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)     

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)     

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)     

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)     

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)     

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.     

Effective storage of granulocytes collected by centrifugation leukapheresis from donors stimulated with granulocyte-colony-stimulating factor

BACKGROUND: Donor stimulation with granulocyte-colony-stimulating factor (G-CSF) has increased the number of neutrophils (PMNs) that can be collected for granulocyte transfusion therapy. Clinical utility, however, has been limited by the inability to store functional PMNs ex vivo. This study was conducted to determine whether granulocyte products from G-CSF-stimulated donors could be effectively stored at reduced temperature (22 degrees C vs. 10 degrees C) with maintenance of functional properties in vitro and in vivo. STUDY DESIGN AND METHODS: Nine normal subjects received G-CSF (600 microg subcutaneously) 12 hours before centrifugation leukapheresis. Granulocyte products were divided and stored for 24 and 48 hours under four conditions: 1) 22 degrees C; 2) 22 degrees C, with supplemental G-CSF (100 ng/mL); 3) 10 degrees C; and 4) 10 degrees C, with supplemental G-CSF. Functional PMN activity during ex vivo storage was assessed in vitro and in vivo by the skin-window technique for granulocytes stored at 10 degrees C for 24 hours. RESULTS: Surface expression of CD11b/CD18, CD14, CD16, CD32, and CD64 was maintained during 48-hour storage at reduced temperature. Inducible respiratory burst activity, bactericidal activity, and fungicidal activity were preserved during storage for 48-hour storage at 10 degrees C. Proinflammatory cytokine production was decreased in product stored at 10 degrees C. Supplemental G-CSF ex vivo did not substantially improve functional activity during storage. After storage at 10 degrees C for 24 hours, in vitro chemotactic potential was maintained, and transfused granulocytes retained capacity to circulate and migrate appropriately in vivo. CONCLUSIONS: Granulocyte product collected by centrifugation leukapheresis from G-CSF-stimulated donors can be effectively stored at subphysiologic temperature for 24 hours with preservation of functional activity. Storage at 10 degrees C appears to be slightly superior to storage at 22 degrees C.     

Leukotriene production in human neutrophils primed by recombinant human granulocyte/macrophage colony-stimulating factor and stimulated with the complement component C5A and FMLP as second signals

Neutrophils (PMN) preincubated with recombinant human granulocyte/macrophage colony-stimulating factor (rhGM-CSF) for 2 h and then stimulated with the chemotactic factors, C5a or FMLP, produce substantial amounts of the lipoxygenase products 5-Hete, LTB4, and omega-oxidised LTB4 metabolites (4.36 +/- 0.95 (SEM) pM (n = 21) LTB4 and LTB4 metabolites/10(6) PMN). No lipoxygenase metabolites are detected by HPLC and RIA if purified PMN are stimulated by either GM-CSF or chemotactic factors in the absence of exogenous arachidonate. The priming effect of GM-CSF upon chemotactic factor induced generation of lipid mediators is a relatively slow process, clearly evident after 1 h and optimal after 2 h. Leukotriene generation is measurable with 0.8 U GM-CSF/10(6) PMN and is maximal with 80 U (10(-11)-10(-9) M). Upon activation of primed PMN with chemotactic factors, leukotriene synthesis is induced very rapidly. Already 2.5 min after activation the major lipoxygenase metabolites present are 20-OH LTB4 and 20-COOH LTB4. Our study shows that the synthesis of lipoxygenase metabolites from endogeneous AA can be initiated in PMN through receptor mediated processes by the appropriately timed combination of biological soluble inflammatory mediator peptides. Furthermore, these results indicate that GM-CSF not only enhances effector cell functions but can qualitatively change the mediator profile formed after activation with a second triggering signal. Such a mechanism might be important in amplifying inflammatory responses. Alternatively, lipid mediators formed might also have an intracellular or autocoid role and be responsible for the enhancement of other PMN functions like oxygen radical release.     

