Original Paper Int Arch Allergy Immunol 1992;97:194-199
Asthma and Allergy Center, Huntington Hospital, Pasadena, Calif., and Department of Pathology, University of Michigan, Ann Arbor, Mich., USA
Key Words
Adenylate cyclase Neutrophils Complement C5a Superoxide
Effect of Adenylate Cyclase Activators on C5a-lnduced Human Neutrophil Aggregation, Enzyme Release and Superoxide Production
Abstract
The effect of adenylate cyclase activators on C5a- and f-Met-Leu-Phe-induced human neutrophil aggregation, enzyme release and superoxide production was investigated. C5a-stimulated superoxide production was markedly inhibited by adenylate cyclase activators, and the order of potency was PGE| > isoprotere nol > epinephrine > PGF2((, which correlated with intracellular cAMP levels. However, neutrophil aggregation was inhibited by PGE,, PGE2, isoproterenol and epinephrine only at concentrations greater than 10-6 M. Lysozyme release was inhibited only via PGEs in the presence of the phosphodiesterase inhib itor, methylisobutylxanthine. These results suggest that in the human neutro phil: (1) C5a-induced superoxide production is more sensitive to regulation by cAMP than neutrophil aggregation or enzyme release, and (2) the type of re ceptor occupied as well as the threshold level of cAMP are important in the regulation of neutrophil aggregation and enzyme release stimulated by C5a.
Introduction
The complement activation product C5a is known as a physiologically important neutrophil activator inducing chemotaxis, aggregation, lysosomal enzyme release and superoxide production [1-3]. Both lysosomal enzyme and oxygen metabolites derived from superoxide have been shown to cause cell and tissue injury in many experimental systems [4,5]. Several studies have demonstrated suppression of neu trophil functions through the elevation of intracellular
cAMP induced by ß-adrenergic or prostaglandin (PG) re ceptor occupancy [6, 7]. However, some investigators [8] showed that PGEs and PGI2 had no effect on superoxide production with phorbol myristic acetate (PMA)-treated rabbit neutrophils. In contrast a marked inhibitory effect of PGEs and PGI2 on superoxide production by opsonized zymosan- or f-Met-Leu-Phe (fMLP)-treated neutrophils has been observed, and other investigators [9] have noted that ß-adrenergic receptor occupancy may be less effective than PG receptor occupancy on fMLP-induced lysosomal enzyme release. These results suggest that the inhibition Correspondence to: M. Michael Glovsky, MD Asthma and Allergy Center. Huntington Hospital 39 Congress Street. Suite 301 Pasadena, CA 91105 (USA)
©1992 S. Karger AG, Basel 1018-2438/92/0973-0194 $ 2.75/0
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Shoji Nagata3 Donald. K. Kebo3 Steven Kunkelh M. Michael Glovskya
Materials and Methods
Buffers used were Hanks’ balanced salt solutions (HBSS) without indicator (Gibco, Santa Clara, Calif.). PG E,, E,, F,n, methylisobutylxanthine, isoproterenol, bovine serum albumin, cytochrome C, cytochalasin B, HEPES, dimethyl sulfoxide (DMSO), and sodium diatrazoatc (Hypaque) were purchased from Sigma (St. Louis, Mo.). Dcxtran T-500 and Ficoll were obtained from Pharmacia (Piscataway, N.J.). Micrococcus lysodeikticus was purchased from Washington Diagnostics (Freehold, N.J.). C5a was highly purified using immunoabsorbent and molecular sieve chromatogrophy [10], and was shown to be immunologically identical to C5a purified by a conventional multistep method and ho mogeneous on SDS-polyacrylamidc gels. Neutrophil Preparations Isolation of human neutrophils was carried out by sedimentation on dcxtran and Ficoll Hypaque as described previously [3]. The cell preparation contained more than 95% neutrophils and the functional characteristics of chemotaxis, enzyme release, and superoxide gener ation were normal in neutrophils isolated by this procedure. Neutrophil Aggregation Neutrophil aggregation was assayed using a Dual Sample Aggregometer (DP-247E, Sienco, Inc., Morrison, Calif.) with a modifica tion of the method described previously [3]. Briefly, 450 pi of a neu trophil suspension (5 x 106/ml) was preincubated with reagents at 37 °C for 2 min in siliconized glass cuvettes in the aggregometer, and then incubated with 50 pi of C5a (2 x 10"* M) for 3 min. The degree of aggregation was determined by the percent transmission of light. The percent transmission of the original sample was set to 0%. 100% transmission was given by the cell-free supernatant obtained after in cubation. Enzyme Release Neutrophils (2 x 107/ml) were suspended in a buffer containing 5 mg/ml cytochalasin B in HBSS with 10 mAf HEPES 1 mg/ml glucose/1 mg/ml bovine scrum albumin, and incubated for 5 min at 37 °C. This was followed by the addition of 1 x 10'7A/ C5a for another 5 min at 37 °C. The reactions were terminated by placing the tubes in an ice bath followed by centrifugation at 400 g for 15 min at 4°C. The supernatant containing released enzymes was assayed for ly sozyme. Lysozyme activity was assayed in the supernatant by deter mining the rate of lysis of M. lysodeikticus [11]. Total release was de termined by incubation in a buffer containing 0.5% Triton X-100. The data are expressed as percent maximum enzyme release.
