Glycogen metabolism in stored granulocytes

Optimal function of transfused granulocytes (PMNs) requires adequate glycogen metabolism. Previous studies in our laboratory suggested that stored PMNs had decreased glycogen. We report here the glycogen content and chemotaxis of stored PMNs, and the ability of fresh and stored PMNs to use glycogen as the fuel source for chemotaxis. PMNs were prepared from 8 fresh units of blood drawn into citrate-phosphate-dextrose-adenine, suspended at 2 or 8 X 10(7) PMN per ml in autologous plasma with or without 15 mM sodium bicarbonate, and stored at 22 to 24 degrees C in transfer packs for 48 hours. Glycogen was measured on resting PMNs, and after challenge with opsonized zymosan and F-Met-Leu-Phe (FMLP). The chemotaxis of fresh and stored PMNs was measured in the presence or absence of extracellular glucose. Fresh PMNs contained 10.3 +/- 0.5 (mean +/- SEM) micrograms of glycogen per 10(6) PMN. Glycogen decreased by 4.2 +/- 0.9 micrograms per 10(6) PMN after challenge with opsonized zymosan and by 1.1 +/- 0.6 micrograms per 10(6) PMN after FMLP. After 48 hours of storage, neutrophil glycogen increased by 18 percent, except in units stored at a concentration of PMN of 8 X 10(7) per ml without sodium bicarbonate. In PMNs from these units stored without bicarbonate, glycogen decreased by 9 percent (p less than .05), and there was a 19 and 55 percent decrease in the ability of PMN from these units to metabolize glycogen after exposure to opsonized zymosan and FMLP, respectively (p less than 0.05). In addition, in PMNs from units stored at a concentration of PMN of 8 X 10(7) per ml without bicarbonate, there was a 47 and 70 percent decrease in chemotaxis at 24 and 48 hours, respectively (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Hydrogen ion maintenance improves the chemotaxis of stored granulocytes

Through technological advances in granulocyte collection, it has become possible to collect neutrophils (PMNs) routinely in high concentration (greater than 5 X 10(7) PMN/ml) for transfusion. Previous studies in this laboratory suggested that storage of neutrophils for transfusion at high PMN concentrations resulted in impaired adenosine triphosphate (ATP) and hydrogen ion maintenance. The studies we report here were designed to assess the effect of PMN storage at concentrations which are usual (2 X 10(7) PMN/ml), intermediate (5 X 10(7) PMN/ml), and high (8 X 10(7) PMN/ml) on chemotactic responses, and to identify variables which are easily measured and might predict the chemotactic function of stored PMNs. Granulocyte concentrates were stored in plastic bags at 2,5, and 8 X 10(7) PMN per ml, with or without 15 mM bicarbonate (HCO3). The random migration (RM) chemotaxis (CTX), ATP, and relative cell size (VOL) of the fresh and stored cells and the pH, glucose, and lactate concentrations in the supernatant medium were measured in the freshly prepared units after 24 and 48 hours storage at room temperature. We found that RM, CTX, ATP, glucose, and pH decreased significantly (p less than .02) following storage for 24 and 48 hours, particularly in units stored at the higher cell concentrations. Cell volume and lactate increased significantly with storage for 24 and 48 hours, and these values were also greater in units stored at the higher cell concentration (p less than .02).(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of secretory events in modulating human neutrophil chemotaxis.

The relationship between neutrophil polymorphonuclear leukocyte (PMN) locomotion and the exocytosis of neutrophil cytoplasmic granules was studied by assessing these processes in cells migrating through micropore filters and by measuring the effects of degranulating stimuli on PMN chemotaxis, orientation, adhesiveness, and ability to bind the chemoattractant f-Met-Leu-[3H]Phe. Studies of cells migrating through cellulose nitrate filters indicated that concentrations of f-Met-Leu-Phe optimal for exocytosis were greater than those optimal for chemotaxis and actually inhibited cell migration. In other studies incubation of PMNs with concentrations of secretagogues causing exocytosis of 30% or greater PMN lysozyme increased cell adhesiveness and inhibited chemotaxis. PMNs that had secreted more than 30% lysozyme appeared round, did not orient in a gradient of chemoattractant, and were capable of significantly less f-Met-Leu-[3H]Phe binding than were control cells. The decreased binding of f-Met-Leu-Phe was not associated with hydrolysis of chemotactic peptide by washed cells, although peptide hydrolysis was caused by cell products secreted extracellularly after vigorous exocytosis. In contrast, when only 10--15% cellular lysozyme was released f-Met-Leu-Phe binding was enhanced significantly and there was no depression of chemotaxis. The data indicate limited exocytosis of intracellular granule contents is associated with increased availability of PMN cehmotactic factor receptors. Vigorous exocytosis is associated with inactivation of chemotactic responsiveness related to increase cell adhesiveness, decreased PMN binding of chemotactic factors, and to hydrolysis of chemoattractants by factors secreted extracellularly.     

