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IMMUNOPHARMACOLOGY AND IMMUNOTOXICOLOGY, 1 3 ( 1 & 2 ) , 1 8 3 - 1 9 8 ( 1 9 9 1 )
EFFECT OF RECOMBINANT HUMAN GRANULOCYTUMACROPHAGECOLONY-STIMULATINGFACTOR ON NEUTROPHILSUPEROXIDE PRODUCTION
Richard M. Schultz Lilly Research Laboratories, Indianapolis, IN 46285
ABSTRACT Recombinant human granulocyte/macrophagecolony-stimulating factor (GM-CSF) induced significant superoxide production in human neutrophils within 30 minutes after addition of stimulus and the response was complete within 2 hr. Other agents known to prime neutrophils, including LPS and tumor necrosis factor-a, lacked activity under the experimental conditions employed. Using a panel of pharmacologic inhibitors, we sought to compare GM-CSF-induced neutrophil superoxide to that produced by cells exposed to N-formyl methionyl-leucyl-phenylalanine(fMet-Leu-Phe) and phorbol 12myristate 13-acetate (PMA). Each stimulant displayed a different profile. Rolipram, a peak IV phosphodiesterase inhibitor, specifically inhibited neutrophil activation by GM-CSF and fMet-Leu-Phe, while superoxide production stimulated by PMA was unaffected. Staurosporine, a protein kinase C (PK-C) inhibitor, suppressed superoxide production induced by all three neutrophil stimulants. Cytochalasin B totally inhibited superoxide induced by GM-CSF under conditions that promote the fMet-Leu-Phe-induced response. Cytochalasin B did not markedly affect PMA-induced superoxide. The results are consistent with the hypothesis that intact PK-C activity is essential for neutrophil superoxide production, but that differences exist in the initial pathways induced by these 183 Copyright 0 1991 by Marcel Dekker, Inc.
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neutrophil activators. Superoxide secretion from GM-CSF-treated neutrophils appears to be a direct, delayed response that requires assembly of microfilaments during exposure to the cytokine.
INTRODUCTION
It is now clear that granulocyte/macrophagecolony-stimulating factor
(GM-CSF)l , originally named for its proliferative effect on bone marrow hematopoietic progenitor cells, enhances a variety of functional changes in mature granulocytes and macrophages. The effects on neutrophils include inhibition of neutrophil motility (NIF-T activity) [l-31, degranulation (4), induction of CDI 1b surface adhesion molecule (5)' phagocytosis (6),and antibody-dependentcell-mediatedcytotoxicity (7). Recent studies have also shown that GM-CSF primes neutrophils for enhanced oxidative metabolism in response to a variety of second signals (8-12). Several investigators have attempted to determine the signal transduction pathways involved in GM-CSF-induced effects on neutrophil function. Tyagi and coworkers showed that GM-CSF causes a weak enhancement of basal diacylglycerol (DAG) levels which occurs slowly ( ~ 3 min), 0 and that it causes a significant enhancement of DAG generation in response to a chemoattractant (14). Sullivan et al. noted that augmentation of the granulocyte's ability to generate superoxide anions, which is induced by priming with GM-CSF, is independent both of the resting transmembrane potential and of alterations in the extent of membrane potential change induced by stimuli such as fMet-Leu-Phe (15). Mege and associates showed that GM-CSF does not prime cytoplasts to
EFFECT OF H U M A N GM-CSF
185
stimulation by fMet-Leu-Phe, suggesting that the granules and/or nucleus are necessary for the priming action (16). Moreover, they presented evidence that the priming by GM-CSF is not mediated by the H-7 sensitive
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protein kinase C, botulinum D-sensitive G-protein, or protein synthesis. Corey and Rosoff observed that GM-CSF primes neutrophils by modulating the activity of at least one pertussis toxin-sensitive G protein not associated with phosphatidylinositol turnover but one involved in arachidonic acid metabolism (17). Gomez-Cambroneroand coworkers demonstrated that GM-CSF, on its own, can initiate several changes in human neutrophils and that these changes are mediated in part by the pertussis toxin-sensitive guanine nucleotide regulatory protein (18). Kapp and his colleagues (13) showed that under certain experimental conditions, GM-CSF was capable of directly activating the oxidative metabolism of human granulocytes. Unlike activation by the major physiological chemoattractants such as fMet-Leu-Phe, complementderived C5a, and leukotriene B4,superoxide production required greater time of incubation (30 to 120 minutes) after addition of GM-CSF. These GM-CSF stimulated cells showed an increased adherence to the substratum developing polarized filopodia and an increased number of intracellular vesicles within 30 min after addition of the stimulus. The authors similarly noted that the neutrophils were adherent to the microplates under their assay conditions and were not kept in suspension (13). The present studies were undertaken to investigate two questions. (i) Does treatment with GM-CSF alone induce superoxide generation in human neutrophil cultures? (ii) Are the effects of GM-CSF, fMet-Leu-Phe, and phorbol myristate acetate (PMA) mediated by similar signal transduction pathways?
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MATERIALS AND METHODS
.-
Prep rations of recombinant human GM-CSF 'ere obtained from
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Genzyme, Boston, MA (lot # 89005; specific act. 5 x 1O7 CFU/mg) and AMGEN, Thousand Oaks, CA (lot #1302-1; specific act. 4 x lo7 unitdmg). Low endotoxin, phenol red-free Hanks' Balanced Salt Solution (HBSS) was obtained from M. A. Bioproducts, Walkersville, MD. Mono-Poly Resolving Medium was purchased from Flow Laboratories, Inc., McLean, VA. Ferricytochrome c (type VI), fMet-Leu-Phe, 3-[4,5-dimethylthiazol-2yl]2,5-diphenyl tetrazolium bromide (MTT), and superoxide dismutase (type I, 3400 U/mg protein) were obtained from Sigma Chemical Co., St. Louis, MO. Fatty acid free, low endotoxin bovine serum albumin fraction V (BSA) was purchased from Miles, Inc., Kankakee, IL. All reagents were shown to be free of detectable endotoxin contamination as measured by the Limulus amebocyte lysate test (Whittaker Bioproducts, Inc., Walkersville, MD).