Superoxide Anion Production The amount of superoxide anion produced was determined by the reduction of ferricytochrome C to ferrocytochromc C by stimulated cells as previously described [12]. Neutrophils (2 x l0 6) were incu bated in the presence of 5 mg/ml cytochalasin B, 100 mAf ferricyto chrome C and 10 nAf C5a for 10 min at 37 °C. The amount of superoxide produced was calculated from the difference in absorbance be tween samples of neutrophils that received superoxide dismutase before activation and those receiving superoxidc dismutase after ac tivation. This difference was divided by the extinction coefficient for the change between ferricytochrome C and ferrocytochromc C to de termine the number of moles of superoxide produced per 2 x 106cells. The data are expressed as mean values from triplicate samples ± standard error of the mean (SEM). At least three different normal donors were utilized in each set of experiments. Radioimmunoassay for Cyclic AMP A Yamasa cAMP kit [13] was used for determination of intracel lular cAMP. Neutrophils (90 ml of 4 x 107 cclls/ml) were incubated with 10 ml of various ligands for5 min at 37 °C and then stopped with 100 ml of 10% trichloroacetic acid. After the centrifugation of the tubes at 3,000 rpm and 4 °C for 15 min, trichloroacetic acid in the supernatc was removed with water-saturated ether. The supernate and AMP standard was succinylated with a succinylating reagent and then 100 ml of a succinylated sample in 0.3 M imidasol buffer, l2SIsuccinylated cAMP and monoclonal anti-cAMP imidasol serum were mixed and incubated overnight at 4 °C for 24 h. 500 gl of activated charcoal was added and the tubes centrifuged at 3,000 rpm for 5 min. The levels of cAMP were computed from the standard curve bound/ reference versus log cAMP. Results
Neutrophil aggregation, lysozyme release and superox ide production were compared with C5a and fMLP. The EC50 of C5a and fMLP in neutrophil aggregation and lyso zyme release was 2.0 x 10~9 and 4.8 x 10~9 M, and 6.4 x 10-9, and 2.2 x K)-7 M, respectively (fig. la, b). About 60% of the total granule content of lysozyme were released in the presence of C5a (1()~7 M) and fMLP (20-6 M). The concen trations of C5a and fMLP required to produce 10 nM of superoxide/2 x 106 neutrophils were 1.5 xlO-8 and 1.4 xlO"9 M, respectively. These results suggest that C5a is relatively less potent than fMLP in superoxide production in con trast to neutrophil aggregation and lysozyme release (fig. lc). Neutrophil aggregation was suppressed by PGEb PGE 2 isoproterenol, epinephrine at high concentrations, while no suppression was observed by PGF2a. Epinepherine and isoproterenol suppressed maximally about 25% in C5a-induced aggregation at 10~5 M. The concentrations of PGE] and PGE2 required to induce 50% inhibition of neutrophil aggregation were 6.8 x 10"6 and 8.2 x 1()-8 M, respectively (fig- 2). 195
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of neutrophil functions by PGs and [1-receptor agonists may be dependent on the agonist used for stimulation, the species tested and the assay of neutrophil function. Whether neutrophil responses stimulated by C5a would be similarly inhibited is unclear. In this study, we investigated the effect of [I-adrenergic agonists, PGEs and PGF2a on C5a-induced neutrophil ag gregation, lysozyme release and superoxidc production.
Fig. 2. Inhibition of neutrophil aggregation by prostaglandins, epinephrine (EPI) and isoproterenol (ISO). Human neutrophils were preincubated (2 min, 37 °C) with PGE, (O), PGE, ( • ) , PGF^ (A), EPI (□ ) and ISO (■ ) at the concentrations indicated, and then incubated with C5a (2 x 10“x M). Maximal aggregation (100%) correspondend to 7.0% transmission shown in control.
Superoxide production
Fig. 1. Dosc-responsc curve of C5a- and fMLP-induccd neutro phil aggregation (a), lysozyme release (b) and superoxide production (c). Each assay system is described in Methods.
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Fig. 3. Inhibition of C5a-induced lysozyme release of neutrophils by prostaglandins and isoproterenol (ISO) with and without methyl isobutylxanthine (MIX). Neutrophils were preincubatcd (5 min, 37 °C) with cytochalasin B (5 pg/ml), in the absence of MIX and ISO (□ ), PGE, (O) and PGE2 (A ) at various concentrations. C5a was added at a concentration of 1 x 10~s A/ and further incubated for 5 min. After centrifugation, supernatants were removed and assayed for lysozyme as described in Methods. Maximal release was 30% of the total amount of enzyme release by Triton X-100. Data represent mean values ± SEM. Each experiment shown is a representative ex ample from at least three separate experiments.
Nagata/Kebo/Kunkel/Glovsky
Modification of C5a-Induccd Neutrophil Functions by Adenylate Cyclase Activators
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In the absence of methylisobutylxanthine, PGE!, PGE2, isoproterenol and epinephrine showed almost no inhibi tion of lysozyme release from the human neutrophil, while in the presence of methylisobutylxanthine, PGEj and PGE2gave significant inhibition at the concentration from ltp8 to 10"5M in a dose-responsive manner. The concentra tions of PGE2 and PGEj for 50% inhibition of lysozyme re lease were 3 x 10-6 and 6.8 x 10-6 M, respectively. Isoprote renol showed little inhibition in lysozyme release even
Fig. 4. Inhibition of C5a-induced Superoxide production by epine phrine (EPI), isoproterenol (ISO) and prostaglandins. Human neu trophils (2 x 106 cells/ml) were incubated at 37 °C for 5 min with cytochrom C, and cytochalasin B in the presence of EPI (□ ), ISO (■ ), PGE, (O), PGE, ( • ) , and PGF, (A). The cells were then exposed to C5a(l x lO^Ai) at 37 °C for 10 min and the amount of superoxide gen erated was measured as described in Methods. The amount of super oxide released in the presence of KP8 M C5a was about 9 nAf/2 x KP cells/10 min. Each experiment shown is a representative example from at least three separate experiments.