Exudation primes human and guinea pig neutrophils for subsequent responsiveness to the chemotactic peptide N-formylmethionylleucylphenylalanine and increases complement component C3bi receptor expression.

After circulating in the vascular system a short time, polymorphonuclear leukocytes (PMN) migrate to extravascular sites in response to chemotactic stimuli. Prestimulation of PMN in vitro by secretagogues has been shown to increase their number of N-formylmethionylleucylphenylalanine (fmet-leu-phe) and complement component C3bi (CR3) receptors. We investigated whether the same phenomenon occurred in vivo, comparing characteristics of human skin chamber and guinea pig peritoneal exudate and blood PMN. Exudate PMN of both species contained approximately 28% less of the specific granule marker vitamin B12-binding protein (P less than 0.01) but a similar amount of the azurophil granule marker beta-glucuronidase. The total number of fmet-leu-phe receptors was 5.9 times higher in guinea pig exudate than in blood PMN (P less than 0.01) and 2.9 times higher in human exudate than in blood PMN (P less than 0.02). All exudate PMN and most blood PMN preparations showed a high affinity receptor (Kd approximately 2.3 X 10(-8) M) and a low affinity receptor (approximately 1.5 X 10(-7) M). The upregulation of fmet-leu-phe receptors in exudate PMN correlated with an improved responsiveness to fmet-leu-phe induced membrane depolarization, oxidative metabolism, and chemotaxis. In addition, the concentration of fmet-leu-phe that produced a half-maximal response of chemotaxis, superoxide production, and membrane potential depolarization was 10-fold lower in exudate PMN than in blood PMN. Human exudate PMN had a twofold increased C3bi receptor expression compared with blood PMN. Thus, a preferential loss of specific granules is associated with increased number of high and low affinity fmet-leu-phe receptors and increased C3bi receptor expression not only in vitro, but also in vivo. The data indicate that exudation primes PMN for their subsequent responsiveness to fmet-leu-phe, a modification that may be crucial for efficient antimicrobial host defense.     

Neutrophil heterogeneity in patients with blunt trauma

To determine whether neutrophil (PMN) dysfunction observed in patients with blunt trauma could be explained by alterations in PMN functional subpopulations and to further study the origins of PMN heterogeneity, we studied PMN subpopulations in 18 patients with severe blunt trauma by using a micropore filter chemotactic assay and a mouse monoclonal antibody (31D8 Mab). A major PMN subpopulation binds 31D8 Mab avidly (31D8 "bright") and depolarizes and responds chemotactically to formyl peptide (fMLP) and C5a; a minor PMN subpopulation binds 31D8 Mab weakly (31D8 "dull") and fails to depolarize and responds poorly to fMLP and C5a. Fourteen patients with trauma had marked alteration of PMN 31D8 expression compared with healthy controls 52% +/- 20% versus 92% +/- 4% bright PMNs, respectively (p less than 0.01). These patients also had significantly decreased PMN chemotaxis and increased band counts compared with controls 30 +/- 10 micron versus 53 +/- 19 micron (p less than 0.01) and 34% +/- 14% versus 6% +/- 1% (p less than 0.01), respectively. Four patients with less-severe injuries had unaltered 31D8 PMN expression and normal PMN chemotaxis. In patients whose band counts exceeded 20%, there was a strong correlation between the number of bands and the percentage of 31D8 dull PMNs. PMNs that weakly express the 31D8 antigen appear to be less mature than PMNs that strongly express the antigen regardless of cell morphology (i.e., bands, multilobed cells). The data suggest that the decreased PMN chemotaxis and increased infection rate in patients with blunt trauma is caused partly by an increase in the number of poorly functioning 31D8 dull PMNs.     