Neutrophil Preparation. Heparinized human venous blood was obtained from consenting healthy adult volunteers. Neutrophils were isolated by centrifugation over a cushion of Mono-Poly Resolving Medium as previously described (19). The purified neutrophils (94%) were washed and resuspended in phenol red-free HBSS containing BSA at 1 mg/ml
(2.0x 106 cells/ml). oxide Production. We modified the method of Pick and Mizel (20) for measuring superoxide production by neutrophils. Neutrophil suspensions in phenol red-free HBSS containing BSA (1 mg/ml) were added in 80-pl
EFFECT OF HUMAN GM-CSF
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aliquots (2 x 1O5 neutrophils/well) to 96-well flat-bottom tissue culture plates (Costar, Cambridge, MA). Subsequently 100-pl aliquots of a ferricytochrome c solution (2.97 mg/ml) in phenol red-free HBSS were Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by University of Toronto on 01/13/15 For personal use only.
added to each well containing neutrophils. Ten microliters of test compounds at various concentrations were added to each well followed by the addition of 10 pl of stimulant (either fMet-Leu-Phe, PMA, or GM-CSF). In some experiments, neutrophils were pretreated with test compounds for 1 hr before addition of activating agent and ferricytochrome c. The plates were incubated at 37 C for various times after addition of neutrophil stimulant. Optical density at 550 nm was determined on a micro ELISA reader (Model MR600; Dynatech Laboratories, Alexander, VA). The reference wavelength was set at 490 nm to compensate for changes in absorbance due to the presence of cells (21). The specificity of the cytochrome c reduction was controlled by the inclusion in some sample wells of superoxide dismutase (300 U/ml final concentration). When the test compounds were solubilized in dimethyl sulfoxide (DMSO), the highest concentration of DMSO in the wells did not exceed 0.3%, which did not interfere with the assay. The results were expressed in nM cytochrome C reduced per 1 x 1O6 neutrophils after subtraction of absorbance readings for wells containing superoxide dismutase.
..
MTT !Tetrazolium) Toxicity Assay. The method of Mossman (22) was used to measure drug toxicity for neutrophils. Briefly, 96-well plates were set up exactly as above (Superoxide Production) with additions of neutrophils and test compounds. Plates were incubated for 1 hr at 37 C in a humidified atmosphere of 5% C02-in-air. MTT was dissolved in phenol red-free
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HBSS at 2 mg/ml and filtered to remove a small amount of insoluble
residue. Following incubation of neutrophil plates, 100-pl of stock MTT solution was added to all wells of an assay, and plates were incubated at
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37 C for 2 hr. Acid-isopropanol (100 pI of 0.04 N HCI in isopropanol) was added to all wells and mixed thoroughly to dissolve the dark blue crystals. After a few minutes, the plates were read on a Dynatech MR600 Microelisa reader, using a test wavelength of 570 nm and a reference wavelength of 630 nm.
RESULTS
GM-CSF induction of human neutrcghil u p e r o x m . Various recombinant human cytokines, including GM-CSF, G-CSF, and TNF-a, were tested for their ability to induce human neutrophil superoxide production. As shown in Figure 1, recombinant human GM-CSF stimulated neutrophils even at a concentration of 1 U/ml and showed a concentration related response up to 1000 U/ml. Recombinant human TNF-a produced barely detectable activity at similar concentrations, whereas G-CSF was totally inactive at all levels tested. Similarly, endotoxic lipopolysaccharidealone failed to induce significant superoxide production at concentrations ranging from 10 to O.OOlpg/ml (data not shown).
Kapp and colleagues showed that GM-CSF required greater time of incubation for superoxide production as compared to neutrophil stimulation by the major physiologic chemoattractants such as fMet-Leu-Phe, complement-derived C5a, and leukotriene B4. We exposed human neutrophils to either GM-CSF (100 U/ml) or fMet-Leu-Phe
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EFFECT O F HUMAN GM-CSF
huGM-CSF huTNF
c] huG-CSF Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by University of Toronto on 01/13/15 For personal use only.
l -
100
1
10
Conc.
0.1
0.01
(unitdml)
Figure 1. Effects of various recombinant human cytokines on human neutrophil superoxide production. Neutrophils were treated with different concentrations of GM-CSF, TNF-a, or G-CSF, and the SODinhibitable part of cytochrome-C reduction was measured after 1 hr of incubation at 37 C. Values represent the mean 2 SEM of 3 experiments utilizing 3 donors.
(1 x 1O-7M) for various times (10 to 90 min.) at 37C and compared the
amount of superoxide formed. As shown in Fig. 2, neutrophils required 30 minutes of incubation with GM-CSF before significant superoxide
was produced, and the response continued to increase for an additional 60 min. This stimulation pattern was in contrast to effects induced by fMet-Leu-Phe, where significant levels of superoxide were formed within 10 minutes of incubation, and the response was complete by 50 min.
CpmDarison of various compounds on neutrophil
m. We tested
various pharmacologic agents for their ability to inhibit neutrophil
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S CH ULTZ
20
0
fMLP GM-CSF
L L Q)
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n
0
lo:@0
E L
C
0
I
10
f
20
30
40
50
60
90
Minutes of Incubation
Figure 2. Time course of superoxide production induced by fMet-Leu-Phe (1 x 1O-7M) and recombinant human GM-CSF (10 U/ml). Values represent the mean k SEM of triplicate determinations.
superoxide induced by GM-CSF, fMet-Leu-Phe, and PMA. We used a protein kinase C (PK-C) inhibitor (staurosporine), a peak IV phosphodiesterase inhibitor (rolipram), and cytochalasin B. As shown in Table 1, each stimulant showed a different profile of responses with these compounds. Staurosporine inhibited superoxide release by all three stimulants, although neutrophil activation by GM-CSF and PMA were more sensitive to the inhibitory action of staurosporine. Rolipram selectively inhibited superoxide induced by GM-CSF and fMet-Leu-Phe. Cytochalasin B totally inhibited neutrophil activation by GM-CSF under conditions that promote the fMet-Leu-Phe response. Inhibitory concentrations of these compounds were not toxic for neutrophils as measured by the MTT tetrazolium assay.
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EFFECT O F HUMAN GM-CSF
Table 1. Effects of Various Compounds on Human Neutrophil Superoxide Production Induced by Human GM-CSF, fMet-Leu-Phe, and PMA. ~~
Compound
Conc. (Clg/ml)
Inh. Superoxide Release GM-CSF fMet-Leu-Phe PMA oo /
Staurosporine
0.1 0.01 0.001
98+Ia 76 f 7 22 f 8
89 f 6 OkO 5+4
Rolipram
1.o 0.1 0.01
69 f 8 45f1 21 + 5
81 + 8 69+3 52f11
Cytochalasin B
1.0 0.1
97 f 3 37 7
165% stim. 147% stim.
