Immunology 1978 36 733
Generation and secretion of eosinophilotactic activity from human polymorphonuclear neutrophils by various mechanisms of cell activation
W. KONIG*, N. FRICKHOFEN & H. TESCH Institute of Medical Microbiology Johannes Gutenberg Universitdt 65 Mainz, Hochhaus Augustusplatz, FRG
Received 8 June 1978; acceptedfor publication 17 August 1978
Summary. An eosinophil chemotactic factor(s) (ECF) can be generated from human polymorphonuclear neutrophils by the calcium ionophore, phagocytosis, arachidonic acid and hypotonic lysis. In kinetic studies it is observed that peak ECF activity is released prior to the maximum of lysosomal enzyme release with the calcium ionophore, phagocytosis and arachidonic acid, while under conditions of hypotonic exposure ECF activity appears after the maximum of enzyme release. The ECF obtained by hypotonic exposure shows a fluctuating pattern with sharp peaks and steep fall-offs in activity. The ECF-release for each stimulus is temperature dependent; extracellular calcium is required when the ionophore or phagocytosis are used as stimuli, while with arachidonic acid and hypotonic exposure no extracellular calcium is necessary for ECF-release. On Sephadex G-25 each preparation of ECF eluted in the low molecular weight range at 500
daltons. Eosinophils can be deactivated and crossdeactivated with the various ECF-preparations indicating either a molecular identity or a common mode of action on eosinophils.
INTRODUCTION Recent interest has focused on the immunobiological role of the eosinophil leucocyte (Parish, 1970; Kay, 1970; Greene & Colley, 1974). Since eosinophils have been recognized to exert specific killer functions on parasites (Butterworth, Sturrock, Houba & Rees, 1975; Mahmoud, Warren & Peters, 1975) and to be prominent participants at sites of inflammation (Goetzl, 1976), the mechanisms of their chemo-attraction are of major importance. It has been shown that with their enzymes, the eosinophil leucocytes are able to counteract the mediators of inflammation such as histamine (Zeiger, Twang & Colten, 1976), the slowreacting substance of anaphylaxis (Wasserman, Goetzl & Austen, 1975) and the platelet aggregating factor (Kater, Goetzl & Austen, 1976). In recent years, several eosinophilotactic factors have been described which are mast cell (Clark, Gallin & Kaplan, 1975; Kay, Stechschulte & Austen, 1971; Goetzl & Austen, 1975) and serum derived products (Ward, Cochrane & Muller-Eberhard, 1976). Among these, ECF-A has been studied best with regard to its
-
Abbreviations: AA, arachidonic acid; ECF, eosinophil chemotactic factor; Ion, ionophore; Zx, opsonized zymosan; Eos, eosinophils; HETE, 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid; HHT, 12-L-hydroxy-5,8,10-heptadecatrienoic acid; HPF, high power field; PMN, polymorphonuclear neutrophil; SRS, slow reacting substance; TACM, T: tris hydroxyaminomethan; A: albumin; SDS, sodium dodecylsulphate. *Correspondence: Dr W. Konig, Institute for Medical Microbiology, 65 Mainz, Augustusplatz, FRG 0079-2805/79/0400-0733$02.00 ©0 1979 Blackwell Scientific Publications
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structure and its eosinophilotactic properties (Goetzl & Austen, 1975). ECF-A is preformed within mast cells and consists of two tetrapeptides. On sonication or lysis of the cells, the total eosinophilotactic activity can be recovered from the supernatant. In addition to the tetrapeptides, oligopeptides have been shown with eosinophilotactic activity which are released from mast cells during the allergic reaction (Boswell, Austen & Goetzl, 1977). The eosinophilotactic peptides account in part for the eosinophilotactic activity of stimulated mast cells. Furthermore, lipid products such as HETE and HHT, derived from arachidonic acid after incubation with the microsomal fraction of platelets have been shown to attract eosinophils and neutrophils by chemokinesis and chemotaxis (Goetzl, Woods & Forman, 1977; Goetzl & Gorman, 1978). In the past, we demonstrated that human neutrophils generate and release an eosinophil chemotactic factor after stimulation with the calcium ionophore A 23187 (Czarnetzki, Konig & Lichtenstein, 1976) and during phagocytosis of opsonized zymosan (Konig