Functional characteristics of neutrophils collected and stored after administration of G-CSF

BACKGROUND: Granulocyte transfusion may be used in neutropenic patients with severe bacterial or fungal infections that are unresponsive to antibiotic therapy. However, the inability to store granulocyte concentrates limits their clinical usefulness. STUDY DESIGN AND METHODS: Neutrophil chemotaxis and NADPH oxidase activity and the integrity of the neutrophil NADPH oxidase system were examined after apheresis collection and during storage to 48 hours. Neutrophils were mobilized in vivo by G-CSF, collected by apheresis techniques, and stored in apheresis bags in the presence and absence of additional G-CSF. For all experiments, cells were further purified by standard techniques of dextran sedimentation and hypotonic RBC lysis. RESULTS: Neutrophil chemotaxis was preserved to 24 hours of storage but was not affected by the G-CSF added to storage units. The NADPH oxidase system was also preserved as a functioning complex, and both cytosolic proteins and membrane-associated proteins were normal to 48 hours. However, there were divergent responses by intact cells to activating stimuli and reduced oxidase activity in the cell-free system. G-CSF did not appear to significantly affect NADPH oxidase activity or NADPH oxidase system integrity during storage. CONCLUSION: Neutrophils collected after the administration of G-CSF retained functional and biochemical characteristics for at least 24 hours of storage, which suggests additional effects of G-CSF mobilization beyond enhancing PMN yields and the possibility of storage of these components after collection.     

Characteristics of stored granulocytes collected from donors stimulated with dexamethasone

Two hours after normal donors were given intravenous dexamethasone, their leukocytes were collected by intermittent flow centrifugation. Neutrophils were tested immediately after collection and following storage at 4 to 6 C for 24, 48, 72 and 96 hours. Tests included total leukocyte and absolute neutrophil counts, plasma glucose concentrations, the percentage of phagocytic neutrophils, the ability of phagocytes to accumulate particles, candidacidal activity, bactericidal capacity and chemotaxis. Total leukocyte and absolute neutrophil counts in the stored suspensions were decreased after 48 hours (p = .005). Plasma glucose levels in the suspensions declined at first, then stabilized at 48 hours of storage probably because of loss of cellular integrity. Chemotaxis, candidacidal activity, phagocytosis and dye exclusion showed statistically significant decreases at 24 hours. Chemotaxis deteriorated rapidly, with a mean 63 per cent functional loss at 48 hours. We conclude that treatment of donors with dexamethasone does not extend the storage limits of granulocyte concentrates used for clinical transfusions. Based on these and our previous observations, unless the storage changes should be shown to be reversible, granulocyte concentrates should probably not be stored more than 24 hours before transfusion.     

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.     

Fibrin regulates neutrophil migration in response to interleukin 8, leukotriene B4, tumor necrosis factor, and formyl-methionyl-leucyl- phenylalanine

We have examined the capacity of four different chemoattractants/cytokines to promote directed migration of polymorphonuclear leukocytes (PMN) through three-dimensional gels composed of extracellular matrix proteins. About 20% of PMN migrated through fibrin gels and plasma clots in response to a gradient of interleukin 8 (IL-8) or leukotriene B4 (LTB4). In contrast, < 0.3% of PMN migrated through fibrin gels in response to a gradient of tumor necrosis factor alpha (TNF) or formyl-methionyl-leucyl-phenylalanine (FMLP). All four chemoattractants stimulated PMN to migrate through gels composed of collagen IV or of basement membrane proteins (Matrigel), or through filters to which fibronectin or fibrinogen had been adsorbed. PMN stimulated with TNF or FMLP adhered and formed zones of close apposition to fibrin, as measured by the exclusion of a 10-kD rhodamine-polyethylene glycol probe from the contact zones between PMN and the underlying fibrin gel. By this measure, IL-8- or LTB4-treated PMN adhered loosely to fibrin, since 10 kD rhodamine-polyethylene glycol permeated into the contact zones between these cells and the underlying fibrin gel. PMN stimulated with FMLP and IL-8, or FMLP and LTB4, exhibited very little migration through fibrin gels, and three times as many of these cells excluded 10 kD rhodamine-polyethylene glycol from their zones of contact with fibrin as PMN stimulated with IL-8 or LTB4 alone. These results show that PMN chemotaxis is regulated by both the nature of the chemoattractant and the composition of the extracellular matrix; they suggest that certain combinations of chemoattractants and matrix proteins may limit leukocyte movements and promote their localization in specific tissues in vivo.     