+
1OOfO 81 f 4 36f3
4+4 8+4 6f1
31
+6
7 f2
aMean f SE of triplicate experiments involving 3 determinations each.
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DISCUSSION
In this report, we reproduced the findings of Kapp and colleagues
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that recombinant human GM-CSF directly activates the oxidative metabolism of human neutrophils under experimental conditions of increased cellular adherence to the substratum (13). In this regard, human GM-CSF has previously been shown to induce the surface adhesion molecule, Mac-I (CDI 1b/CD18) on human neutrophils (5, 23). It is interesting to note that Nathan (24, 25) has recently shown that neutrophils exposed to polystyrene surfaces coated with serum, cultured endothelium, or extracellular matrix proteins, such as fibronectin and laminin, secrete massive amounts of hydrogen peroxide in response to cytokines or chemotactic factors that elicit little secretion in suspension. Hydrogen peroxide production was characterized by a pronounced lag period of 15 to 90 min and was quite prolonged in duration (approximately 1 to 3.5 hr depending on the substrate). These studies demonstrated the requirement for coincident adherence to substrate and addition of a soluble stimulus for potentiation of the neutrophil respiratory burst. Furthermore, Shappell and coworkers (26) observed that CDIIWCDI 8 mediated adherence-dependent hydrogen peroxide production by human and canine neutrophils. It seems likely that neutrophil adhesion is involved in the GM-CSF-induced neutrophil superoxide response, since the time course was similar to that observed in Nathan's studies and involved coincident neutrophil adherence. Moreover, GM-CSF treatment of neutrophil suspensions does not directly induce superoxide production, but rather "primes" for the enhanced
EFFECT OF HUMAN GM-CSF
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production of superoxide anion in response to a variety of second signals (8-12).
Most studies on the neutrophil respiratory burst record short-term Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by University of Toronto on 01/13/15 For personal use only.
observations (1-15 minutes) after stimulation of cells in suspension. Extended studies of adherent neutrophils give a different view of the respiratory burst than short-term studies of cells in suspension. Some of these differences include the identity of triggering vs priming factors, the role of the cytoskeleton, the kinetics of signal transduction, and the duration and magnitude of production of reactive oxygen intermediates. It is possible that the prolonged adherence to a surface during exposure to GM-CSF may constitute an important control mechanism in the host. The mechanism by which adhesion potentiates the respiratory burst elicited by GM-CSF is presently unknown. In the present study, we show that cytochalasin B selectively inhibits superoxide production by GM-CSF. Indeed, as shown in Table 1, cytochalasin B has diametrically opposite effects on the responses of adherent neutrophils to GM-CSF and fMet-Leu-Phe. Similarly, Nathan showed that cytochalasins abolished the secretion of hydrogen peroxide by adherent neutrophils in response to TNF-a (24). It is possible that microfilaments may be needed for internalization of GM-CSF-receptor complexes, and that internalization of these complexes is necessary for initiating superoxide production. The increased mobilization of GM-CSF receptors to the plasma membrane in response to microfilament-dependent cell spreading or capacitation of the triggering capacity of GM-CSF receptors by association with the cytoskeleton are alternative hypotheses that should be considered.
SCHULTZ
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Further studies are necessary to determine whether assembly of microfilaments produce changes in the number, affinity, internalization, and/or signalling capacity of GM-CSF receptors. We observed that GM-CSF-
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treated neutrophils fail to spread with concurrent cytochalasin B treatment, but retain their ability to attach to albumin-coated plates. Much evidence indicates that a different sequence of biochemical reactions is involved in the stimulation of NADPH oxidase by different neutrophil agonists (reviewed in (27). The peak IV phosphodiesterase inhibitor, rolipram has been shown to inhibit the neutrophil respiratory burst, presumably by elevating CAMP levels (28). In the present study, rolipram inhibited neutrophil superoxide production by GM-CSF and fMet-Leu-Phe, but not by PMA. The Ca2+- and phospholipid-dependentprotein kinase C has a crucial role in signal transduction for a variety of biologically active substances which activate cellular function and proliferation. Several studies suggest that PK-C is involved in the activation of the neutrophil respiratory burst (27, 29). We observed that the PK-C inhibitor, staurosporine inhibits neutrophil superoxide production induced by all three neutrophil stimulants (GM-CSF, fMet-Leu-Phe, and PMA). This data is consistent with the hypothesis that intact PK-C activity is essential for neutrophil superoxide production, although differences exist in the initial pathways induced by these neutrophil activators. Our results further suggest that neutrophils adherent to intra- or extravascular surfaces may undergo a prolonged respiratory burst after exposure to GM-CSF. The relevance of GM-CSF-induced neutrophil activation to the pathogenesis of inflammatory diseases remains to be determined.
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EFFECT OF HUMAN GM-CSF
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FOOTNOTES Abbreviations used: GM-CSF, granulocyte/macrophage colonyst imulating f act0r ; f Met-Leu- Phe, N-fo rmy 1 methionyl-leucylphenylalanine; PMA, phorbol 12-myristate 13-acetate; PK-C, protein kinase C; DAG, diacylglycerol; HBSS, Hanks' balanced salt solution; MTT, 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyl tetrazolium bromide; SOD, superoxide dismutase; BSA, bovine serum albumin; G-CSF, granulocyte colony-stimulating factor; and TNF-a, tumor necrosis factor-a. References 1) Gasson, J. C., Weisbart, R. H., Kaufman, S. E., Clark, S. C., Hewick, R. M., Wong, G. G., and Golde, D. W., Purified human granulocytemacrophage colony-stimulating factor: Direct action on neutrophils, Science, 226:1339, 1984. 2) Weisbart, R. H., Golde, D. W., Clark, S . C., Wong, G. G., and Gasson, J. C., Human granulocyte-macrophagecolony-stimulatingfactor is a neutrophil activator, Nature, 314:361, 1985. 3) Kharazmi, A., Nielsen, H., and Bendtzen, K., Modulation of human neutrophil and monocyte chemotaxis and superoxide responses by recombinant TNF-alpha and GM-CSF, Immunobiol., 177:363, 1988. 4) Richter, J., Anderson, T., and Olsson, I., Effect of tumor necrosis factor and granulocyte/macrophagecolony-stimulatingfactor on neutrophil degranulation, J. Immunol., 142:3199, 1989. 5) Socinski, M. A., Cannistra, S. A., Sullivan, R., E!ias, F.,,Antman, K., Schnipper, L., and Griffin, J. D.,Granulocyte-macrophage colonystimulating factor induces the expression of the CD11b surface adhesion molecule on human granulocytes in vivo , Blood, 72: 691, 1988. 6) Fleischmann, J., Golde, D. W., Weisbart, R. H., and Gasson, J. C., Granulocyte-macrophagecolony stimulating factor enhances phagocytosis of bacteria by human neutrophils, Blood, 68:708, 1986.