Czarnetzki & Lichtenstein, 1976). Quite recently, we observed that human PMNs but not human lymphocytes generate and release an eosinophilotactic activity on stimulation with arachidonic acid (Konig, Tesch & Frickhofen, 1978), which is indistinguishable from the ionophore or phagocytosis induced ECF. With each stimulus, the ECFs proved to be of low molecular weight (500 daltons) and highly specific for guinea-pig and human eosinophils; in contrast to HETE and HHT, the ECFs do not attract guinea-pig or human neutrophils. Furthermore, ECF is not preformed within human PMNs, and no ECF activity can be obtained from sonicated cells (Konig, Czarnetzki & Lichtenstein, 1978). We now, however, present evidence, that potent ECF activity can be recovered from human PMNs during their exposure to hypotonic buffer. An intact cell membrane is required for each stimulus (ionophore, phagocytosis, arachidonic acid, hypotonic lysis) to generate and release ECF. The kinetics of ECF release with each stimulus appear to be similar in that after an early rise in ECF activity a steep fall off occurs at later times of secretion suggesting a mechanism of inactivation. Quite recently, we obtained evidence by subcellular fractionation studies of unstimulated PMNs that a cell derived inactivator is present within the azurophilic granules of human PMNs which most likely inactivates ECF by binding (Frickhofen & Konig, 1978). In addition, we
demonstrated by equilibrium gradient centrifugation that ECF activity in stimulated PMNs cosediments
with the microsomal fractions and most likely with the plasma membrane. The purpose of this paper is to analyse carefully the ECF release pattern from human PMNs as compared to the release pattern of granular and microsomal (acid, alkaline PNP phosphatase, Mg2 + ATPase, alkaline phosphodiesterase I) enzymes. These studies will help to understand the molecular basis of activation for human PMNs and to clarify the origin and the release process of ECF induced by various stimuli. It will be demonstrated that eosinophilotactic activity can be recovered prior to the release of granular enzymes with the calcium ionophore, phagocytosis and arachidonic acid, while under conditions of hypotonic exposure, ECF activity appears after the release of the granular enzyme markers.
MATERIALS AND METHODS
Commercial source of reagents Glycogen, phenol reagent, H202, casein, vitamin B12, glucose (E. Merck, Darmstadt, W. Germany). Heparin grade II, horseradish peroxidase type II, zymosan A, f-glycerophosphate, phenolphthalein glucuronic acid, phenolphthalein, lysozyme, Micrococcus lysdeikticus, p-nitrophenol, adenosine 5'-triphosphate, thymidine 5'-monophospho-p-nitrophenylester, pyruvic acid, calf thymus DNA type I (Sigma, Munchen, W. Germany)' Ficoll 400, Sephadex G-25, blue dextran 2000 (Pharmacia, Uppsala, Sweden). o-tolidine, triton X-100 (Serva, Heidelberg, W. Germany); dextran = Macrodex 6% (Knoll, Ludwigshafen, W. Germany); sodium metrizoate 75% (Nyegaard & Co., Oslo, Norway); sterile glucose 5% (Fresenius, Bad-Homburg, W. Germany); ethyl acetate (Riedel de Haen Hannover, W. Germany);
p-nitrophenyl-phosphate (Boehringer, Mannheim, W. Germany); [C14]-histamine (Amersham Buchler, W. Germany); human serum albumin (Behringwerke, Marburg, W. Germany). Reagents not further listed were purchased from E. Merck, Darmstadt, W. Germany. Preparations of cells Human leucocytes were obtained from heparinized blood of healthy donors and separated on a Ficoll metrizoate gradient followed by dextran sedimentation (Boyum, 1968). This method has been previously described and leads to more than 95% pure PMNs. As target cells for the chemotactic assay,
Eosinophilotactic activity from human polymorphnuclear neutrophils guinea-pig peritoneal exudates rich in eosinophils (35-70%) were obtained by injecting human serum intraperitoneally at weekly intervals (Czarnetzki et al., 1976; Konig et al., 1976). Neutrophil-rich exudates (more than 95%) were obtained from guinea-pigs 15-18 h after injection of 0 1% glycogen (Ward et al., 1976). Two to three hours after intraperitoneal injection of sterile 5% glucose, the cells were harvested from the peritoneal cavity. The total yield ranged between 1-3 x 108 neutrophils per guinea-pig.