The anti-inflammatory effects of circulating fatty acids in obstructive jaundice: similarities with pregnancy-induced immunosuppression

Rheumatoid arthritis (RA) is ameliorated during both obstructive jaundice and pregnancy. Previous studies of polymorphonuclear leukocyte (PMN) function during pregnancy have shown reductions in the stimulated release of arachidonic acid (AA) and leukotriene B4 (LTB4), and lower NADPH oxidase activity. These changes may account for the amelioration of RA. The cause of this reduction in PMN function appears to be a progressive change in circulating fatty acids (FA), with a reduction in polyunsaturated FA, predominantly AA. The NADPH oxidase responsible for the respiratory burst has a direct requirement for polyunsaturated FA, particularly AA. We investigated whether the same changes in PMN function and FA, occur during obstructive jaundice. Patients with biliary obstructions were investigated before and after surgical correction (n=14). Obstructive jaundice caused significant changes in the proportions of serum and cellular FA. There was a striking reduction in polyunsaturated FA, particularly AA (48% in serum, p<0.001; 42% in PMNs, p<0.001) and an increase in mono-unsaturated oleic acid (24% in serum, p<0.001; 15% in PMNs, p<0.005). Similar changes occurred in mononuclear cell FA. Jaundice also caused a significant reduction in PMN function. Respiratory burst activity was reduced by between 32% and 38% in response to physiological and non-physiological stimuli, and there were similar significant reductions in the release of AA and LTB4. These changes in stimulated PMN function were evident whether or not the cells were first primed with tumour necrosis factor alpha (TNFalpha). Incubation of PMNs from healthy donors in pooled serum from patients with obstructive jaundice caused a reduction of 32% in cellular AA and 38% in NADPH oxidase activity. These findings support the idea that circulating FA can regulate PMN inflammatory responsiveness. The FA-induced attenuation in PMN activity in both jaundice and pregnancy may explain their ameliorating effects upon RA.     

Heterogeneity of human polymorphonuclear leukocyte receptors for leukotriene B4. Identification of a subset of high affinity receptors that transduce the chemotactic response

Human polymorphonuclear (PMN) leukocytes bound [3H]leukotriene B4 ([3H]-LTB4) specifically, as assessed by the displacement of 88% or more of the bound radioactivity by a 15,000-fold higher concentration of nonradioactive LTB4 or by micromolar concentrations of structural isomers of LTB4. The specific binding of [3H]LTB4 by PMN leukocytes was characterized by rapid association and dissociation, and was saturable at 800 nM LTB4. The results of computer analyses of the concentration dependence of binding of [3H]LTB4 were consistent with the expression of two classes of receptors having respective mean affinities of 3.9 X 10(-10) M and 6.1 X 10(-8) M and mean densities of 4.4 X 10(3) and 2.7 X 10(5) per PMN leukocyte. Structural isomers of LTB4 inhibited the binding of [3H]LTB4 to PMN leukocytes at concentrations similar to those required to elicit chemotaxis, while chemotactic peptides did not inhibit binding. PMN leukocytes that were deactivated by prior exposure to LTB4 lost high affinity binding sites selectively and concurrently with a reduction in the chemotactic response to LTB4. Chemotactic deactivation altered, but did not eliminate, the low affinity receptors for LTB4 and reduced only minimally the lysosomal degranulation elicited by LTB4. The high affinity receptors for LTB4 on normal human PMN leukocytes appear to transduce the chemotaxis evoked by LTB4 without substantially modifying lysosomal degranulation.     

Bacterial lipopolysaccharide primes human neutrophils for enhanced release of arachidonic acid and causes phosphorylation of an 85-kD cytosolic phospholipase A2.