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7) Vadas, M. A., Nicola, N. A., and Metcalf, D., Activation of antibodydependent cell-mediated cytotoxicity of human neutrophils and eosinophils by separate colony-stimulatingfactors, J. Immunol.,
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130:795, 1983. 8) Weisbart, R. H., Kwan, L., Golde, D. W., and Gasson, J. C., Human GM-CSF primes neutrophils for enhanced oxidative metabolism in response to the major physiological chemoattractants, Blood, 69:18, 1987. 9) Edwards, S. W., Holden, C. S., Humphreys, J. M., and Hart, C. A., Granulocyte-macrophagecolony-stimulatingfactor (GM-CSF) primes the respiratory burst and stimulates protein biosynthesis in human neutrophils, FEBS Letters, 256:62, 1989. 10) English, D., Broxmeyer, H. E., Gabig, T. G., Akard, L P., Williams, D. E., and Hoffman, R.,Temporal adaptation of neutrophil oxidative responsivenessto n-formyl-methionyl-leucyl-phenylalanine, J. Immunol., 141:2400, 1988. 11) Weisbart, R. H., Golde, D. W., and Gasson, J. C., Biosynthetic human GM-CSF modulates the number and affinity of neutrophil f-met-leu-phe receptors, J. Immunol., 137:3584, 1986. 12) Wong,.G. G., Witek, J. S., Temple, P. A., Wilkens, K. M., Leary, A. C., Luxemburg, D. P., Jones, S. S., Brown, E. L., Kay, R. M., Orr, E. C., Shoemaker, C., Golde, D. W., Kaufman, R. J., Hewick, R. M., Wang, E. A,, and Clark, S.C., Human GM-CSF: Molecular cloning of the complementary DNA and purification of the natural and recombinant proteins, Science, 228:810, 1985. 13) Kapp,.A., Zeck-Kapp, G., Danner, M., and Luger, T. A., Human granulocytemacrophage colony stimulating factor: An effective direct activator of human polymorphonuclear neutrophilic granulocytes, J. Invest. Dermatol., 91 :49,1988. 14) Tyagi, S. R., Winton, E. F., and Lambeth, J. D., Granulocyte/macrophage colony-stimulating factor primes human neutrophils for increased diacylglycerol generation in response to chemoattractant, FEBS Letters, 257:188, 1989.
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15) Sullivan, R., Fredette, J. P., Leavitt, J. L., Gadenne, A.-S., Griffin, J. D., and Simons, E. R., Effects of recombinant human granulocytemacrophage colony-stimulatingfactor (GM-CSF) on transmembrane electical potentials in granulocytes: Relationship between enhancement of ligand-mediateddepolarization and augmentation of superoxide anion production, J. Cell. Physiol., 139:361, 1989. 16) Mege, J.-L., Gomez-Cambronero,J., Molski, T. F. P., Becker, E. L., and Sha'afi, R. I., Effect of granulocyte-macrophage colony-stimulating factor on superoxide production in cytoplasts and intact human neutrophils: Role of protein kinase and G-proteins, J. Leukocyte Biol., 46:161, 1989. 17) Corey, S. J. and Rosoff, P. M., Granulocytre-macrophage colonystimulating factor primes neutrophils by activating a pertussus toxinsensitive G protein not associated with phosphatidylinositolturnover, J. Biol. Chem., 264:14165, 1989. 18) Gomez-Cambronero,J., Yamazaki, M., Metwally, F., Molski, T. F. P., Bonak, V. A., Huang, C.-K., Becker, E. L., and Sha'afi, R. I., Granulocytemacrophage colony-stimulating factor and human neutrophils: Role of guanine nucleotide regulatory proteins, Proc. Natl. Acad. Sci. USA, 86:3569, 1989. 19) Ferrante, A. and Thong, Y. H.,Optimal conditions for simultaneous purification of mononuclear and polymorphonuclear leucocytes from human peripheral blood by the hvpaque-ficoll method, J. Immunol. Methods, 36:109, 1980. 20) Pick, E.and Mizel, D., Rapid microassays for the measurement of superoxide and hydrogen peroxide production by macrophages in culture using an automatic enzyme immunoassay reader, J. Immunol. Methods, 46:211,1981. 21) Rajkovic, I. A. and Williams, R., Rapid microassays of phagocytosis, bacterial killing, superoxide, and hydrogen peroxide production by human neutrophils in vitro , J. Immunol. Methods, 78:35, 1985. 22) Mosrnann, T., Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays, J. Immunol. Methods, 65:55, 1983.
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23) Devereux, S., Bull, H. A., Campos-Costa, D., Saib, R., and Linch, D. C., Granulocyte-macrophage colony stimulating factor induced changes in cellular adhesion molecule expression and adhesion to endothelium. In vitro and in vivo studies in man, Br. J. Haematol., 71:323, 1989. 24) Nathan, C. F., Neutrophil activation on biological surfaces. Massive secretion of hydrogen peroxide in response to products of macrophages and lymphocytes, J. Clin. Invest., 80:1550, 1987. 25) Nathan, C. F., Respiratory burst in adherent neutrophils: Triggering by colony-stimulating factors CSF-GM and CSF-G, Blood, 73: 301, 1989. 26) Shappell, S. B., Toman, C., Anderson, D. C., Taylor, A. A., Entman, M. L., and Smith, C. W., Mac-1 (CD11b/CD18) mediates adherence-dependent hydrogen peroxide production by human and canine neutrophils, J. lmmunol., 144: 2702, 1990. 27) Rossi, F., The 02--forming NADPH oxidase of the phagocytes: nature, mechanisms of activation and function, Biochimica et Biophysica Acta, 853:65, 1986. 28) Nielson, C. P., Heaslip, R. J., and Vestal, R. E., Selective CAMP phosphodiesterase inhibitors reduce the polymorphonuclear leukocyte respiratory burst, Pharmacologist, 31 : 148, 1989. 29) Berkow, R. L., Dodson, R. W., and Kraft, A. S., The effect of a protein kinase C inhibitor, H-7, on human neutrophil oxidative burst and degranulation, J. Leukocyte Biol., 41: 4-41, 1987.