Hydroxyethyl starch reduces the chemotaxis of white cells through endothelial cell monolayers

BACKGROUND: Polymorphonuclear leukocytes (PMNs) play a tremendous role during inflammatory processes. PMNs have to pass a monolayer of endothelial cells to migrate into the extravascular space. Hydroxyethyl starch (HES) is frequently used as a volume expander in critically ill patients. STUDY DESIGN AND METHODS: The aim of this study was to investigate whether HES influences the chemotaxis of PMNs through endothelial cell monolayers by using a test system that allows the simultaneous treatment of both cell types. Human umbilical endothelial cells were cultured on microporous membrane filters. PMNs were isolated and PMN chemotaxis was studied. RESULTS: The number of untreated PMNs that migrated through untreated endothelial cell monolayers in response to a chemoattractant was used as a control and set as 100 percent. In clinically relevant concentrations, HES was able to significantly decrease PMN chemotaxis through endothelial cell monolayers, showing a dose-dependent effect (0.1 mg/mL: 99.6 +/- 10.9%, p = NS compared to control; 1 mg/mL: 82.4 +/- 8.3%, p<0.05 compared to control; 10 mg/mL: 62.9 +/- 11.7%, p<0.05).In this assay, both cell types (PMNs and endothelial cells in the monolayer) were treated simultaneously, which simulated the clinical situation after an intravenous injection of HES. The treatment of one cell type, PMNs (89.6 +/- 8.8%, p<0.05) or endothelial cells in the monolayer (76.2 +/- 9.4%, p<0.05), suggests that the influence on endothelial cells is greater. CONCLUSION: HES is able to significantly reduce the chemotaxis of PMNs through endothelial cell monolayers. The possible clinical consequence of a moderate reduction in endothelium-mediated PMN chemotaxis in critically ill patients remains to be evaluated.     

A functional granulocyte colony-stimulating factor receptor is required for normal chemoattractant-induced neutrophil activation

Granulocyte colony-stimulating factor (G-CSF) is a hematopoietic growth factor that is widely used to treat neutropenia. In addition to stimulating polymorphonuclear neutrophil (PMN) production, G-CSF may have significant effects on PMN function. Because G-CSF receptor (G-CSFR)-deficient mice do not have the expected neutrophilia after administration of human interleukin-8 (IL-8), we examined the effect of the loss of G-CSFR on IL-8-stimulated PMN function. Compared with wild-type PMNs, PMNs isolated from G-CSFR-deficient mice demonstrated markedly decreased chemotaxis to IL-8. PMN emigration into the skin of G-CSFR-deficient mice in response to IL-8 was also impaired. Significant chemotaxis defects were also seen in response to N-formyl-methionyl-leucyl-phenylalanine, zymosan-activated serum, or macrophage inflammatory protein-2. The defective chemotactic response to IL-8 does not appear to be due to impaired chemoattractant receptor function, as the number of IL-8 receptors and chemoattractant-induced calcium influx, actin polymerization, and release of gelatinase B were comparable to those of wild-type PMNs. Chemoattractant-induced adhesion of G-CSFR-deficient PMNs was significantly impaired, suggesting a defect in beta2-integrin activation. Collectively, these data demonstrate that selective defects in PMN activation are present in G-CSFR-deficient mice and indicate that G-CSF plays an important role in regulating PMN chemokine responsiveness.     

The comparative responses of human polymorphonuclear leukocytes obtained by counterflow centrifugal elutriation and Ficoll-Hypaque density centrifugation. I. Resting volume, stimulus-induced superoxide production, and primary and specific granule release

Standard isolation techniques for the human polymorphonuclear leukocyte (PMN) involve sequential exposure of cells to the nonphysiologic environments of dextran, Ficoll-Hypaque (FH) gradient centrifugation, and hypotonic conditions. It has been suggested that these may be harmful to the recovered PMN. Counterflow centrifugal elutriation (CCE) allows separation of human PMNs while the cells are continuously bathed in a physiologic and isotonic buffer. To investigate whether preparative technique may alter PMN activation, we compared PMNs obtained by these two methods for stimulus-induced superoxide production and release of primary and specific granule contents. Resting PMN volume was also evaluated. We observed that PMNs obtained using the CCE method were larger and released significantly more superoxide and specific granule contents than PMNs obtained by the standard FH technique. The possible origins for these differences are discussed.     