Fig. 5. Increase in intracellular cAMP levels in human neutrophils stimulated by epinephrine (EPI, • ) , isoproterenol (ISO, A ), PGE, and PGF2a. Data represent ratio of increase to basal intracellular cAMP levels (1.5 pmol/5 min/5 x 105 cells). Maximal increase in cAMP corresponds to 7.8 pmol/5 min/5 x 10s cells. Each value shown is a representative example from at least two separate experiments.
with methylisobutylxanthine. Epinephrine and PGF2 had no inhibitory effect on C5a-induced lysozyme release with or without methylisobutylxanthine (fig. 3). The dose responses for epinephrine, isoproterenol PGE!, PGE2 and PGF in the inhibition of C5a-induced Su peroxide production from human neutrophils are shown in figure 4. PGE! and PGE2 inhibited superoxide produc tion at concentrations from KP10 to KP5 M, and suppressed about 80% superoxide production at KP5 M. The inhibi tion of superoxide production by isoproterenol and epine phrine was observed at relatively higher concentrations (KP8 to IO"5 M) than PGE , and PGE2, and was about 70% at KP5 M. PGF^ has no enhancing effect on superoxide production at any concentration tested in this study. How ever, it suppressed superoxide production at high concen trations (ICE6 to KP5 M). An order of potency of PGEi, PGE2 > isoproterenol > epinephrine > PGF^, was observed, which was similar to that for neutrophil aggregation. The half maximal concentrations of PGE!, PGE2, iso proterenol, and epinephrine for inhibition of superoxide production were 2 x HP9, 6.4 x HP9, 6.3 x HP8, and 5.8 x HP7 Af, respectively.
PGE], isoproterenol and epinephrine increased the in tracellular cAMP levels in the human neutrophil in a dosedependent fashion (fig. 5). PGF2 also increased cAMP on ly at higher concentration (HP6 to 1(P5 A/). The degree of potency of these agents was PGE[ > isoproterenol > epi nephrine > PGF2a. The cAMP levels in neutrophils seem to correspond well with the inhibitory effect of superoxide production. The increment of intracellular cAMP stimu lated by PGF2(I at a high concentration may explain the in hibition of superoxide production by PGF2(I at UP*’ to 1(P5 M. Methylisobutylxanthine has little effect on the intracel lular cAMP level at 10"* M. Discussion
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Chemotactic peptides such as C5a can stimulate neu trophils in a manner that results in neutrophil accumu lation, lysosomal enzyme release and production of super oxide anion ( 0 2), H20 2 and other products [2, 5]. There are a number of studies about neutrophil functions and ac tivation mechanisms after stimulation by fMLP. However, despite its potent chemotactic activity and physiological
198
on cytosolic Ca2+ and that the increase in cellular cAMP inhibits phosphatidyl inositol metabolism, preventing the formation of diacylglycerol, and suggesting that under these conditions, protein kinase C is insufficiently activa ted. This is due to the absence of diacylglycerol, consid ered to be the physiological activator of the ubiquitous Ca2+-dependent protein kinase C [14,15]. Leukotriene B4, C5a, and fMLP have been shown to bind to rabbit neutrophil membrane GTPase, which is as sociated with an N[ regulatory protein [16]. This suggests that GTP hydrolysis is coupled with activating signals for chemotaxis, aggregation, enzyme release and superoxide formation. The N,' regulatory protein could either downregulate adenylate cyclase or stimulate protein kinase C. The latter possibility could be associated with Oj produc tions and neutrophil activation. Since PMA directly binds to protein kinase C to produce Oj and lysosomal enzyme release, this may explain the lack of PG or cAMP inhibi tion of PMA-induced signals. GTP is a known stimulator of adenylate cyclase and binds to a Ns regulatory protein. GTP addition would thus activate adenylate cyclase and result in increased cAMP. GTPase would block such an ef fect. Whether these mechanisms are operative or other secondary pathways are stimulated which induce phos pholipid catabolism and activation of protein kinase C by diacylglycerol remain to be determined. Another factor of relevance to the differences in fMLP- and C5a-induced 0 2 production is the relative numbers of receptors per neutrophil. fMLP has 2,000 re ceptors per neutrophil, whereas C5a has 100,000 to 300,000 receptors per cell [17,18], Thus at V,oo the concentration of C5a, fMLP would saturate its receptors. However in ag gregation and enzyme release C5a is more potent than fMLP. A lesser number of receptor sites occupied are as sociated with greater activity than that found with fMLP. This implies that C5a binding to its receptor is more effi cient than fMLP in aggregation and lysozyme release or that different biochemical pathways are involved. It is also necessary to understand C5a and fMLP cata bolism on the neutrophil surface. Other mechanisms of importance with different agonists on neutrophil function may require dissection of the component pathways. Thus, for the neutrophil to move towards a chemical gradient, or chemotaxis, it has to reorient its position in the vascula ture. Next, alterations in shape and contractions of cytoskcletal elements need to be accomplished. These events are repeated until the cell reaches its destination. Aggre gation is less understood than chemotaxis, yet a regulation of adherance molecules is likely to be required. Superox ide production may require less complex physiologic and
Nagata/Kcbo/Kunkel/Glovsky
Modification of C5a-Induced Neutrophil Functions by Adenylate Cyclase Activators