Production of leukotriene B4 (LTB4) by human neutrophils (PMN) in response to different stimuli is increased after pretreatment with lipopolysaccharides (LPS). We have analyzed the steps in arachidonic acid (AA) metabolism affected by LPS by examining release of AA and its metabolites from [3H]AA prelabeled PMN. Pretreatment of PMN for 60 min with up to 1 microgram/ml of LPS alone had no effect, but release of [3H]AA was stimulated up to fivefold during subsequent stimulation with a second agent. In the absence of LPS-binding protein (LBP), priming was maximal after pretreatment of PMN with 10 ng of LPS/ml for 60 min; in the presence of LBP maximal priming occurred within 45 min at 0.1 ng of LPS/ml and within 15 min at 100 ng of LPS/ml. Treatment of PMN with 10 ng of LPS/ml also increased uptake of opsonized zymosan by up to 60%. Phospholipids are the source of released [3H]AA. No release was observed from [14C]oleic acid (OA)-labeled PMN suggesting that phospholipolysis may be specific for [3H]AA-labeled phospholipid pools. Cytosol from PMN primed with LPS contains two to three times the phospholipase A2 (PLA2) activity of control PMN, against 1-palmitoyl-[2-14C]arachidonoyl-phosphatidylcholine. This activity is Ca2+ dependent and dithiothreitol resistant. LPS priming is accompanied by reduced migration during SDS-PAGE of an 85-kD protein, identified as a cytosolic PLA2. The extent and kinetics of this effect of LPS on cPLA2 parallel the priming of [3H]AA release, both depending on LPS concentration either with or without LBP. These findings suggest that priming by LPS of AA metabolism by PMN includes phosphorylation of an AA-phospholipid-selective cytosolic PLA2 that is dissociated from activation until a second stimulus is applied.     

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.     

以海藻糖为添加剂冷藏保存血小板悬液的研究

本研究探讨以海藻糖(trehalose)为添加剂的血小板悬液冷藏保存方法。采用核素标记法检测血小板生存时间,用比浊法测定血小板聚集率,诱导剂为终浓度11.2μmol/L的ADP。采集兔心脏血,按常规方法制备浓缩血小板悬液(PCs),在悬液中加入50mg/ml的海藻糖,37℃水浴4小时后,放于4-8℃冰箱保存,冷藏12天后,测定血小板聚集功能和输入自身体内后的生存时间。结果表明:常温和冷藏储存24小时后的PC血小板聚集率分别为(75.3±9.8)%和(80.5±12.5)%;输入兔体内24、48和72小时时的血小板存活率分别为(78.1±7.9)%、(65.4±6.7)%、(57.5±7.2)%和(5.1±2.5)%、(2.8±2.0)%、(0.9±0.8)%。加入海藻糖的PC冷藏保存12天后,血小板聚集率为(77.8±9.5)%;输入体内24、48和72小时时的血小板存活率分别为(75.7±11.0)%、(67.0±8.5)%、(56.8±8.0)%,与常温保存24小时对照相比,两者相近,P值均>0.05。结论:海藻糖能保护冷藏储存的兔血小板,延长冷藏血小板在体内的生存时间,经海藻糖冷藏储存的兔血小板功能完好。     

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.     

Local versus regional procurement and distribution of granulocytes

Granulocyte concentrates obtained by discontinuous flow centrifugation (DFC) and continuous flow centrifugation (CFC) were studied. The DFC granulocytes were obtained from a regional center and stored for 24 hours prior to transfusion. The CFC granulocytes were obtained locally and transfused within a few hours. Even at 24 hours, DFC granulocytes had significantly reduced bactericidal capacity, chemiluminescence, nitroblue tetrazolium reduction, chemotaxis and random mobility. Granulocyte kinetics utilizing DF32P and skin windows demonstrated the ability of DFC granulocytes to circulate and migrate into the tissues despite the in vitro abnormalities. Until effective storage techniques for granulocyte preservation became available, rapid transportation and processing from regional centers or local procurement of granulocytes is necessary to transfuse functional granulocytes.