Lipid complexing decreases amphotericin B inflammatory activation of human neutrophils compared with that of a desoxycholate-suspended preparation of amphotericin B (Fungizone).

Amphotericin B (AmB) has toxic effects and alters neutrophil (polymorphonuclear leukocyte [PMN]) function. A lipid-complexed formulation of AmB (AmB-LC) has been reported (A. S. Janoff, L. T. Boni, M. C. Popescu, S. R. Minchey, P. R. Cullis, T. D. Madden, T. Taraschi, S. M. Gruner, E. Shyamsunder, M. W. Tate, R. Mendelsohn, and D. Bonner, Proc. Natl. Acad. Sci. USA 85:6122-6126, 1988) to be less toxic than a desoxycholate-suspended preparation of AmB (AmB-des; Fungizone). In this study we compared the effects of AmB-des and AmB-LC on in vitro PMN function. Neither form of AmB stimulated PMN chemiluminescence, but AmB-des (2 micrograms/ml) nearly tripled PMN chemiluminescence in response to f-Met-Leu-Phe (fMLP), a phenomenon known as priming. Because AmB stimulates monocytes to release cytokines which can affect PMN function, we studied the effects of AmB on PMNs in mixed leukocyte cultures. AmB-des (1 to 2 micrograms/ml) increased the chemiluminescence of PMNs plus mixed mononuclear leukocytes (MNLs) to fMLP. The activity was about three times that of PMNs plus MNLs and seven times the activity of PMNs stimulated with fMLP in the absence of MNLs. Cell-free AmB-des (2 micrograms/ml)-stimulated, MNL-conditioned medium primed pure PMNs to a level equal to that of whole MNLs treated with AmB-des. AmB-LC was much less potent. AmB-LC (20 micrograms/ml) increased fMLP-stimulated chemiluminescence to two times that of PMNs plus MNLs without AmB-LC. AmB-des (2 micrograms/ml) (but not AmB-LC [2 micrograms/ml]) increased nitroblue tetrazolium reduction by PMNs in whole blood from 31 to 52% of positive cells. Neither form of AmB increased Mac-1 (the CD11b/CD18 integrin) expression of pure PMNs. AmB-des (0.5 to 2 micrograms/ml) (but not AmB-LC [< or = 40 micrograms/ml]) nearly doubled PMN Mac-1 expression in the presence of MNLs, and cell-free AmB-des (2 micrograms/ml)-stimulated, MNL-conditioned medium stimulated PMN Mac-1 to 125% of the control level. AmB-des (0.2 to 2 micrograms/ml) (but not AmB-LC [< or = 40 micrograms/ml]) decreased chemotaxis of pure PMNs to fMLP by as much as 35% and that of PMNs in the presence of MNLs by as much as 50%. Desoxycholate by itself had no effect on PMN function. These differences in activity between AmB-des and AmB-LC may explain the lessened toxicity observed with AmB-LC.     

Effects of amodiaquine, chloroquine, and mefloquine on human polymorphonuclear neutrophil function in vitro.

This study concerns the in vitro interaction with human polymorphonuclear neutrophils (PMNs) of amodiaquine, chloroquine, and mefloquine, three antimalarial drugs currently in use for the treatment and prophylaxis of malaria. It was found that mefloquine (100 and 50 micrograms/ml) significantly altered PMN viability while the other two drugs did not. Neutrophil chemotaxis was impaired by chloroquine (100 micrograms/ml) and mefloquine (greater than 10 micrograms/ml) but not by amodiaquine. Phagocytosis was decreased by about 50% in the presence of chloroquine (100 micrograms/ml) or mefloquine (10 micrograms/ml). The three antimalarial drugs altered neutrophil oxidative metabolism as assessed by luminol-amplified chemiluminescence. The strongest effect was observed with mefloquine, which abolished almost completely the neutrophil burst at concentrations of greater than 10 micrograms/ml whatever the stimulus used. This effect was not reversed by washing. Chloroquine and amodiaquine also impaired this PMN response by approximately 80 and 50%, respectively, but only at the highest concentration used (100 micrograms/ml). In the case of amodiaquine, the neutrophil response was restored by washing, except for stimulation with opsonized particles. After washing, the depressive effect of chloroquine was reversed completely in the case of phorbol myristate acetate stimulation and partly in the case of opsonized particle stimulation, but the formylmethionyl-leucyl-phenylalanine-induced response was not restored. These data show that although they are structurally related, amodiaquine and chloroquine exhibit qualitatively and quantitatively different depressive effects on PMN function and probably interfere at different points of cell activation, although the precise mechanisms are as yet unresolved.     