IMMUNOPHARMACOLOGY AND IMMUNOTOXICOLOGY, 1 3 ( 1 & 2 ) , 1 8 3 - 1 9 8 ( 1 9 9 1 )
EFFECT OF RECOMBINANT HUMAN GRANULOCYTUMACROPHAGECOLONY-STIMULATINGFACTOR ON NEUTROPHILSUPEROXIDE PRODUCTION
Richard M. Schultz Lilly Research Laboratories, Indianapolis, IN 46285
ABSTRACT Recombinant human granulocyte/macrophagecolony-stimulating factor (GM-CSF) induced significant superoxide production in human neutrophils within 30 minutes after addition of stimulus and the response was complete within 2 hr. Other agents known to prime neutrophils, including LPS and tumor necrosis factor-a, lacked activity under the experimental conditions employed. Using a panel of pharmacologic inhibitors, we sought to compare GM-CSF-induced neutrophil superoxide to that produced by cells exposed to N-formyl methionyl-leucyl-phenylalanine(fMet-Leu-Phe) and phorbol 12myristate 13-acetate (PMA). Each stimulant displayed a different profile. Rolipram, a peak IV phosphodiesterase inhibitor, specifically inhibited neutrophil activation by GM-CSF and fMet-Leu-Phe, while superoxide production stimulated by PMA was unaffected. Staurosporine, a protein kinase C (PK-C) inhibitor, suppressed superoxide production induced by all three neutrophil stimulants. Cytochalasin B totally inhibited superoxide induced by GM-CSF under conditions that promote the fMet-Leu-Phe-induced response. Cytochalasin B did not markedly affect PMA-induced superoxide. The results are consistent with the hypothesis that intact PK-C activity is essential for neutrophil superoxide production, but that differences exist in the initial pathways induced by these 183 Copyright 0 1991 by Marcel Dekker, Inc.
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neutrophil activators. Superoxide secretion from GM-CSF-treated neutrophils appears to be a direct, delayed response that requires assembly of microfilaments during exposure to the cytokine.
INTRODUCTION
It is now clear that granulocyte/macrophagecolony-stimulating factor
(GM-CSF)l , originally named for its proliferative effect on bone marrow hematopoietic progenitor cells, enhances a variety of functional changes in mature granulocytes and macrophages. The effects on neutrophils include inhibition of neutrophil motility (NIF-T activity) [l-31, degranulation (4), induction of CDI 1b surface adhesion molecule (5)' phagocytosis (6),and antibody-dependentcell-mediatedcytotoxicity (7). Recent studies have also shown that GM-CSF primes neutrophils for enhanced oxidative metabolism in response to a variety of second signals (8-12). Several investigators have attempted to determine the signal transduction pathways involved in GM-CSF-induced effects on neutrophil function. Tyagi and coworkers showed that GM-CSF causes a weak enhancement of basal diacylglycerol (DAG) levels which occurs slowly ( ~ 3 min), 0 and that it causes a significant enhancement of DAG generation in response to a chemoattractant (14). Sullivan et al. noted that augmentation of the granulocyte's ability to generate superoxide anions, which is induced by priming with GM-CSF, is independent both of the resting transmembrane potential and of alterations in the extent of membrane potential change induced by stimuli such as fMet-Leu-Phe (15). Mege and associates showed that GM-CSF does not prime cytoplasts to
EFFECT OF H U M A N GM-CSF
185
stimulation by fMet-Leu-Phe, suggesting that the granules and/or nucleus are necessary for the priming action (16). Moreover, they presented evidence that the priming by GM-CSF is not mediated by the H-7 sensitive
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protein kinase C, botulinum D-sensitive G-protein, or protein synthesis. Corey and Rosoff observed that GM-CSF primes neutrophils by modulating the activity of at least one pertussis toxin-sensitive G protein not associated with phosphatidylinositol turnover but one involved in arachidonic acid metabolism (17). Gomez-Cambroneroand coworkers demonstrated that GM-CSF, on its own, can initiate several changes in human neutrophils and that these changes are mediated in part by the pertussis toxin-sensitive guanine nucleotide regulatory protein (18). Kapp and his colleagues (13) showed that under certain experimental conditions, GM-CSF was capable of directly activating the oxidative metabolism of human granulocytes. Unlike activation by the major physiological chemoattractants such as fMet-Leu-Phe, complementderived C5a, and leukotriene B4,superoxide production required greater time of incubation (30 to 120 minutes) after addition of GM-CSF. These GM-CSF stimulated cells showed an increased adherence to the substratum developing polarized filopodia and an increased number of intracellular vesicles within 30 min after addition of the stimulus. The authors similarly noted that the neutrophils were adherent to the microplates under their assay conditions and were not kept in suspension (13). The present studies were undertaken to investigate two questions. (i) Does treatment with GM-CSF alone induce superoxide generation in human neutrophil cultures? (ii) Are the effects of GM-CSF, fMet-Leu-Phe, and phorbol myristate acetate (PMA) mediated by similar signal transduction pathways?
186
SCHULTZ
MATERIALS AND METHODS
.-
Prep rations of recombinant human GM-CSF 'ere obtained from
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Genzyme, Boston, MA (lot # 89005; specific act. 5 x 1O7 CFU/mg) and AMGEN, Thousand Oaks, CA (lot #1302-1; specific act. 4 x lo7 unitdmg). Low endotoxin, phenol red-free Hanks' Balanced Salt Solution (HBSS) was obtained from M. A. Bioproducts, Walkersville, MD. Mono-Poly Resolving Medium was purchased from Flow Laboratories, Inc., McLean, VA. Ferricytochrome c (type VI), fMet-Leu-Phe, 3-[4,5-dimethylthiazol-2yl]2,5-diphenyl tetrazolium bromide (MTT), and superoxide dismutase (type I, 3400 U/mg protein) were obtained from Sigma Chemical Co., St. Louis, MO. Fatty acid free, low endotoxin bovine serum albumin fraction V (BSA) was purchased from Miles, Inc., Kankakee, IL. All reagents were shown to be free of detectable endotoxin contamination as measured by the Limulus amebocyte lysate test (Whittaker Bioproducts, Inc., Walkersville, MD).
