The effect of storage on degranulation by human neutrophils

We investigated the possibility that the functional impairment in neutrophil (PMN) chemotaxis which occurs during granulocyte concentrate storage might be due to autotoxicity from the release of neutrophil granule contents during storage. Preliminary experiments confirmed that the exposure of fresh PMNs to the intracellular contents of disrupted PMNs, decreased the subsequent chemotaxis of the fresh PMNs by 63 +/- 5 percent compared to control PMNs (p less than .01). Freshly harvested neutrophils were stored at low (2 X 10(7) PMN/ml) or high cell concentration (8 X 10(7) PMN/ml) with or without 15 mM sodium bicarbonate (in order to maintain pH). Prior to storage, and 24 and 48 hours after storage at 22 to 24 degrees C, we measured the cell and unit plasma content of lactate dehydrogenase (LDH), beta-glucuronidase, and lysozyme. These enzymes served as markers for cell lysis, and primary and specific neutrophil granule contents, respectively. We also measured the effect on neutrophil chemotaxis of adding a protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), to the storage medium. In addition, we measured the ability of PMNs to degranulate in response to an inflammatory stimulus before and after storage. The cell content of granule markers was largely unchanged during storage, except in the case of the units at a concentration of 8 X 10(7) PMN per ml stored without bicarbonate. In these units, lysozyme activity decreased by 15 +/- 7 percent after 48 hours of storage (p less than 0.02 vs. fresh PMNs). Likewise, the plasma content of LDH, beta-glucuronidase, and lysozyme increased significantly during storage, especially in units of high cell concentration stored without bicarbonate.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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)     

Chemotaxin receptor cycling in fresh and stored granulocytes

The authors reported previously that stored granulocytes (PMN) had decreased receptor affinity (Kd) for and chemotaxis (CTX) to the chemotactic peptide F-Met-Leu-Phe (FMLP), but the evidence did not favor a significant role for altered FMLP receptor affinity in causing diminished CTX of stored PMN. Since recruitment and/or recycling of FMLP receptors is required for normal CTX, the hypothesis that stored PMN might have abnormal FMLP receptor cycling was tested. The effect of storage on the proportion of high- and low-affinity FMLP receptors was also investigated. Units of PMN were tested within 4 hours of collection and after 24 and 48 hours of storage at 22 degrees C, unagitated, in 150-ml transfer packs. In comparison to fresh PMN, there was no alteration in the Kd of the high-affinity FMLP receptor of PMN stored for 24 to 48 hours; however, the Kd of the low-affinity receptor increased (fresh PMN = 36 +/- 5 nM; 24 hours = 107 +/- 19; 48 hours = 121 +/- 22; p less than 0.01 for both 24 and 48 h versus fresh PMN). Likewise, while the number of high-affinity receptors increased (fresh PMN = 25,000 +/- 6,000 receptors/PMN; 24 hours = 95,000 +/- 21,000; 48 hours = 161,000 +/- 40,000; p less than 0.01 for both 24 and 48 hours versus fresh PMN). No abnormality was found in the ability of stored PMN to down-regulate FMLP receptors in the presence of ligand or to reexpress FMLP receptors after a 15-minute incubation in the absence of ligand.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Function of irradiated polymorphonuclear leukocytes obtained by buffy- coat centrifugation

Several studies suggest that transfusion of polymorphonuclear leukocytes (PMNs) may be beneficial in the treatment of septic neonatal patients. Because of expense, donor availability, and the technical effort involved in obtaining PMNs by intermittent or continuous flow leukapheresis, buffy coat centrifugation of whole blood has been suggested as an alternative source. An in vitro study was performed to determine whether PMNs collected by this method have adequate oxidative and migratory function measured by chemiluminescence (CL) and chemotaxis under agarose (CT), respectively. Whole blood samples from six adult volunteers were drawn into citrate-phosphate-dextrose-adenine-one and stored at 4 degrees C for 0 to 48 hours. One-half of each sample was irradiated with 1500 rads. PMNs isolated from the buffy coat of these samples had greater than 80 percent normal CT and CL following 0 to 28 hours of storage in whole blood. Irradiation caused no depression in function. Units of whole blood yielded 1.11 +/- 0.40 X 10(9) PMNs per unit. This study indicates that transfusion of radiated PMNs obtained from stored whole blood that is less than 28 hours old is reasonable to use in studies involving PMN transfusions.     