Buffers The medium used for washing the cells and for mediator release unless stated otherwise was a Tris buffer (pH 7 35, 0-025 M) with NaCl (120 mM), KCI (4mM) CaCl2 (0-6 mM) and Mg chloride (1 -0 mM). This buffer is referred to as TCM buffer; when human serum albumin (0 3 mg/ml) was added, we refer to it as TACM buffer. Preparation of ECF Methods for the release of ECF induced by phagocytosis, by the calcium ionophore or by arachidonic acid have been described previously (Konig et al., 1976; Czarnetzki et al., 1976; Konig et al., 1978). Briefly, I x 107 PMNs/ml were incubated with the calcium ionophore (a gift of Dr Hamill of the Eli Lilly Research Laboratories, Indianapolis, Indiana, USA) at 5 x 10-6M, with zymosan (Nutritional Biochemical Corporation, Cleveland, Ohio) coated with complement (Zx) or with arachidonic acid (Sigma GmbH., Munich, West Germany) at a concentration of 0-35 mM. Zymosan was coated with complement (Zx) as has been described. Briefly, zymosan (200 mg) was suspended in 3 ml human serum and incubated for 30 min at 37°. The particles were then washed in TCM buffer and used at 3 mg/ml final concentration. To study the kinetics of ECF release, human PMNs (I x 107/ml) were incubated with different stimuli (calcium ionophore, Zx or arachidonic acid) in TACM buffer. After various times, cells were centrifuged and the supernatant assayed for ECF activity. The generation and release of ECF in hypotonic buffer was carried out by using either a 001 M or 0005 M Tris-HCl buffer at pH 7 5 without the addition of Ca2 + chloride and Mg chloride. The conductivity of these buffers was 0 4 mS (0-005 M) and 0-7 mS (0 01 M). After the incubation, I ml of TCM buffer two-fold more concentrated than listed previously (26 inS) was added to restore isotonicity. In case, ECF activity was analysed within the cell, the cell pellets were washed,
735
resuspended in isotonic TCM buffer (1 ml) and then sonicated for 1 min at 4° (Mullard Equipment, Ltd, England). The conductivity of the buffers was measured using the Radiometer, Copenhagen instrument.
Chemotaxis The method for eosinophil chemotaxis has been described in detail (Czarnetzki et al., 1976). Briefly, 2-5 x 106 guinea-pig peritoneal cells containing 35-70% eosinophils were placed above a nitrocellulose filter (Sartorius Membranfilter, GmbH., Gottingen, West Germany, 8 gm pore size, 13 mm diameter) in a modified Boyden Chamber. The chemotactic factor was placed below the filter. After 3 h of incubation, eosinophils that had migrated through the filter were counted at 100 x magnification. Five highpower fields (HPF) were evaluated. Buffer control in each experiment never exceeded five to ten eosinophils per 5 HPF. Neutrophil chemotaxis was performed using 3 gm filter and casein (1-10 mg/ml) as a positive control (Ward et al., 1976). Assay conditions were the same as described for eosinophil chemotaxis. The precision coefficient of the chemotactic assay varied between 8-12%. The chemokinetic properties of the chemotactic factors was assayed by placing the factor either above the filter or above and below the filter. Random migration was then assayed after 3 h of incubation. Deactivation of cells The experiments follow those described by Wasserman et al. (Wasserman, Whitmer, Goetzl & Austen, 1975). Guinea-pig peritoneal cells containing 50% eosinophils were incubated with either the phagocytosis, ionophore, arachidonic acid or hypotonic lysis induced ECF for 15 min at 37°. After incubation, the cells were washed repeatedly and then exposed to phagocytosis, ionophore, arachidonic acid or hypotonic lysis induced ECF to measure their chemotactic response towards the various ECFs. In each experiment, an aliquot of cells was suspended in TACM buffer instead of ECF and served as a positive control.