Neutrophil Preparation. Heparinized human venous blood was obtained from consenting healthy adult volunteers. Neutrophils were isolated by centrifugation over a cushion of Mono-Poly Resolving Medium as previously described (19). The purified neutrophils (94%) were washed and resuspended in phenol red-free HBSS containing BSA at 1 mg/ml
(2.0x 106 cells/ml). oxide Production. We modified the method of Pick and Mizel (20) for measuring superoxide production by neutrophils. Neutrophil suspensions in phenol red-free HBSS containing BSA (1 mg/ml) were added in 80-pl
EFFECT OF HUMAN GM-CSF
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aliquots (2 x 1O5 neutrophils/well) to 96-well flat-bottom tissue culture plates (Costar, Cambridge, MA). Subsequently 100-pl aliquots of a ferricytochrome c solution (2.97 mg/ml) in phenol red-free HBSS were Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by University of Toronto on 01/13/15 For personal use only.
added to each well containing neutrophils. Ten microliters of test compounds at various concentrations were added to each well followed by the addition of 10 pl of stimulant (either fMet-Leu-Phe, PMA, or GM-CSF). In some experiments, neutrophils were pretreated with test compounds for 1 hr before addition of activating agent and ferricytochrome c. The plates were incubated at 37 C for various times after addition of neutrophil stimulant. Optical density at 550 nm was determined on a micro ELISA reader (Model MR600; Dynatech Laboratories, Alexander, VA). The reference wavelength was set at 490 nm to compensate for changes in absorbance due to the presence of cells (21). The specificity of the cytochrome c reduction was controlled by the inclusion in some sample wells of superoxide dismutase (300 U/ml final concentration). When the test compounds were solubilized in dimethyl sulfoxide (DMSO), the highest concentration of DMSO in the wells did not exceed 0.3%, which did not interfere with the assay. The results were expressed in nM cytochrome C reduced per 1 x 1O6 neutrophils after subtraction of absorbance readings for wells containing superoxide dismutase.
..
MTT !Tetrazolium) Toxicity Assay. The method of Mossman (22) was used to measure drug toxicity for neutrophils. Briefly, 96-well plates were set up exactly as above (Superoxide Production) with additions of neutrophils and test compounds. Plates were incubated for 1 hr at 37 C in a humidified atmosphere of 5% C02-in-air. MTT was dissolved in phenol red-free
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HBSS at 2 mg/ml and filtered to remove a small amount of insoluble
residue. Following incubation of neutrophil plates, 100-pl of stock MTT solution was added to all wells of an assay, and plates were incubated at
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37 C for 2 hr. Acid-isopropanol (100 pI of 0.04 N HCI in isopropanol) was added to all wells and mixed thoroughly to dissolve the dark blue crystals. After a few minutes, the plates were read on a Dynatech MR600 Microelisa reader, using a test wavelength of 570 nm and a reference wavelength of 630 nm.
RESULTS
GM-CSF induction of human neutrcghil u p e r o x m . Various recombinant human cytokines, including GM-CSF, G-CSF, and TNF-a, were tested for their ability to induce human neutrophil superoxide production. As shown in Figure 1, recombinant human GM-CSF stimulated neutrophils even at a concentration of 1 U/ml and showed a concentration related response up to 1000 U/ml. Recombinant human TNF-a produced barely detectable activity at similar concentrations, whereas G-CSF was totally inactive at all levels tested. Similarly, endotoxic lipopolysaccharidealone failed to induce significant superoxide production at concentrations ranging from 10 to O.OOlpg/ml (data not shown).
Kapp and colleagues showed that GM-CSF required greater time of incubation for superoxide production as compared to neutrophil stimulation by the major physiologic chemoattractants such as fMet-Leu-Phe, complement-derived C5a, and leukotriene B4. We exposed human neutrophils to either GM-CSF (100 U/ml) or fMet-Leu-Phe
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EFFECT O F HUMAN GM-CSF
huGM-CSF huTNF
c] huG-CSF Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by University of Toronto on 01/13/15 For personal use only.
l -
100
1
10
Conc.
0.1
0.01
(unitdml)
Figure 1. Effects of various recombinant human cytokines on human neutrophil superoxide production. Neutrophils were treated with different concentrations of GM-CSF, TNF-a, or G-CSF, and the SODinhibitable part of cytochrome-C reduction was measured after 1 hr of incubation at 37 C. Values represent the mean 2 SEM of 3 experiments utilizing 3 donors.
(1 x 1O-7M) for various times (10 to 90 min.) at 37C and compared the
amount of superoxide formed. As shown in Fig. 2, neutrophils required 30 minutes of incubation with GM-CSF before significant superoxide
was produced, and the response continued to increase for an additional 60 min. This stimulation pattern was in contrast to effects induced by fMet-Leu-Phe, where significant levels of superoxide were formed within 10 minutes of incubation, and the response was complete by 50 min.
CpmDarison of various compounds on neutrophil
m. We tested
various pharmacologic agents for their ability to inhibit neutrophil
190
S CH ULTZ
20
0
fMLP GM-CSF
L L Q)
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n
0
lo:@0
E L
C
0
I
10
f
20
30
40
50
60
90
Minutes of Incubation
Figure 2. Time course of superoxide production induced by fMet-Leu-Phe (1 x 1O-7M) and recombinant human GM-CSF (10 U/ml). Values represent the mean k SEM of triplicate determinations.
superoxide induced by GM-CSF, fMet-Leu-Phe, and PMA. We used a protein kinase C (PK-C) inhibitor (staurosporine), a peak IV phosphodiesterase inhibitor (rolipram), and cytochalasin B. As shown in Table 1, each stimulant showed a different profile of responses with these compounds. Staurosporine inhibited superoxide release by all three stimulants, although neutrophil activation by GM-CSF and PMA were more sensitive to the inhibitory action of staurosporine. Rolipram selectively inhibited superoxide induced by GM-CSF and fMet-Leu-Phe. Cytochalasin B totally inhibited neutrophil activation by GM-CSF under conditions that promote the fMet-Leu-Phe response. Inhibitory concentrations of these compounds were not toxic for neutrophils as measured by the MTT tetrazolium assay.
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EFFECT O F HUMAN GM-CSF
Table 1. Effects of Various Compounds on Human Neutrophil Superoxide Production Induced by Human GM-CSF, fMet-Leu-Phe, and PMA. ~~
Compound
Conc. (Clg/ml)
Inh. Superoxide Release GM-CSF fMet-Leu-Phe PMA oo /
Staurosporine
0.1 0.01 0.001
98+Ia 76 f 7 22 f 8
89 f 6 OkO 5+4
Rolipram
1.o 0.1 0.01
69 f 8 45f1 21 + 5
81 + 8 69+3 52f11
Cytochalasin B
1.0 0.1
97 f 3 37 7
165% stim. 147% stim.
