a paracrine manner

OBJECTIVE: Polymorphonuclear leukocytes (PMN) and tumor necrosis factor-alpha (TNF-alpha) play prominent roles in acute respiratory distress syndrome, ischemia-reperfusion injury, trauma, and sepsis. Whereas direct effects of TNF-alpha on PMN function and viability are well documented, little data are available addressing the ability of PMN to communicate with each other in response to cytokine stimulation. Therefore, the aim of this study was to determine whether TNF-alpha can modulate PMN function by inducing PMN to secrete products upon stimulation, which would affect other PMN in vitro in a manner independent of cell contact. METHODS: PMN were purified daily from blood obtained from a pool of 22 healthy volunteers. Conditioned media (CM-TNF) was prepared by incubating PMN in Hanks' balanced salt solution plus TNF-alpha for 1-4 hrs. Freshly isolated PMN were resuspended in CM-TNF and analyzed for 1) phagocytosis of opsonized Escherichia coli, 2) oxidative metabolism as measured as an index of DCF-DA activation, and 3) migration to chemoattractants through Transwell inserts. RESULTS: CM-TNF decreased PMN phagocytotic activity by 8% to 15% and completely suppressed oxidative metabolism but did not modulate the expression of receptors associated with these functions. CM-TNF suppressed the migration of PMN to two biologically relevant agents, N-formyl-methionyl-leucyl-phenylalanine and leukotriene B4, by approximately 65%, but had no effect on PMN migration to interleukin-8. This suppression was observed for migration across plastic filters as well as extracellular matrix proteins. CONCLUSION: Our data demonstrate that PMN stimulated with TNF-alpha suppress the immunologic function and migration of other PMN independent of cell-cell contact and suggest that TNF-alpha may participate in a negative feedback loop by inducing a PMN-derived factor that counteracts its activity.     

Mechanisms of intralipid effect on polymorphonuclear leukocytes

The effect of Intralipid, a lipid emulsion used in total parenteral nutrition, on polymorphonuclear leukocytes (PMNL) was studied in vitro. Preincubation of PMNL with Intralipid was found to depress oxidative metabolic and phagocytic functions. Preincubation of PMNL with intralipid also caused a decrease in Fc receptors. However, when Intralipid was added at the start of metabolic assays, it stimulated the respiratory burst. We interpret these findings as suggesting that Intralipid, acting as a particulate stimulus, activated and exhausted metabolic pathways involved in the respiratory burst and phagocytosis and that it also decreased numbers or availability of Fc receptors on PMNL. Individual components of Intralipid did not induce these changes. This dysfunction may contribute to the infections seen in patients receiving parenteral hyperalimentation with Intralipid supplementation.     

Effect of various agonists on nitric oxide generation by human polymorphonuclear leukocytes

Nitric oxide generation is involved in a range of diseases involving polymorphonuclear leukocytes. The aim of this study was to determine whether human polymorphonuclear leukocytes are able to generate nitric oxide and to investigate the time course of its generation after stimulation with 10⁻⁷ MN-formyl-methionyl-leucylphenylalanine, 60 ng/ml phorbol myristate acetate, 10⁻⁷ M concanavalin A, and 10⁻⁷ M platelet activating factor. Stimulation of human polymorphonuclear leukocytes withN-formyl-methionyl-leucyl-phenylalanine and phorbol myristate acetate caused sustained nitric oxide generation, reaching maximal values of 1,105±361 nM (n=32) and 628±119 nM (n=30), respectively. Platelet activating factor did not affect nitric oxide production (maximal value 29±7 nM,n=8), whereas concanavalin A caused only a slight increase (102±24 nM,n=8) when compared with resting cells control (26±6 nM,n=8). Human polymorphonuclear leukocytes were able to respond to both consecutive and alternateN-formyl-methionyl-leucyl-phenylalanine and phorbol myristate acetate stimulation with nitric oxide generation. Nitric oxide generation was inhibited by specific inhibitors (N ^ω-nitro-l-arginine andN ^ω-monomethyl-l-arginine) and restored withl-arginine. We provide, to our knowledge, the first direct evidence that human neutrophils generate nitric oxide.     