Dissociation of human neutrophil activation events by prolonged treatment with amiloride

Human polymorphonuclear leukocytes (PMNs) can be stimulated to release granule contents and to produce superoxide anion. These functional responses are associated with cellular alkalinization and influx of Na+ in exchange for H+. Amiloride is a potassium-sparing diuretic that will inhibit stimulus-induced Na+-H+ exchange and prevent an increase in cell pH. Amiloride has been shown to inhibit a number of protein kinases including the calcium phospholipid-dependent protein kinase. Because PMA, which binds and activates C-kinase, is a potent stimulus of the PMN, this study was undertaken to investigate the effect of prolonged incubation of PMNs with amiloride on PMN stimulation by the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP), the calcium ionophore A23187, opsonized zymosan particles, and the tumor promoter phorbol myristate acetate (PMA). Our results demonstrate that amiloride inhibits superoxide anion production by FMLP, A23187, and opsonized zymosan by causing a slower rate of release and lower maximal release without altering lag time. In contrast, amiloride, despite an inhibition of 22Na+ influx, did not affect superoxide anion production stimulated by PMA. PMN degranulation, phagocytosis, arachidonic acid release, and early influx of calcium were unaffected by preincubation with amiloride. These data suggest that PMN superoxide release induced by FMLP, A23187, and opsonized zymosan is likely modulated by amiloride-sensitive Na+-H+ exchange; and phorbol ester-induced superoxide anion release and degranulation by any stimulant do not appear to be modulated by inhibition of an amiloride-sensitive Na+-H+ exchange.     

Adherence of fresh and stored granulocytes to endothelial cells. Effect of storage temperature

Granulocyte (PMN) concentrates collected for transfusion to septic, neutropenic patients are stored in the blood bank for various periods of time before they are given. Current methods of blood bank storage of PMN concentrates are associated with impaired in vitro PMN chemotaxis (CTX) and in vivo recovery and circulation kinetics after 24 hours of storage. This suggested the possibility that PMN may become hyperadherent during storage. To test this hypothesis, PMN concentrates were harvested and stored at both 22 and 6 degrees C and their adherence properties to relevant biologic surfaces, endothelial cell (EC) monolayers, and extracellular matrix (ECM) derived from endothelium were measured. Adherence was measured within 4 hours of collection and after 24 and 48 hours of storage. The aggregation properties of fresh and stored PMN were also studied. The adherence of fresh, unstimulated PMN to EC and ECM (31 +/- 5% and 34 +/- 4%, respectively) increased significantly after storage for 24 hours (EC = 41 +/- 8%; ECM = 43 +/- 4%) at 22 degrees C. F-Met-Leu-Phe (FMLP) stimulated the adherence of fresh PMN (EC = 37 +/- 4%; ECM = 42 +/- 4%; p less than 0.05). The adherence of PMN stored at 22 degrees C was further stimulated by FMLP (EC = 46 +/- 6%; ECM = 50 +/- 4%). PMN stored at 6 degrees C had significantly higher adherence than PMN stored at 22 degrees C, and the percentage of increase in adherence induced by FMLP was attenuated in PMN stored at 6 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stimulus-response coupling in fresh and stored granulocytes

Granulocytes stored in the blood bank prior to transfusion undergo progressive decrements in their ability to circulate and migrate in vivo and to migrate in vitro (chemotaxis). The pathogenesis of granulocyte (PMN) chemotaxis (CTX) dysfunction after room-temperature storage of PMN is unclear. Previous work in the authors' laboratory and others led to the hypothesis that intracellular transmission of chemotactic signals, referred to as stimulus-response coupling (SRC), might be abnormal in stored PMN. This report presents an investigation of the ability of fresh and stored PMN to generate and respond to leukotriene-B4 (LTB4), the chief intracellular amplifier of SRC for CTX. PMN were sampled from concentrates within 4 hours of collection and after 24 and 48 hours of storage in transfer packs at room temperature (RT). Fresh, stimulated PMN synthesized 202 +/- 51 ng of LTB4 and 110 +/- 11 ng of 5-hydroxyeicosatetraenoic acid (HETE) per 10(7) PMN. Synthesis of LTB4, but not HETE was significantly decreased after 24 hours' storage, and LTB4 and HETE synthesis decreased after 48 hours. The incubation of stored PMN with arachidonic acid (AA) maintained levels of LTB4 synthesis in PMN stored for 24 but not 48 hours. Also, the CTX defect of stored PMN to F-Met-Leu-Phe (FMLP) was not improved by the supplementation of PMN with exogenous sources of LTB4 or AA.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Effects of adenosine on functions of polymorphonuclear leukocytes from patients with septic shock