+
1OOfO 81 f 4 36f3
4+4 8+4 6f1
31
+6
7 f2
aMean f SE of triplicate experiments involving 3 determinations each.
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DISCUSSION
In this report, we reproduced the findings of Kapp and colleagues
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that recombinant human GM-CSF directly activates the oxidative metabolism of human neutrophils under experimental conditions of increased cellular adherence to the substratum (13). In this regard, human GM-CSF has previously been shown to induce the surface adhesion molecule, Mac-I (CDI 1b/CD18) on human neutrophils (5, 23). It is interesting to note that Nathan (24, 25) has recently shown that neutrophils exposed to polystyrene surfaces coated with serum, cultured endothelium, or extracellular matrix proteins, such as fibronectin and laminin, secrete massive amounts of hydrogen peroxide in response to cytokines or chemotactic factors that elicit little secretion in suspension. Hydrogen peroxide production was characterized by a pronounced lag period of 15 to 90 min and was quite prolonged in duration (approximately 1 to 3.5 hr depending on the substrate). These studies demonstrated the requirement for coincident adherence to substrate and addition of a soluble stimulus for potentiation of the neutrophil respiratory burst. Furthermore, Shappell and coworkers (26) observed that CDIIWCDI 8 mediated adherence-dependent hydrogen peroxide production by human and canine neutrophils. It seems likely that neutrophil adhesion is involved in the GM-CSF-induced neutrophil superoxide response, since the time course was similar to that observed in Nathan's studies and involved coincident neutrophil adherence. Moreover, GM-CSF treatment of neutrophil suspensions does not directly induce superoxide production, but rather "primes" for the enhanced
EFFECT OF HUMAN GM-CSF
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production of superoxide anion in response to a variety of second signals (8-12).
Most studies on the neutrophil respiratory burst record short-term Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by University of Toronto on 01/13/15 For personal use only.
observations (1-15 minutes) after stimulation of cells in suspension. Extended studies of adherent neutrophils give a different view of the respiratory burst than short-term studies of cells in suspension. Some of these differences include the identity of triggering vs priming factors, the role of the cytoskeleton, the kinetics of signal transduction, and the duration and magnitude of production of reactive oxygen intermediates. It is possible that the prolonged adherence to a surface during exposure to GM-CSF may constitute an important control mechanism in the host. The mechanism by which adhesion potentiates the respiratory burst elicited by GM-CSF is presently unknown. In the present study, we show that cytochalasin B selectively inhibits superoxide production by GM-CSF. Indeed, as shown in Table 1, cytochalasin B has diametrically opposite effects on the responses of adherent neutrophils to GM-CSF and fMet-Leu-Phe. Similarly, Nathan showed that cytochalasins abolished the secretion of hydrogen peroxide by adherent neutrophils in response to TNF-a (24). It is possible that microfilaments may be needed for internalization of GM-CSF-receptor complexes, and that internalization of these complexes is necessary for initiating superoxide production. The increased mobilization of GM-CSF receptors to the plasma membrane in response to microfilament-dependent cell spreading or capacitation of the triggering capacity of GM-CSF receptors by association with the cytoskeleton are alternative hypotheses that should be considered.
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Further studies are necessary to determine whether assembly of microfilaments produce changes in the number, affinity, internalization, and/or signalling capacity of GM-CSF receptors. We observed that GM-CSF-
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treated neutrophils fail to spread with concurrent cytochalasin B treatment, but retain their ability to attach to albumin-coated plates. Much evidence indicates that a different sequence of biochemical reactions is involved in the stimulation of NADPH oxidase by different neutrophil agonists (reviewed in (27). The peak IV phosphodiesterase inhibitor, rolipram has been shown to inhibit the neutrophil respiratory burst, presumably by elevating CAMP levels (28). In the present study, rolipram inhibited neutrophil superoxide production by GM-CSF and fMet-Leu-Phe, but not by PMA. The Ca2+- and phospholipid-dependentprotein kinase C has a crucial role in signal transduction for a variety of biologically active substances which activate cellular function and proliferation. Several studies suggest that PK-C is involved in the activation of the neutrophil respiratory burst (27, 29). We observed that the PK-C inhibitor, staurosporine inhibits neutrophil superoxide production induced by all three neutrophil stimulants (GM-CSF, fMet-Leu-Phe, and PMA). This data is consistent with the hypothesis that intact PK-C activity is essential for neutrophil superoxide production, although differences exist in the initial pathways induced by these neutrophil activators. Our results further suggest that neutrophils adherent to intra- or extravascular surfaces may undergo a prolonged respiratory burst after exposure to GM-CSF. The relevance of GM-CSF-induced neutrophil activation to the pathogenesis of inflammatory diseases remains to be determined.
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EFFECT OF HUMAN GM-CSF
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FOOTNOTES Abbreviations used: GM-CSF, granulocyte/macrophage colonyst imulating f act0r ; f Met-Leu- Phe, N-fo rmy 1 methionyl-leucylphenylalanine; PMA, phorbol 12-myristate 13-acetate; PK-C, protein kinase C; DAG, diacylglycerol; HBSS, Hanks' balanced salt solution; MTT, 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyl tetrazolium bromide; SOD, superoxide dismutase; BSA, bovine serum albumin; G-CSF, granulocyte colony-stimulating factor; and TNF-a, tumor necrosis factor-a. References 1) Gasson, J. C., Weisbart, R. H., Kaufman, S. E., Clark, S. C., Hewick, R. M., Wong, G. G., and Golde, D. W., Purified human granulocytemacrophage colony-stimulating factor: Direct action on neutrophils, Science, 226:1339, 1984. 2) Weisbart, R. H., Golde, D. W., Clark, S . C., Wong, G. G., and Gasson, J. C., Human granulocyte-macrophagecolony-stimulatingfactor is a neutrophil activator, Nature, 314:361, 1985. 3) Kharazmi, A., Nielsen, H., and Bendtzen, K., Modulation of human neutrophil and monocyte chemotaxis and superoxide responses by recombinant TNF-alpha and GM-CSF, Immunobiol., 177:363, 1988. 4) Richter, J., Anderson, T., and Olsson, I., Effect of tumor necrosis factor and granulocyte/macrophagecolony-stimulatingfactor on neutrophil degranulation, J. Immunol., 142:3199, 1989. 5) Socinski, M. A., Cannistra, S. A., Sullivan, R., E!ias, F.,,Antman, K., Schnipper, L., and Griffin, J. D.,Granulocyte-macrophage colonystimulating factor induces the expression of the CD11b surface adhesion molecule on human granulocytes in vivo , Blood, 72: 691, 1988. 6) Fleischmann, J., Golde, D. W., Weisbart, R. H., and Gasson, J. C., Granulocyte-macrophagecolony stimulating factor enhances phagocytosis of bacteria by human neutrophils, Blood, 68:708, 1986.