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importance, C5a has rarely been studied in a chemically pure form and usually with rabbit and not human neutro phils. In the present study, C5a showed more potent activity on neutrophil aggregation and lysozyme release than fMLP whereas C5a was less potent in superoxide produc tion compared to fMLP (fig. 1). This suggested that differ ent mechanisms may be involved in superoxide production versus exocytosis of granular enzymes and aggregation. PGE,, PGE2, epinephrine and isoproterenol inhibited the C5a-stimulated neutrophil aggregation only at high concentrations and these inhibitions were enhanced by mcthylisobutylxanthinc (data not shown). The C5a-induced lysozyme release was little inhibited by isoprotere nol and PGE,, and PGE2 alone; however, PGE, and PGE2 showed apparent inhibition of lysozyme release at more than 10"7 M when methylisobutylxanthine was added. In contrast to neutrophil aggregation and lysozyme release, superoxide production was markedly inhibited by these agents without methylisobutylxanthine. Previous studies using human and rabbit neutrophils had shown that isoproterenol, PGE2 and PGI2 at concen trations sufficient to increase intracellular cAMP levels in hibited lysosomal enzyme release and superoxide produc tion by fMLP or opsonized zymosan [6,8], while relatively high concentrations of fMLP (1()~7 M) and PMA resulted in less inhibition of lysosomal enzyme release by PGE, [8]. Superoxide production is inhibited by either PGE, or iso proterenol in the absence of methylisobutylxanthine, while lysozyme release is inhibited only via the PGE re ceptor and then only in the presence of methylisobutyl xanthine [9], These findings observed in fMLP-stimulated neutrophil enzyme release or superoxide production arc consistent with our results using C5a as neutrophil stim ulator. The order of potency for adenylate cyclase activators tested was PGE,, PGEL > isoproterenol > epinephrine > PGF 2 „, correlating with increased intracellular levels of cAMP. These results supported the hypothesis that super oxide production is more sensitive to the regulation by cAMP than enzyme release. There are two main hypotheses to explain the mecha nism of inhibition of neutrophil function by cAMP: (1) cAMP affects ionized cytosolic Ca2+ homeostasis, possibly by blocking Ca2+ mobilization from intracellular pools and/or by enhancing Ca2+ sequestration, and (2) cAMP in terferes with phospholipid turnover by inhibiting phos pholipase C activity [14]. Some investigators have demon strated that cAMP inhibition of fMLP-dependcnt metab olic responses in human neutrophils is not due to its effect
metabollic pathways than aggregation, chemotaxis, and lysozomal enzyme release, and thus be more easily blocked by cAMP. In conclusion, C5a-induccd human PMN Superoxide production was blocked by PGE, PGE2 > isoproteronal > epinephrine > PGF2. This mechanism is correlated with intracellular cAMP levels and does not require treatment with methyl xanthines. In contrast to Os production, neu trophil aggregation and lysozyme release is inhibited by
PGE, and PGE2 and only at high molar concentration. Since C5a receptor occupancy is linked to GTPase, and regulatory proteins, aggregation and enzyme release arc likely to be controlled by complex metabolic events. These include phospholipid catabolism, Ca2+ mobilization and oxydative metabolism. The precise sequences by which these events occur and the relative control of stimulatory and inhibitory signals on adenylate cyclase, phospholi pases and oxydases remains to be determined.
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References
8 Fantone JC, Marasco WA, Elgas U. Ward PA: Stimulus specificity of prostaglandin inhibition of rabbit polymorphonuclear leukocyte lysoso mal enzyme release and superoxide anion pro duction. Am J Pathol 1984;115:9-16. 9 Lad PM, Goldberg BJ, Smiley PA, Olson CV: Receptor-specific threshold effects of cyclic AMP are involved in the regulation of enzyme release and superoxidc production from hu man neutrophils. Biochim Biophys Acta 1985; 846:286-295. 10 Manderino GL, Sarerca AF, Kunkel SL, Ward PA, Hirata AA, Showed H: Purification of hu man C5a-des-Arg by immunoabsorbent and molecular sieve chromatography. J Immunol Methods 1982;53:41-50. 11 Showed HF, Glovksy MM, Ward PA: C3a-induced lysosomal enzyme secretion from human neutrophils. Lack of inhibition by f-Met-LeuPhe antagonists and inhibition by arachidonic acid antagonist. Int Arch Allergy Appl Immu nol 1982;67:227-232. 12 Simchowitz L, Spilberg I, Atkinson FP: Evi dence that the functional responses of human neutrophils occur independently of transient elevations in cyclic AMP levels. J Cyclic Nucle otide Res 1983:9:35^47. 13 Honma M, Satoh T, Takezawa J, Ui M: An ul trasensitive method for the simultaneous de termination of cyclic AMP and cyclic GMP in small volume samples from blood and tissue. Biochem Med 1977;18:257-273.
14 De Togni P, Cabrini G, Di Virgilio F: Cyclic AMP inhibition of fMLP dependent metabolic responses in human neutrophils is not due to its effects on cytosolic Ca**. Biochcm J 1984;224: 629-635. 15 Nishizuka Y: The role of protein kinase C in cell surface signal transduction and tumor pro motion. Nature 1984;308:693-698. 16 Feltner DE, Smith RH, Marasko WA: Charac terization of the plasma membrane bound GTPase from rabbit neutrophils. I. Evidence for an Ni-like protein coupled to the formyl peptide, C5a and leukotrienc B; receptors. J Immunol 1986;137:1961-1970. 17 Williams LT, Snydcrman R, Pike MC, Lefkowitz RJ: Specific receptor sites for chemotactic peptides on human polymorphonuclear leuko cytes. Proc Natl Acad Sci USA 1977:74:12041208. 18 Chcnoweth DE, Hugli TE: Demonstration of specific C5a receptor on intact human poly morphonuclear leukocytes. Proc Natl Acad Sci USA 1978;75:3943-3937.