Inasmuch as polymorphonuclear leukocytes (PMNs) play a major role in antibacterial defense but can also cause substantial tissue injury, drugs are needed which are able to attenuate tissue-toxic PMN reactions without inhibiting bactericidal mechanisms. Adenosine as a retaliatory metabolite is produced in response to metabolically unfavorable conditions like inflammation. However, it is not known whether adenosine can selectively downregulate adverse PMN reactions in sepsis. In this prospective clinical study, we characterized the effects of adenosine ex vivo on PMN functions in patients with septic shock ([SS] n = 33) and healthy volunteers ([HV] n = 33). The PMNs were primed by tumor necrosis factor-alpha (TNF-alpha) and subsequently stimulated with N-formyl methionyl-leucyl-phenylalanine (fMLP) to test for the formation of hydrogen peroxide (H2O2) in response to soluble inflammatory stimuli. The PMNs were also challenged by opsonized zymosan particles to assess adhesion, phagocytosis, and the associated H2O2 production.As compared with HV, PMNs from SS patients showed strongly enhanced tissue-toxic H2O2 production elicited by TNF-alpha/fMLP. Increasing concentrations of adenosine dose-dependently reduced this tissue-toxic H2O2 production in both groups with a half-maximal inhibitory concentration of 25 nmol/L and 114 nmol/L in HV and SS patients, respectively. This 4.6-fold decrease in the adenosine-mediated inhibition of PMNs from patients with septic shock was compensated by a 3-fold increase in the plasma concentrations of the nucleoside (HV, 42.5 +/- 2.9 nmol/L vs. SS, 125.6 +/- 18.2 nmol/L; mean +/- SEM). When the effects of adenosine were tested at a very high A2A receptor saturating concentration of 10 mol/L, neither adhesion, phagocytosis, nor the associated H2O2 production induced by opsonized zymosan was affected in both groups. These results were confirmed by the highly selective A2A agonist, CGS21680.Thus, adenosine or A2A agonists may be useful to selectively inhibit the potentially tissue-toxic H2O2 production elicited by soluble inflammatory mediators in patients with septic shock.     

Glycolytic enzymes of stored granulocytes

A marked reduction of granulocyte chemotactic function accompanies the storage of granulocyte concentrates. Since chemotaxis is energy dependent, we studied energy metabolism in stored neutrophils. We and others have reported that stored neutrophils have a defect in their energy metabolism. We found that defective adenosine triphosphate maintenance in stored neutrophils was occult in resting cells, but was unmasked by an energy-intensive stimulus, phagocytosis. In studies reported here, we sought to determine if defective adenosine triphosphate maintenance during granulocyte storage was related to altered glycolytic enzyme activity. We studied the activity of glycolytic enzymes in fresh and stored, resting and stimulated (opsonized zymosan) neutrophils. The following enzyme activities showed no major changes during storage, in resting or stimulated neutrophils: hexokinase, phosphofructokinase, aldolase, glucose phosphate isomerase, triose phosphate isomerase, glyceraldehyde phosphate dehydrogenase, phosphoglycerate kinase, phosphoglyceromutase, enolase, lactate dehydrogenase, glucose-6-phosphate dehydrogenase, glutathione reductase, and glutathione peroxidase. In contrast, pyruvate kinase activity consistently increased during storage. In 6 units, pyruvate kinase activity increased by 75 percent after 24 hours of storage at room temperature and by 198 percent after 48 hours. The storage-associated increase in pyruvate kinase activity was not inhibited by cycloheximide. Stimulation of neutrophils by phagocytosis of opsonized zymosan also produced striking increases in the pyruvate kinase activity of both fresh and stored cells. Additional studies indicated that the increases in pyruvate kinase activity observed during storage and after phagocytosis were associated with an increase in the availability of pyruvate kinase activity in the supernatant fraction of neutrophil sonicates. Total pyruvate kinase activity in sonicates of neutrophils was unchanged by storage or particle ingestion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional effects of monoclonal antibodies (mAbs) against human polymorphonuclear (PMN) adherence antigens

Two mouse monoclonal antibodies (mABs), 25.31 raised against an subunit epitope of LFA1 antigen and Mol against an epitope of the complement receptor type 3 (CR3) were used for investigating their effects on human polymorphonuclear (PMN) functions. The two mABs have an inhibitory effect on PMN adherence. Furthermore, the PMN adherence strength depends upon the support and the adherence induces the capping process of these antigens. Other PMN functions dependent upon adherence were also altered by these two mAbs: random locomotion and that directed by formyl-methionyl-leucyl-phenylalanine (FMLP) or by activated serum, degranulation induced by opsonized or non opsonized zymosan but not by phorbol myristate acetate (PMA), iodination, K562 cell cytotoxicity. Luminol enhanced chemiluminescence of PMN was diminished by both mAbs when PMN were stimulated either by opsonized zymosan or by PMA. Our results confirm other workers' findings, and they are consistent with PMN functional abnormalities observed in children with congenital LFA1, Mol antigens defect.     

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.     

Effects of a chemotactic factor, N-formylmethionyl peptide, on adherence, superoxide anion generation, phagocytosis, and microtubule assembly of human polymorphonuclear leukocytes.