Defective energy metabolism in stored granulocytes

Previous studies suggested that a general metabolic defect might be responsible for impaired chemotaxis of stored granulocytes (PMN). We studied PMN energy metabolism in fresh and stored PMN. Adenosine triphosphate (ATP) decreased slightly in unstimulated PMN after 48 hours of storage at room temperature. Exposure of fresh PMN to opsonized zymosan resulted in a 12 percent decrease in ATP. In contrast, PMN stored for 24 or 48 hours at room temperature and then exposed to opsonized zymosan, showed significant decreases in ATP. Incubation of fresh or stored, stimulated (opsonized zymosan) or resting PMN with potassium cyanide resulted in no change in the pattern of ATP decrements during storage. Likewise, incubation of fresh or stored, stimulated or resting PMN in the presence or absence of extracellular glucose, resulted in no change in the pattern of ATP decrements during storage. Preincubation of PMN with 2-deoxy-glucose obscured the differences in ATP maintenance between fresh and stored PMN. ATP decrements were also observed in unstimulated PMN which had been stored at high cell concentrations. Decrements in ATP correlated stronglywith decreased glucose (r = .82) and pH (r = .89) in the storage medium. These decrements were partially reversed by incubation of PMN in fresh buffer. The ATP decrements in stored, concentrated granulocyte preparations thus were prevented by maintenance of pH. ATP decrements were not prevented by additional glucose. Thus, in PMN stored for 24 or 48 hours at room temperature, there was a defect in ATP maintenance which was occult in unstimulated cells, but was evoked by the energy-dependent process, phagocytosis. These findings are compatible with a major role for glycolysis and glycogenolysis in maintaining ATP in fresh and stored PMN. Storage of PMN at high cell concentrations was accompanied by impaired ATP maintenanceand decreased pH in the storage medium, both of which were preventable by addition of alkali to the PMN concentrate prior to storage.     

Effect of ibuprofen on neutrophil migration in vivo in cystic fibrosis and healthy subjects

Long-term treatment with ibuprofen twice daily, at doses that achieve peak plasma concentration (Cmax) >50 microg/ml, slows progression of lung disease in patients with cystic fibrosis (CF). Previous data suggest that Cmax >50 microg/ml is associated with a reduction in neutrophil (PMN) migration into the lung and that lower concentrations are associated with an increase in PMN migration. To estimate the threshold concentration at which ibuprofen is associated with a decrease in PMN migration in vivo, we measured the PMN content of oral mucosal washes in 35 healthy (age 19-40 years) and 16 CF (age 18-32 years) subjects who took ibuprofen twice daily for 10 days in doses that achieved Cmax 8 to 90 microg/ml. Cmax >50 microg/ml was associated with a 31 +/- 7% (mean +/- S.E.M.) reduction in PMNs in CF (n = 11, p < 0.001) and 25 +/- 6% reduction in PMNs in healthy subjects (n = 16, p < 0.001). Increasing concentrations above 50 microg/ml was not associated with a greater decrease in PMNs. The reduction in PMN migration was consistently present 12 h after a dose, but not after 24 h. Cmax <50 microg/ml was associated with an increase in PMNs of approximately 40%. These results suggest that Cmax >50 microg/ml and twice daily dosing of ibuprofen are required to decrease PMN migration, and reinforce the current recommendation that pharmacokinetics should be performed in CF patients prescribed ibuprofen.     