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1 Fernandez HN, Henson PN. Otani A, Hugli TE: Chemotactic response to human C3a and C5a anaphylatoxin. J Immunol 1978:120:109119. 2 Webster RO, Hong SR, Johnston RB. Henson PM Jr: Biological effect of human complement fragment C5a and C5a-des-Arg on neutrophil function. Immunopharmacology 1980:2:201219. 3 Nagata S, Glovsky MM. Kunkel S: Anaphylatoxin-induced neutrophil chemotaxis and ag gregation-limited aggregation and specific de sensitization induced by human C3a and syn thetic C3a octapeptide. Int Arch Allergy Appl Immunol 1987;82:4-9. 4 Fantone JC. Ward PA: A review: Role of oxy gen-derived free radicals and metabolites in leukocyte-dependent inflammatory reactions. Am J Pathol 1980;107:395-418. 5 Ward PA, Till GO, Kunkel R. Beauchamp C: Evidence for role of hydroxyl radical in com plement and neutrophil-dependent tissue in jury. J Clin Invest 1983;72:789-801. 6 Bussc WW, Sosman JM: Isoproterenol inhibi tion of isolated human neutrophil function. J Allergy Clin Immunol 1984;73:404-410. 7 Rivkin I, Rosenblatt J, Becker EL: The role of cyclic AMP in the chemotactic responsiveness and spontaneous motility of rabbit peritoneal neutrophils: The inhibition of neutrophil movement and elevation of cyclic AMP level by catecholamines, prostaglandins, theophylline and cholera toxin. J Immunol 1975;115:1126— 1134.
Asthma and Allergy Center, Huntington Hospital, Pasadena, Calif., and Department of Pathology, University of Michigan, Ann Arbor, Mich., USA
Key Words
Adenylate cyclase Neutrophils Complement C5a Superoxide
Effect of Adenylate Cyclase Activators on C5a-lnduced Human Neutrophil Aggregation, Enzyme Release and Superoxide Production
Abstract
The effect of adenylate cyclase activators on C5a- and f-Met-Leu-Phe-induced human neutrophil aggregation, enzyme release and superoxide production was investigated. C5a-stimulated superoxide production was markedly inhibited by adenylate cyclase activators, and the order of potency was PGE| > isoprotere nol > epinephrine > PGF2((, which correlated with intracellular cAMP levels. However, neutrophil aggregation was inhibited by PGE,, PGE2, isoproterenol and epinephrine only at concentrations greater than 10-6 M. Lysozyme release was inhibited only via PGEs in the presence of the phosphodiesterase inhib itor, methylisobutylxanthine. These results suggest that in the human neutro phil: (1) C5a-induced superoxide production is more sensitive to regulation by cAMP than neutrophil aggregation or enzyme release, and (2) the type of re ceptor occupied as well as the threshold level of cAMP are important in the regulation of neutrophil aggregation and enzyme release stimulated by C5a.
Introduction
The complement activation product C5a is known as a physiologically important neutrophil activator inducing chemotaxis, aggregation, lysosomal enzyme release and superoxide production [1-3]. Both lysosomal enzyme and oxygen metabolites derived from superoxide have been shown to cause cell and tissue injury in many experimental systems [4,5]. Several studies have demonstrated suppression of neu trophil functions through the elevation of intracellular
cAMP induced by ß-adrenergic or prostaglandin (PG) re ceptor occupancy [6, 7]. However, some investigators [8] showed that PGEs and PGI2 had no effect on superoxide production with phorbol myristic acetate (PMA)-treated rabbit neutrophils. In contrast a marked inhibitory effect of PGEs and PGI2 on superoxide production by opsonized zymosan- or f-Met-Leu-Phe (fMLP)-treated neutrophils has been observed, and other investigators [9] have noted that ß-adrenergic receptor occupancy may be less effective than PG receptor occupancy on fMLP-induced lysosomal enzyme release. These results suggest that the inhibition Correspondence to: M. Michael Glovsky, MD Asthma and Allergy Center. Huntington Hospital 39 Congress Street. Suite 301 Pasadena, CA 91105 (USA)
©1992 S. Karger AG, Basel 1018-2438/92/0973-0194 $ 2.75/0
Downloaded by: Karolinska Institutet, University Library 130.237.122.245 - 1/13/2019 5:38:11 AM
Shoji Nagata3 Donald. K. Kebo3 Steven Kunkelh M. Michael Glovskya
Materials and Methods
Buffers used were Hanks’ balanced salt solutions (HBSS) without indicator (Gibco, Santa Clara, Calif.). PG E,, E,, F,n, methylisobutylxanthine, isoproterenol, bovine serum albumin, cytochrome C, cytochalasin B, HEPES, dimethyl sulfoxide (DMSO), and sodium diatrazoatc (Hypaque) were purchased from Sigma (St. Louis, Mo.). Dcxtran T-500 and Ficoll were obtained from Pharmacia (Piscataway, N.J.). Micrococcus lysodeikticus was purchased from Washington Diagnostics (Freehold, N.J.). C5a was highly purified using immunoabsorbent and molecular sieve chromatogrophy [10], and was shown to be immunologically identical to C5a purified by a conventional multistep method and ho mogeneous on SDS-polyacrylamidc gels. Neutrophil Preparations Isolation of human neutrophils was carried out by sedimentation on dcxtran and Ficoll Hypaque as described previously [3]. The cell preparation contained more than 95% neutrophils and the functional characteristics of chemotaxis, enzyme release, and superoxide gener ation were normal in neutrophils isolated by this procedure. Neutrophil Aggregation Neutrophil aggregation was assayed using a Dual Sample Aggregometer (DP-247E, Sienco, Inc., Morrison, Calif.) with a modifica tion of the method described previously [3]. Briefly, 450 pi of a neu trophil suspension (5 x 106/ml) was preincubated with reagents at 37 °C for 2 min in siliconized glass cuvettes in the aggregometer, and then incubated with 50 pi of C5a (2 x 10"* M) for 3 min. The degree of aggregation was determined by the percent transmission of light. The percent transmission of the original sample was set to 0%. 