白介素-10对肺泡巨噬细胞致炎效应的调节作用

目的 :探讨白介素 10 (IL 10 )对肺泡巨噬细胞致炎效应的影响。方法 :内毒素 (L PS)刺激体外培养的小鼠肺泡巨噬细胞 ,观察 IL 10对肺泡巨噬细胞释放细胞因子的影响。结果 :肺泡巨噬细胞受 10 mg/LL PS刺激后 6、12和 2 4小时 ,肿瘤坏死因子α(TNFα)、IL 1β和 IL 6释放达峰值 ,浓度分别为 (1790±985 ) ng/L、(986± 35 7) ng/L 和 (42 33± 6 5 7) ng/L,与 L PS刺激 0时比较 ,P均 <0 .0 5。IL 10在 L PS刺激后 8小时持续升高〔(2 38± 87) ng/L〕,2 4小时达到峰值 ,浓度为 (16 0 0± 5 2 1) ng/L(P<0 .0 5 )。 15 0 μg/L IL 10单克隆抗体抑制内源性 IL 10释放后 ,导致 TNFα、IL 1β、IL 6释放明显增加 ,分别达 (16 89± 36 4) ng/L、(12 0 0± 2 5 3) ng/L 和 (5 2 6 9± 112 7) ng/L。给予外源性重组 IL 10 (5 0 μg/L) ,则明显抑制 L PS诱导的TNFα、IL 1β、IL 6释放 ,浓度分别降低 5 2 %、84%和 39%。结论 :IL 10对炎症性细胞因子释放具有明显抑制作用 ,补充外源性 IL 10有助于控制肺泡巨噬细胞的异常炎症反应。     

Reversible phagocytosis in rabbit polymorphonuclear leukocytes.

We have studied the fate of inert phagocytized particles in rabbit neutrophils. Neutrophils release significant quantities of preingested oil emulsion. Roughly 50% of an ingested load is released in 40 min at 37 degrees C. By electron microscopy the process of release appears to be by exocytosis: particles appear extruded through a network of processes often accompanied by membranous vesicles. Exocytosis is temperature and glucose dependent but unlike phagocytosis does not require divalent cations. From Coulter counter measurements virtually the entire cell population appears to undergo the phagocytosis-exocytosis sequence. Neutrophils undergoing exocytosis remain intact as determined by direct counts, electron microscopy, and absence of lactate dehydrogenase release. Moreover, by sequentially feeding differently labeled particles, it is shown that the processes of phagocytosis and exocytosis can occur concurrently. Indeed, it is found that ingestion accelerates release. The implications of these phenomena for membrane recycling, lysosomal enzyme release, and the killing of microorganisms are briefly discussed.     

Natural and antibody-dependent cellular cytotoxicity of polymorphonuclear leukocytes

Cytotoxic activity of polymorphonuclear leukocytes (PMN) in the peripheral blood of patients with various diseases was demonstrated to K562 cells (natural cytotoxicity, NC) and the antibody-coated P815 cells (antibody-dependent cellular cytotoxicity, ADCC), using a 51Cr-release method. The NC values of normal PMN were lower than those of normal lymphocytes with mean values of 5.0% and 30%, respectively. The NC values of patients' PMN were also lower in malignancy, chronic hepatitis and connective tissue diseases. The ADCC values of normal PMN were moderately high with a mean value of 16.0%, which was almost a half of normal lymphocytes. Higher ADCC values of PMN were found in patients with chronic hepatitis, SLE and Behcet's disease, and in these cases the ADCC values of their lymphocytes were extremely low. The supernatants of PMN mix-cultured with unlabeled K562 or the antibody-coated P815 cells were fairly cytotoxic to both cells, though the similar supernatants of lymphocytes were cytotoxic only to K562 cells, but not to the antibody-coated P815 cells.     

Intracellular localization of Rickettsia tsutsugamushi in polymorphonuclear leukocytes

Rickettsia tsutsugamushi (Gilliam strain) was serially propagated in BHK-21 cell cultures and incubated with guinea pig peritoneal polymorphonuclear leukocytes to study the ultrastructural features of rickettsial uptake and entry into the leukocytes. Significant numbers of rickettsiae were phagocytized selectively by these leukocytes within 30 min. About one-half of these rickettsiae remained sequestered in phagosomes but the other one-half were free from the phagosome and localized directly in the polymorphonuclear leukocyte cytoplasm. Various stages of rickettsial release from the phagosomes were observed. Once free within the polymorphonuclear leukocyte cytoplasm, the rickettsiae were preferentially localized in the glycogen-packed areas which are devoid of lysosomes and other cytoplasmic organelles. This study indicates that rickettsiae phagocytized by polymorphonuclear leukocytes can escape from the phagosome into the cytoplasm.     