Cytochalasin B facilitates the inhibition of human polymorphonuclear leukocyte generation of superoxide by verapamil

Several functions of the human neutrophil are dependent upon extracellular calcium for optimal activation. We studied the calcium dependency of human polymorphonuclear leukocyte (PMN) superoxide generation in response to opsonized zymosan particles and its modulation by the calcium channel antagonist verapamil. PMNs were isolated from anticoagulated blood after Ficoll-Hypaque density centrifugation. The isolated PMNs were incubated with opsonized zymosan particles. The superoxide anion generated was measured by a cytochrome C reduction assay. When PMNs were incubated with opsonized zymosan in a calcium-free buffer, there was 0.45 +/- 0.06 nmol of cytochrome C reduced per 1 X 10(6) PMN per minute. This increased to 0.76 +/- 0.12 nmol per 1 X 10(6) PMN per minute when 0.6 mmol/L Ca++ was present. Moreover, if the PMNs were incubated with cytochalasin B (5 micrograms/ml), the generation of superoxide anion was further enhanced. If the same experiments were conducted in the presence of verapamil, 100 mumol/L, superoxide generation was inhibited by 51.5% +/- 8.4%. For verapamil to inhibit superoxide generation, cytochalasin B was necessary. Our results suggest that verapamil-sensitive calcium channels may exist in human PMNs, and the demonstration of their presence is cytochalasin B-dependent.     

Effects of fibrinogen derivatives upon the inflammatory response. Studies with human fibrinopeptide B.

Fibrin formation and turnover are intimately associated with inflammation and wound healing. To explore whether fibrin(ogen)-derived peptides exert direct effects upon cells involved in inflammation and tissue repair we examined the capacity of human fibrinopeptide B (hFpB), a thrombin-derived proteolytic cleavage product of the fibrinogen B beta-chain, to stimulate neutrophils (PMN), monocytes, and fibroblasts. hFpB caused directed cell migration of PMN and fibroblasts that was optimal at approximately 10(-8) M. This chemotactic activity was blocked by preincubating hFpB with antiserum to hFpB. hFpB was not chemotactic for monocytes. The chemotactic potency of hFpB for PMN was equivalent to that of anaphylatoxin from the fifth component of human complement (C5a), leukotriene B4 (LTB4), and formyl-methionyl-leucyl-phenylalanine (fMLP), and for fibroblasts its chemotactic activity was comparable to that of platelet-derived growth factor. hFpB did not interact with PMN receptors for C5a, LTB4, or fMLP as (a) desensitization with 10(-7) M hFpB abolished chemotaxis to hFpB but had no effect upon chemotaxis to C5a, LTB4, or fMLP and (b) induction of chemotactic responses to fMLP and LTB4 in neutrophilic leukemic cells (HL-60 cells) by incubation with dimethylsulfoxide did not extend to hFpB. Like fMLP, hFpB caused a rapid, dose-dependent increase in PMN cytoskeletal associated actin, but unlike fMLP, hFpB did not cause PMN aggregation, release of lysosomal enzymes (lysozyme and beta-glucuronidase), or the production of superoxide anion. These results suggest that hFpB may have a role in recruiting PMN and fibroblasts at sites of fibrin deposition and turnover. The capacity of hFpB to cause PMN chemotaxis without causing concurrent release of lysosomal enzymes or the production of superoxide anion is further evidence for the complexity of PMN responses to chemotactic agents.     

胸部撞击伤时游离Ca2+浓度与中性粒细胞凋亡及肺损伤的关系

目的 :动态观察兔胸部撞击伤时中性粒细胞 (PMN)凋亡的发生以及与肺损伤之间的关系。方法 :制备兔胸部撞击伤模型 ,分离纯化肺灌洗液中的 PMN,应用流式细胞术测定 PMN凋亡、坏死、存活细胞比例及呼吸爆发功能的变化 ,并且观察与乳酸脱氢酶 (L DH)和胞浆游离 Ca2 变化之间的关系。结果 :肺灌洗液中PMN的凋亡延迟持续至 12小时 ,在伤后各时间点活细胞增多 ;而肺灌洗液 PMN呼吸爆发从伤后 2小时即显著增强 ,8小时达到峰值 ;同时肺灌洗液 L DH的升高在伤后 4～ 2 4小时显著高于正常对照组 ;伤后 PMN胞浆游离 Ca2 有短暂升高。结论 :胸部撞击伤时 ,PMN在肺组织中大量扣押 ,且正常的凋亡途径发生障碍 ,造成PMN持续处于激活状态及毒性内容物的持续释放 ,与肺组织损伤有密切关系 ,并且 PMN凋亡延迟可能与胞浆游离 Ca2 的短暂升高有关     