100% transmission was given by the cell-free supernatant obtained after in cubation. Enzyme Release Neutrophils (2 x 107/ml) were suspended in a buffer containing 5 mg/ml cytochalasin B in HBSS with 10 mAf HEPES 1 mg/ml glucose/1 mg/ml bovine scrum albumin, and incubated for 5 min at 37 °C. This was followed by the addition of 1 x 10'7A/ C5a for another 5 min at 37 °C. The reactions were terminated by placing the tubes in an ice bath followed by centrifugation at 400 g for 15 min at 4°C. The supernatant containing released enzymes was assayed for ly sozyme. Lysozyme activity was assayed in the supernatant by deter mining the rate of lysis of M. lysodeikticus [11]. Total release was de termined by incubation in a buffer containing 0.5% Triton X-100. The data are expressed as percent maximum enzyme release.
Superoxide Anion Production The amount of superoxide anion produced was determined by the reduction of ferricytochrome C to ferrocytochromc C by stimulated cells as previously described [12]. Neutrophils (2 x l0 6) were incu bated in the presence of 5 mg/ml cytochalasin B, 100 mAf ferricyto chrome C and 10 nAf C5a for 10 min at 37 °C. The amount of superoxide produced was calculated from the difference in absorbance be tween samples of neutrophils that received superoxide dismutase before activation and those receiving superoxidc dismutase after ac tivation. This difference was divided by the extinction coefficient for the change between ferricytochrome C and ferrocytochromc C to de termine the number of moles of superoxide produced per 2 x 106cells. The data are expressed as mean values from triplicate samples ± standard error of the mean (SEM). At least three different normal donors were utilized in each set of experiments. Radioimmunoassay for Cyclic AMP A Yamasa cAMP kit [13] was used for determination of intracel lular cAMP. Neutrophils (90 ml of 4 x 107 cclls/ml) were incubated with 10 ml of various ligands for5 min at 37 °C and then stopped with 100 ml of 10% trichloroacetic acid. After the centrifugation of the tubes at 3,000 rpm and 4 °C for 15 min, trichloroacetic acid in the supernatc was removed with water-saturated ether. The supernate and AMP standard was succinylated with a succinylating reagent and then 100 ml of a succinylated sample in 0.3 M imidasol buffer, l2SIsuccinylated cAMP and monoclonal anti-cAMP imidasol serum were mixed and incubated overnight at 4 °C for 24 h. 500 gl of activated charcoal was added and the tubes centrifuged at 3,000 rpm for 5 min. The levels of cAMP were computed from the standard curve bound/ reference versus log cAMP. Results
Neutrophil aggregation, lysozyme release and superox ide production were compared with C5a and fMLP. The EC50 of C5a and fMLP in neutrophil aggregation and lyso zyme release was 2.0 x 10~9 and 4.8 x 10~9 M, and 6.4 x 10-9, and 2.2 x K)-7 M, respectively (fig. la, b). About 60% of the total granule content of lysozyme were released in the presence of C5a (1()~7 M) and fMLP (20-6 M). The concen trations of C5a and fMLP required to produce 10 nM of superoxide/2 x 106 neutrophils were 1.5 xlO-8 and 1.4 xlO"9 M, respectively. These results suggest that C5a is relatively less potent than fMLP in superoxide production in con trast to neutrophil aggregation and lysozyme release (fig. lc). Neutrophil aggregation was suppressed by PGEb PGE 2 isoproterenol, epinephrine at high concentrations, while no suppression was observed by PGF2a. Epinepherine and isoproterenol suppressed maximally about 25% in C5a-induced aggregation at 10~5 M. The concentrations of PGE] and PGE2 required to induce 50% inhibition of neutrophil aggregation were 6.8 x 10"6 and 8.2 x 1()-8 M, respectively (fig- 2). 195
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of neutrophil functions by PGs and [1-receptor agonists may be dependent on the agonist used for stimulation, the species tested and the assay of neutrophil function. Whether neutrophil responses stimulated by C5a would be similarly inhibited is unclear. In this study, we investigated the effect of [I-adrenergic agonists, PGEs and PGF2a on C5a-induced neutrophil ag gregation, lysozyme release and superoxidc production.
Fig. 2. Inhibition of neutrophil aggregation by prostaglandins, epinephrine (EPI) and isoproterenol (ISO). Human neutrophils were preincubated (2 min, 37 °C) with PGE, (O), PGE, ( • ) , PGF^ (A), EPI (□ ) and ISO (■ ) at the concentrations indicated, and then incubated with C5a (2 x 10“x M). Maximal aggregation (100%) correspondend to 7.0% transmission shown in control.