FMLP promoted microtubule assembly in PMNs at concentrations which were chemotactic for the cells. At higher concentrations than those required for chemotaxis, FMLP enhanced the adherence of PMNs to nylon glass fibers. With colchicine, PMN adherence was inhibited, but upon exposure to FMLP, PMN adherence could be restored. The effect of FMLP on PMN adherence was transitory and was no longer evident by 5 min. At concentrations similar to those employed in the adherence studies. FMLP induced the cells to briefly generate O-2, since ferricytochrome C reduction was no longer evident by 5 min. Pretreatment of the PMNs with cytochalasin B enhanced the release of O-2 by PMNs exposed to FMLP. On the other hand, there was no effect of FMLP on phagocytosis of C3-coated particles. These results suggest that FMLP induces responsive cells to develop a hyperadherent plasma membrane which is largely independent on microtubule control. Since oligopeptides similar of AMLP are formed in bacteria, it is likely that the action of N-formylated peptides is important in regulating the inflammatory response.     

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.     

Purine catabolism in polymorphonuclear neutrophils. Phorbol myristate acetate-induced accumulation of adenosine owing to inactivation of extracellularly released adenosine deaminase.

Since physiological concentrations (0.1-1 microM) of adenosine influence the functions of human polymorphonuclear neutrophils (PMNs), we investigated the metabolism of adenosine in suspensions of stimulated and unstimulated PMNs. Stimulation with phorbol myristate acetate (PMA, 1 microM), but not by zymosan (0.5 mg/ml) or N-formyl-methionyl-leucyl-phenylalanine (fMLP, 1 microM), provoked an accumulation of endogenous adenosine at a rate of 2.3 +/- 1.0 amol/cell per minute. A similar accumulation was observed with both unstimulated and stimulated PMNs after the addition of deoxycoformycin (dCF, 1-100 microM), an inhibitor of adenosine deaminase. Exogenous adenosine (10 microM) was deaminated at a rate of 9.8 +/- 3.7 amol/cell per minute in control or zymosan or fMLP-stimulated PMN suspensions. This deamination was nearly completely suppressed when the PMNs had been stimulated with PMA. In contrast, the activity of adenosine deaminase in PMN lysates (231 +/- 72 amol/cell per minute) was not modified by PMA stimulation. alpha, beta-Methyleneadenosine 5'-diphosphate (AMPCP, 2.5 mM), an inhibitor of membranous ecto-5'-nucleotidase, profoundly inhibited endogenous adenosine accumulation under all conditions. PMA stimulation also provoked an inactivation of extracellular adenosine deaminase, purine nucleoside phosphorylase, and lactate dehydrogenase in PMN suspensions. We concluded that PMNs, even when not stimulated, continuously produce adenosine by dephosphorylation of extracellularly released adenylates; and that stimulation of PMNs by PMA causes adenosine accumulation owing to the inactivation of adenosine deaminase released by broken cells.     

High teicoplanin uptake by human neutrophils

The cellular/extracellular (C/E) concentration ratio of teicoplanin in polymorphonuclear leukocytes (PMNs) increased rapidly with time (C/E 60 +/- 13 at 20 min). The C/E ratio was time- and concentration-dependent. At 20 min and an initial concentration of 75 +/- 16 micrograms/ml the cellular drug concentration was 4,700 +/- 1,300 micrograms/ml. The mechanism of drug uptake was by an active process and transported (cellular) drug retained its antimicrobial activity. Washing removed 42% of cellular drug. Teicoplanin inhibited PMN chemotaxis at very high concentrations and PMN microbicidal activity at lower concentrations.     

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.     

Decreased affinity for a chemotactic factor in stored granulocyte concentrates

Previous studies in this laboratory demonstrated decreased migration of neutrophils after storage for 24 hours at room temperature. The current work was undertaken to identify a possible mechanism for decreased migration after storage. Initial studies ruled out the possibility that chemotaxis was decreased due to impaired ability to sense a chemotactic factor gradient since chemokinesis was decreased in addition to chemotaxis. Dose-response curves to the synthetic chemotactic agent Formyl-Methionyl-Leucyl-Phenylalanine (FMLP) showed decreased response to submaximal chemokinetic stimuli in stored neutrophils. This suggested the possibility of altered FMLP receptor binding in stored neutrophils. Neutrophil FMLP receptors were measured in 11 fresh and stored granulocyte concentrates. Although there was a small increase in total FMLP receptors per neutrophil after storage, the affinity of FMLP receptors in fresh neutrophils was significantly greater than that in neutrophils stored 24 hours at room temperature. Thus, decreased migration toward submaximal chemotactic stimuli in stored neutrophils may be due to altered membrane FMLP binding. However, decreased migration of stored neutrophils to maximal stimuli cannot be explained by altered FMLP binding affinity.