Neutrophil chemotaxis and adherence in vitro and localization in vivo in rabbits with Staphylococcus aureus abscesses

Decreased neutrophil (PMN) function may contribute to an altered host defense system in hosts with bacterial abscesses but has not been well correlated to in vivo outcome. We have found that blood PMNs from rabbits with chronic (2-week) experimental Staphylococcus aureus abscesses have decreased chemotaxis in response to an S. aureus supernatant (370 +/- 130 microns migration vs 570 +/- 180 microns, p less than 0.05) and decreased adherence (11.5% +/- 13.2% vs 32.9% +/- 18.6%, p less than 0.005) compared with PMNs from animals with acute (24-hour) abscesses. No differences were found in chemokinesis, random migration, and chemotaxis in response to zymosan-activated serum. No plasma inhibitors of PMN chemotaxis or inhibitors of chemotaxins were found. Although animals with chronic abscesses had higher levels of circulating chemotaxins, in both groups of animals abscess chemotaxin levels were greater than the plasma chemotaxin level. Animals with a concomitant chronic abscess had less PMN influx into an acute abscess but also less bacterial growth within the abscess than animals without a concomitant chronic abscess. We conclude that rabbits with chronic staphylococcal abscesses have decreased chemotaxis and adherence measured in vitro and decreased PMN localization in vivo. In this model, these functions were not associated with increased bacterial proliferation in vivo.     

Human leukocytic pyrogen induces release of specific granule contents from human neutrophils.

The ability of highly purified human leukocytic pyrogen (LP) to induce neutrophil lysosomal protein release is described. Human peripheral blood neutrophils isolated by Ficoll-Hypaque and dextran sedimentation were exposed to purified human LP. The specific granule-associated proteins, lysozyme and lactoferrin were selectively released, whereas primary granule (beta-glucuronidase) and cytoplasmic (lactic dehydrogenase) enzyme markers were not. Optimum release was observed after 45 min in the presence of Ca++ and Mg++. Cytochalasin B (5 microgram/ml) had no effect on LP-induced lysosomal enzyme release. Since the pyrogenicity of LP is dependent on prostaglandin synthesis, the effect of two potent inhibitors of prostaglandin synthesis on lysozyme release was studied. Both indomethacin and naproxen failed to inhibit specific granule protein release. These observations suggest that the concommitance of fever, elevated serum or urine lysozyme and hypoferremia may, in part, be explained by the interaction of LP and peripheral blood neutrophils.     

Stored red blood cells selectively activate human neutrophils to release IL-8 and secretory PLA2

Packed red blood cell (PRBC) transfusion has been invoked previously with immunosuppression and increased infections, but it has now been demonstrated that stored PRBCs (>14 days) can prime PMNs and provoke multiple organ failure. Recently, the role of PMNs in the genesis of MOF has been extended to their release of inflammatory cytokines, notably IL-1, IL-8, TNFalpha, and secretory phospholipase A2 (sPLA2). We hypothesize that stored PRBCs can act as a second event via stimulating the release of inflammatory cytokines from PMNs. Isolated human PMNs were incubated for 24 h in RPMI with either 20% fresh plasma or plasma from 42 day old PRBC (day of outdate) and release of IL-8, IL-1beta, TNFalpha, and sPLA2 were measured. Plasma from stored PRBCs contained small amounts of IL-8, sPLA2, and TNFalpha (102.1 +/-5.6 pg/ml, 87.6+/-6.0 pg/ml and 9.7+/-.7 pg/ml). Levels of IL-1beta were below detection (<1 pg/ml). Day 42 PRBC plasma stimulated significant PMN release of both IL-8 and sPLA2 as compared to both control and day 0 plasma (*P < .05), but PRBC plasma did not stimulate PMN release of either IL-1beta or TNFalpha. Transfused blood is emerging as an inflammatory agent that is capable of producing PMN priming. In this study we have demonstrated that PRBC plasma selectively activates PMNs to release both IL-8 and sPLA2. Thus, transfusion of PRBCs may represent a preventable inflammatory insult via modification of both blood banking and transfusion practices.     