Superoxide production
Fig. 1. Dosc-responsc curve of C5a- and fMLP-induccd neutro phil aggregation (a), lysozyme release (b) and superoxide production (c). Each assay system is described in Methods.
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Fig. 3. Inhibition of C5a-induced lysozyme release of neutrophils by prostaglandins and isoproterenol (ISO) with and without methyl isobutylxanthine (MIX). Neutrophils were preincubatcd (5 min, 37 °C) with cytochalasin B (5 pg/ml), in the absence of MIX and ISO (□ ), PGE, (O) and PGE2 (A ) at various concentrations. C5a was added at a concentration of 1 x 10~s A/ and further incubated for 5 min. After centrifugation, supernatants were removed and assayed for lysozyme as described in Methods. Maximal release was 30% of the total amount of enzyme release by Triton X-100. Data represent mean values ± SEM. Each experiment shown is a representative ex ample from at least three separate experiments.
Nagata/Kebo/Kunkel/Glovsky
Modification of C5a-Induccd Neutrophil Functions by Adenylate Cyclase Activators
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In the absence of methylisobutylxanthine, PGE!, PGE2, isoproterenol and epinephrine showed almost no inhibi tion of lysozyme release from the human neutrophil, while in the presence of methylisobutylxanthine, PGEj and PGE2gave significant inhibition at the concentration from ltp8 to 10"5M in a dose-responsive manner. The concentra tions of PGE2 and PGEj for 50% inhibition of lysozyme re lease were 3 x 10-6 and 6.8 x 10-6 M, respectively. Isoprote renol showed little inhibition in lysozyme release even
Fig. 4. Inhibition of C5a-induced Superoxide production by epine phrine (EPI), isoproterenol (ISO) and prostaglandins. Human neu trophils (2 x 106 cells/ml) were incubated at 37 °C for 5 min with cytochrom C, and cytochalasin B in the presence of EPI (□ ), ISO (■ ), PGE, (O), PGE, ( • ) , and PGF, (A). The cells were then exposed to C5a(l x lO^Ai) at 37 °C for 10 min and the amount of superoxide gen erated was measured as described in Methods. The amount of super oxide released in the presence of KP8 M C5a was about 9 nAf/2 x KP cells/10 min. Each experiment shown is a representative example from at least three separate experiments.
Fig. 5. Increase in intracellular cAMP levels in human neutrophils stimulated by epinephrine (EPI, • ) , isoproterenol (ISO, A ), PGE, and PGF2a. Data represent ratio of increase to basal intracellular cAMP levels (1.5 pmol/5 min/5 x 105 cells). Maximal increase in cAMP corresponds to 7.8 pmol/5 min/5 x 10s cells. Each value shown is a representative example from at least two separate experiments.
with methylisobutylxanthine. Epinephrine and PGF2 had no inhibitory effect on C5a-induced lysozyme release with or without methylisobutylxanthine (fig. 3). The dose responses for epinephrine, isoproterenol PGE!, PGE2 and PGF in the inhibition of C5a-induced Su peroxide production from human neutrophils are shown in figure 4. PGE! and PGE2 inhibited superoxide produc tion at concentrations from KP10 to KP5 M, and suppressed about 80% superoxide production at KP5 M. The inhibi tion of superoxide production by isoproterenol and epine phrine was observed at relatively higher concentrations (KP8 to IO"5 M) than PGE , and PGE2, and was about 70% at KP5 M. PGF^ has no enhancing effect on superoxide production at any concentration tested in this study. How ever, it suppressed superoxide production at high concen trations (ICE6 to KP5 M). An order of potency of PGEi, PGE2 > isoproterenol > epinephrine > PGF^, was observed, which was similar to that for neutrophil aggregation. The half maximal concentrations of PGE!, PGE2, iso proterenol, and epinephrine for inhibition of superoxide production were 2 x HP9, 6.4 x HP9, 6.3 x HP8, and 5.8 x HP7 Af, respectively.