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7) Vadas, M. A., Nicola, N. A., and Metcalf, D., Activation of antibodydependent cell-mediated cytotoxicity of human neutrophils and eosinophils by separate colony-stimulatingfactors, J. Immunol.,
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130:795, 1983. 8) Weisbart, R. H., Kwan, L., Golde, D. W., and Gasson, J. C., Human GM-CSF primes neutrophils for enhanced oxidative metabolism in response to the major physiological chemoattractants, Blood, 69:18, 1987. 9) Edwards, S. W., Holden, C. S., Humphreys, J. M., and Hart, C. A., Granulocyte-macrophagecolony-stimulatingfactor (GM-CSF) primes the respiratory burst and stimulates protein biosynthesis in human neutrophils, FEBS Letters, 256:62, 1989. 10) English, D., Broxmeyer, H. E., Gabig, T. G., Akard, L P., Williams, D. E., and Hoffman, R.,Temporal adaptation of neutrophil oxidative responsivenessto n-formyl-methionyl-leucyl-phenylalanine, J. Immunol., 141:2400, 1988. 11) Weisbart, R. H., Golde, D. W., and Gasson, J. C., Biosynthetic human GM-CSF modulates the number and affinity of neutrophil f-met-leu-phe receptors, J. Immunol., 137:3584, 1986. 12) Wong,.G. G., Witek, J. S., Temple, P. A., Wilkens, K. M., Leary, A. C., Luxemburg, D. P., Jones, S. S., Brown, E. L., Kay, R. M., Orr, E. C., Shoemaker, C., Golde, D. W., Kaufman, R. J., Hewick, R. M., Wang, E. A,, and Clark, S.C., Human GM-CSF: Molecular cloning of the complementary DNA and purification of the natural and recombinant proteins, Science, 228:810, 1985. 13) Kapp,.A., Zeck-Kapp, G., Danner, M., and Luger, T. A., Human granulocytemacrophage colony stimulating factor: An effective direct activator of human polymorphonuclear neutrophilic granulocytes, J. Invest. Dermatol., 91 :49,1988. 14) Tyagi, S. R., Winton, E. F., and Lambeth, J. D., Granulocyte/macrophage colony-stimulating factor primes human neutrophils for increased diacylglycerol generation in response to chemoattractant, FEBS Letters, 257:188, 1989.
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15) Sullivan, R., Fredette, J. P., Leavitt, J. L., Gadenne, A.-S., Griffin, J. D., and Simons, E. R., Effects of recombinant human granulocytemacrophage colony-stimulatingfactor (GM-CSF) on transmembrane electical potentials in granulocytes: Relationship between enhancement of ligand-mediateddepolarization and augmentation of superoxide anion production, J. Cell. Physiol., 139:361, 1989. 16) Mege, J.-L., Gomez-Cambronero,J., Molski, T. F. P., Becker, E. L., and Sha'afi, R. I., Effect of granulocyte-macrophage colony-stimulating factor on superoxide production in cytoplasts and intact human neutrophils: Role of protein kinase and G-proteins, J. Leukocyte Biol., 46:161, 1989. 17) Corey, S. J. and Rosoff, P. M., Granulocytre-macrophage colonystimulating factor primes neutrophils by activating a pertussus toxinsensitive G protein not associated with phosphatidylinositolturnover, J. Biol. Chem., 264:14165, 1989. 18) Gomez-Cambronero,J., Yamazaki, M., Metwally, F., Molski, T. F. P., Bonak, V. A., Huang, C.-K., Becker, E. L., and Sha'afi, R. I., Granulocytemacrophage colony-stimulating factor and human neutrophils: Role of guanine nucleotide regulatory proteins, Proc. Natl. Acad. Sci. USA, 86:3569, 1989. 19) Ferrante, A. and Thong, Y. H.,Optimal conditions for simultaneous purification of mononuclear and polymorphonuclear leucocytes from human peripheral blood by the hvpaque-ficoll method, J. Immunol. Methods, 36:109, 1980. 20) Pick, E.and Mizel, D., Rapid microassays for the measurement of superoxide and hydrogen peroxide production by macrophages in culture using an automatic enzyme immunoassay reader, J. Immunol. Methods, 46:211,1981. 21) Rajkovic, I. A. and Williams, R., Rapid microassays of phagocytosis, bacterial killing, superoxide, and hydrogen peroxide production by human neutrophils in vitro , J. Immunol. Methods, 78:35, 1985. 22) Mosrnann, T., Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays, J. Immunol. Methods, 65:55, 1983.
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23) Devereux, S., Bull, H. A., Campos-Costa, D., Saib, R., and Linch, D. C., Granulocyte-macrophage colony stimulating factor induced changes in cellular adhesion molecule expression and adhesion to endothelium. In vitro and in vivo studies in man, Br. J. Haematol., 71:323, 1989. 24) Nathan, C. F., Neutrophil activation on biological surfaces. Massive secretion of hydrogen peroxide in response to products of macrophages and lymphocytes, J. Clin. Invest., 80:1550, 1987. 25) Nathan, C. F., Respiratory burst in adherent neutrophils: Triggering by colony-stimulating factors CSF-GM and CSF-G, Blood, 73: 301, 1989. 26) Shappell, S. B., Toman, C., Anderson, D. C., Taylor, A. A., Entman, M. L., and Smith, C. W., Mac-1 (CD11b/CD18) mediates adherence-dependent hydrogen peroxide production by human and canine neutrophils, J. lmmunol., 144: 2702, 1990. 27) Rossi, F., The 02--forming NADPH oxidase of the phagocytes: nature, mechanisms of activation and function, Biochimica et Biophysica Acta, 853:65, 1986. 28) Nielson, C. P., Heaslip, R. J., and Vestal, R. E., Selective CAMP phosphodiesterase inhibitors reduce the polymorphonuclear leukocyte respiratory burst, Pharmacologist, 31 : 148, 1989. 29) Berkow, R. L., Dodson, R. W., and Kraft, A. S., The effect of a protein kinase C inhibitor, H-7, on human neutrophil oxidative burst and degranulation, J. Leukocyte Biol., 41: 4-41, 1987.