The structure-activity relations of synthetic peptides as chemotactic factors and inducers of lysosomal secretion for neutrophils

24 di-, tri-, and tetrapeptides have been synthesized as a start of a systematic study of the structural requirements for chemotactic activity and lysosomal enzyme-releasing ability in rabbit neutrophils. All but two of them are N-formyl methionyl peptides. Using the method of Zigmond and Hirsch (10), two representative peptides, F-Met-Leu-Phe and F-Met-Met-Met, were shown to stimulate directed, as well as, random locomotion; thus, they were truly chemotactic. The various peptides showed a wide spread in activity. F-Met-Leu-Phe, the most active peptide studied, had an ED50 for induced migration of 7 X 10(-11) M and for lysozyme and beta-glucuronidase release of 2.4 X 10(-10) M and 2.6 X 10(-10) M, respectively; the least active, Met-Leu-Glu was 26 million times less active in these respects. The relation of activity to structure is exceedingly specific, very small changes in structure making large changes in activity. Moreover, this specificity exhibits a definite regularity and pattern; the activity of a given peptide depends not only on its constituent amino acids but on the position of the amino acid in the peptide chain. Most striking in this last regards is the high activity conferred by phenylalanine when it is in the carboxyl terminal position of a tripeptide, whereas, as the second amino acid from the NH2 terminal end whether in a tripeptide or a dipeptide, it contributes no more to the activity than other amino acids with hydrophobic side chains such as leucine or methionine. The high activity and the specificity and nature of the structural requirements strongly suggest that the primary interaction of peptide and neutrophil leading to either chemotaxis or lysosomal enzyme release is a binding of the peptide with a stereospecific receptor on the neutrophil surface. Whether all chemotactic factors act through the same receptor is not known. An essentially exact correlation exists between the concentrations of the various synthetic peptides required to induce migration and their ability to induce release of lysozyme or beta-glucuronidase. This implies that these two neutrophil functions are triggered by teh same primary interaction; possibly, the binding of the peptides to the same putative receptor. A higher concentration of a given peptide is required to stimulate lysosomal enzyme release than a corresponding migratory response. A slightly but significantly higher concentration of peptide is required to induce beta-glucuronidase secretion than lysozyme release.     

Effects of antifungal agents on the function of human neutrophils in vitro.

Polymorphonuclear leukocytes (PMNs) are an important component of the host defense against fungi. We investigated the influence of five antifungal agents on PMN function and compared them with amphotericin B (AmB). The in vitro effects of AmB, flucytosine, ketoconazole, fluconazole, Sch-39304, and cilofungin (LY121019) on chemotaxis, phagocytosis, oxidative metabolism of PMN as reflected by superoxide anion (O2-) generation, and intracellular killing of Candida albicans blastoconidia were examined. With regard to chemotaxis in response to N-formylmethionyl-leucyl-phenylalanine, as measured by the multiwell chamber method, AmB induced a marked decrease (greater than or equal to 5 micrograms/ml), whereas ketoconazole at 5 micrograms/ml enhance it. Phagocytosis was significantly decreased after pretreatment of PMNs with AmB and Sch-39304 (greater than 5 and 1 to 10 micrograms/ml, respectively). O2- production after stimulation of PMNs with N-formylmethionyl-leucyl-phenyl-alanine was significantly decreased by AmB (greater than 5 micrograms/ml) and enhanced by Sch-39304 (1 to 5 micrograms/ml). In contrast, intracellular killing, as tested by methylene blue staining, was enhanced by ketoconazole (5 micrograms/ml) and Sch-39304 (1 to 5 micrograms/ml). Flucytosine, fluconazole, and cilofungin did not affect PMN function at therapeutic concentrations. The results of this comprehensive study indicate that AmB, flucytosine, cilofungin, and the newer azoles, at safely achievable concentrations, generally do not suppress PMN function at therapeutic enhance selective functions.