PGE], isoproterenol and epinephrine increased the in tracellular cAMP levels in the human neutrophil in a dosedependent fashion (fig. 5). PGF2 also increased cAMP on ly at higher concentration (HP6 to 1(P5 A/). The degree of potency of these agents was PGE[ > isoproterenol > epi nephrine > PGF2a. The cAMP levels in neutrophils seem to correspond well with the inhibitory effect of superoxide production. The increment of intracellular cAMP stimu lated by PGF2(I at a high concentration may explain the in hibition of superoxide production by PGF2(I at UP*’ to 1(P5 M. Methylisobutylxanthine has little effect on the intracel lular cAMP level at 10"* M. Discussion
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Chemotactic peptides such as C5a can stimulate neu trophils in a manner that results in neutrophil accumu lation, lysosomal enzyme release and production of super oxide anion ( 0 2), H20 2 and other products [2, 5]. There are a number of studies about neutrophil functions and ac tivation mechanisms after stimulation by fMLP. However, despite its potent chemotactic activity and physiological
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on cytosolic Ca2+ and that the increase in cellular cAMP inhibits phosphatidyl inositol metabolism, preventing the formation of diacylglycerol, and suggesting that under these conditions, protein kinase C is insufficiently activa ted. This is due to the absence of diacylglycerol, consid ered to be the physiological activator of the ubiquitous Ca2+-dependent protein kinase C [14,15]. Leukotriene B4, C5a, and fMLP have been shown to bind to rabbit neutrophil membrane GTPase, which is as sociated with an N[ regulatory protein [16]. This suggests that GTP hydrolysis is coupled with activating signals for chemotaxis, aggregation, enzyme release and superoxide formation. The N,' regulatory protein could either downregulate adenylate cyclase or stimulate protein kinase C. The latter possibility could be associated with Oj produc tions and neutrophil activation. Since PMA directly binds to protein kinase C to produce Oj and lysosomal enzyme release, this may explain the lack of PG or cAMP inhibi tion of PMA-induced signals. GTP is a known stimulator of adenylate cyclase and binds to a Ns regulatory protein. GTP addition would thus activate adenylate cyclase and result in increased cAMP. GTPase would block such an ef fect. Whether these mechanisms are operative or other secondary pathways are stimulated which induce phos pholipid catabolism and activation of protein kinase C by diacylglycerol remain to be determined. Another factor of relevance to the differences in fMLP- and C5a-induced 0 2 production is the relative numbers of receptors per neutrophil. fMLP has 2,000 re ceptors per neutrophil, whereas C5a has 100,000 to 300,000 receptors per cell [17,18], Thus at V,oo the concentration of C5a, fMLP would saturate its receptors. However in ag gregation and enzyme release C5a is more potent than fMLP. A lesser number of receptor sites occupied are as sociated with greater activity than that found with fMLP. This implies that C5a binding to its receptor is more effi cient than fMLP in aggregation and lysozyme release or that different biochemical pathways are involved. It is also necessary to understand C5a and fMLP cata bolism on the neutrophil surface. Other mechanisms of importance with different agonists on neutrophil function may require dissection of the component pathways. Thus, for the neutrophil to move towards a chemical gradient, or chemotaxis, it has to reorient its position in the vascula ture. Next, alterations in shape and contractions of cytoskcletal elements need to be accomplished. These events are repeated until the cell reaches its destination. Aggre gation is less understood than chemotaxis, yet a regulation of adherance molecules is likely to be required. Superox ide production may require less complex physiologic and
Nagata/Kcbo/Kunkel/Glovsky
Modification of C5a-Induced Neutrophil Functions by Adenylate Cyclase Activators
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importance, C5a has rarely been studied in a chemically pure form and usually with rabbit and not human neutro phils. In the present study, C5a showed more potent activity on neutrophil aggregation and lysozyme release than fMLP whereas C5a was less potent in superoxide produc tion compared to fMLP (fig. 1). This suggested that differ ent mechanisms may be involved in superoxide production versus exocytosis of granular enzymes and aggregation. PGE,, PGE2, epinephrine and isoproterenol inhibited the C5a-stimulated neutrophil aggregation only at high concentrations and these inhibitions were enhanced by mcthylisobutylxanthinc (data not shown). The C5a-induced lysozyme release was little inhibited by isoprotere nol and PGE,, and PGE2 alone; however, PGE, and PGE2 showed apparent inhibition of lysozyme release at more than 10"7 M when methylisobutylxanthine was added. In contrast to neutrophil aggregation and lysozyme release, superoxide production was markedly inhibited by these agents without methylisobutylxanthine. Previous studies using human and rabbit neutrophils had shown that isoproterenol, PGE2 and PGI2 at concen trations sufficient to increase intracellular cAMP levels in hibited lysosomal enzyme release and superoxide produc tion by fMLP or opsonized zymosan [6,8], while relatively high concentrations of fMLP (1()~7 M) and PMA resulted in less inhibition of lysosomal enzyme release by PGE, [8]. Superoxide production is inhibited by either PGE, or iso proterenol in the absence of methylisobutylxanthine, while lysozyme release is inhibited only via the PGE re ceptor and then only in the presence of methylisobutyl xanthine [9], These findings observed in fMLP-stimulated neutrophil enzyme release or superoxide production arc consistent with our results using C5a as neutrophil stim ulator. The order of potency for adenylate cyclase activators tested was PGE,, PGEL > isoproterenol > epinephrine > PGF 2 „, correlating with increased intracellular levels of cAMP. These results supported the hypothesis that super oxide production is more sensitive to the regulation by cAMP than enzyme release. There are two main hypotheses to explain the mecha nism of inhibition of neutrophil function by cAMP: (1) cAMP affects ionized cytosolic Ca2+ homeostasis, possibly by blocking Ca2+ mobilization from intracellular pools and/or by enhancing Ca2+ sequestration, and (2) cAMP in terferes with phospholipid turnover by inhibiting phos pholipase C activity [14]. Some investigators have demon strated that cAMP inhibition of fMLP-dependcnt metab olic responses in human neutrophils is not due to its effect
metabollic pathways than aggregation, chemotaxis, and lysozomal enzyme release, and thus be more easily blocked by cAMP. In conclusion, C5a-induccd human PMN Superoxide production was blocked by PGE, PGE2 > isoproteronal > epinephrine > PGF2. This mechanism is correlated with intracellular cAMP levels and does not require treatment with methyl xanthines. In contrast to Os production, neu trophil aggregation and lysozyme release is inhibited by
PGE, and PGE2 and only at high molar concentration. Since C5a receptor occupancy is linked to GTPase, and regulatory proteins, aggregation and enzyme release arc likely to be controlled by complex metabolic events. These include phospholipid catabolism, Ca2+ mobilization and oxydative metabolism. The precise sequences by which these events occur and the relative control of stimulatory and inhibitory signals on adenylate cyclase, phospholi pases and oxydases remains to be determined.
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References
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