Colchicine analogues: Effect on amyloidogenesis in a murine model and, in vitro, on polymorphonuclear leukocytes

Colchicine has been used in diverse clinical settings such as gout, familial Mediterranean fever, liver cirrhosis, Behcet's disease and pericarditis. It also has an antimitotic potential hitherto unexplored due to its narrow therapeutic toxic ratio. The aim of the present study was to compare the effectiveness and the toxicity of colchicine and three analogues: thiocolchicine, 2,3 dimethyl-colchicine and 3-dimethylthiocolchicine in the blockage of amyloid synthesis in a murine model. 3-demethylthiocolchicine was equipotent to colchicine in the blockage of casein induced amyloidogenesis. However, it was markedly less toxic (LD50 11.3 mg kg-1 vs. 1.6 mg kg-1). Thiocolchicine was toxic (LD50 1.0 mg kg-1) and 2,3 didemethyl-colchicine was far less effective. The effect of 3-dimethylthiocolchicine on polymorphonuclear leukocytes was then compared to colchicine. The effect of this analogue on inhibition of chemotaxis was equivalent to that of colchicine whereas the latter was superior to the analogue in the suppression of phagocytosis (by a ratio of 2:1) and in the inhibition of bactericidal activity (by a ratio of 10:1). Since in therapeutic concentrations the only detectable effect of colchicine on PMNs is inhibition of chemotaxis, our data may point to 3-demethylthiocolchicine as an optional, perhaps superior alternative to colchicine for some of its therapeutic indications.     

还原型谷胱甘肽对糖尿病大鼠肾组织非酶糖基化影响的试验研究

目的探讨还原型谷胱甘肽对糖尿病大鼠肾脏的保护作用及其可能机制。方法以链脲佐菌素诱导糖尿病大鼠模型，给予还原型谷胱甘肽、盐酸氨基胍单独或联合治疗8周，以荧光分光光度计测定肾皮质荧光值即糖基化终末产物（AGEs）含量，行肾组织PAS染色测定平均肾小球截面积（MGAl及体积（MGV），放免法测定尿白蛋白排泄率（UAER），同时检测糖化血红蛋白（HbAIc）、血清肌酐清除率（Cer）、肾重／体重比值、血糖水平等。结果8周时，各组糖尿病大鼠Ccr、UAER、MGA、MGV、肾重／体重比值、AGEs含量均较空白对照组明显增加（P〈0．05或P〈0．01）。还原型谷胱苷肽、盐酸氨基胍单独或联合治疗组Cor、UAER、MGA、MGV、肾重／体重比值、AGEs含量均较糖尿瘸对照组明显降低（P〈0．05或P〈0．01）。结论还原型谷胱苷肽及盐酸氨基胍均可减少肾组织糖基化终末产物生成，对糖尿病大鼠肾脏具保护作用。     

Effect of antibiotics on the phagocytosis and killing of Pseudomonas aeruginosa by rabbit polymorphonuclear leukocytes.

The effect of antibiotics on phagocytosis and killing of Pseudomonas aeruginosa by rabbit polymorphonuclear leukocytes was studied. Carbenicillin and sulbenicillin, when added to an incubation medium at a concentration as low as 1/16 MIC, increased phagocytosis and killing of P. aeruginosa by PMN. Meanwhile, gentamicin and 3',4'-dideoxykanamycin B gave no influence on the PMN activity, and polymyxin B and colistin enhanced the activity only at MIC. The PMN activity was not facilitated even when the cells of P. aeruginosa had been pretreated with antibiotics. The bactericidal activity of PMN decreased after sonification, but was restored following addition of carbenicillin.     

Ultrasonic effects on alveolar macrophages in suspension

Rabbit alveolar macrophages in suspension were exposed to 5 or 10 min of continuous 2-MHz ultrasound with 5, 10, and 15 W/cm² spatial average intensities. Viability as determined by dye exclusion decreased with increasing intensity. Pressure experiments indicated that this was a result of acoustic cavitation. Ultrasound induced clumping of cells and often reduced membrane ruffling. Some cells were disintegrated. Cells that appeared to be otherwise intact had swollen mitochondria with ruptured cristae.     

Intracellular penetration and activity of gemifloxacin in human polymorphonuclear leukocytes

The intracellular penetration and activity of gemifloxacin in human polymorphonuclear leukocytes (PMN) were evaluated. Gemifloxacin reached intracellular concentrations eight times higher than extracellular concentrations. The uptake was rapid, reversible, and nonsaturable and was affected by environmental temperature, cell viability, and membrane stimuli. At therapeutic extracellular concentrations, gemifloxacin showed intracellular activity against Staphylococcus aureus.