Chromatography Gel filtration analysis of eosinophilotactic acitivity was performed on a Sephadex G-25 column (1 6 x 25 cm). Elution was performed in TCM buffer and fractions of 1-5 ml each were collected and assayed for their eosinophilotactic activity. Biological activity was compared to the elution profile of molecular weight
736
W. Konig, N. Frickhofen & H. Tesch
markers such as blue dextran (2 x 106), vitamin B12 (1335) and ['4C]-histamine (111). Blue dextran was determined spectrophotometrically (260 nm) as well as vitamin B12 (360 nm). ['4C]-histamine was measured in a scintillation counter (Packard).
determination were added to one part of the DNA solution and incubated for 10-20 h at 37°. The absorbency was read at 535 nm; calf thymus DNA was used as standard.
RESULTS
Biochemical assays Protein was measured at 280 nm in a Beckman photometer or by the Folin Lowry method (Lowry, Rosebrough, Farr & Randall, 1951). Enzyme determinations were performed as has been described: 1.11.1.7 peroxidase (Baggiolini, Hirsch & de Duve, 1909); 3.1.3.2 acid P-glycerophosphatase (Chen, Toribara & Warner, 1956; Bowers, Finkenstaedt & de Duve, 1967); 3.2.1.31 ,B-glucuronidase (Avila & Convit, 1973); 3.1.1.17 lysozyme (Sigma technical bulletin); 3.1.3.1 alkaline p-nitrophenylphosphatase (Bergmeyer, 1970); 3.1.3.2 acid p-nitrophenylphosphatase (Dingle, 1972); 3.6.1.3. Mg2+ ATPase (Harlan, de Chatelet, Iversion & McCall, 1977); 3.1.4.1. Alkaline phosphodiesterase I (Beaufay, Amar-Costesec, Feytmans, Thisnes-Sempoux, Wibo, Robbi & Berthet, 1974); 1.1.1.27 lactate dehydrogenase (Bretz & Baggiolini, 1974). The incubation volume is always 1 ml except for the lysozyme assay in which 0 05 ml sample was added to 1 25 ml substrate solution. Enzyme activities are expressed as percentage of the total activity calculated from unstimulated cells. Total enzyme activity was recovered after solubilization of the cells in 1% SDS. A solution of Na2HPO4 was used as standard for acid f-glycerophosphatase and Mg ATPase, while paranitrophenol (PNP) served as standard for alkaline PNP phosphatase, acid PNP phosphatase and alkaline phosphodiesterase I; phenolphthalein was used as standard in the,B-glucuronidase assay; horse radish peroxidase served as standard for peroxidase. One unit of lysozyme is defined as the decrease in optical density at 450 nm of 10 0 per min as has been described by Spitznagel, Dalldorf, Leffel, Folds, Welsh, Cooney & Martin, 1974). In all phosphatase assays, the effect of sodium tartrate (10 mM) was investigated to calculate the interference of the lysosomal acid phosphatase. For all enzyme determinations, a Carl Zeiss M4 Q3/PMQ2 spectrophotometer was used. DNA was determined as has been described according to Burton (1956). Briefly, the cell fractions (200-500 pl) were precipitated by 12 ml of ice-cold TCA (10%). The precipitate was re-suspended in NaOH (1-0 N; 0-1 ml) and HCIO4 (5-0 N; 0-4 ml) were added before the determination. Two parts of the Dische reagent which was prepared just before the
Human PMNs were incubated with the calcium ionophore, opsonized zymosan, arachidonic acid or exposed to hypotonic medium. On stimulation with ionophore, opsonized zymosan or arachidonic acid, ECF acitivity rapidly appears in the supernatant in the first 8 min of incubation (Fig. la-c). It appears from our data that the kinetics of ECF release induced by AA are more rapid as compared to stimulation with the ionophore or during phagocytosis. In repeated experiments, however, it was observed that with a less potent AA concentration, ECF activity peaked after 5 min of incubation. With all stimuli, the eosinophil chemotactic activity decreases markedly at later times of secretion and shows a fluctuating pattern with a second peak of activity. With the three stimuli, the generation and release of ECF occurs under non-cytotoxic conditions, as was determined by the enzyme LDH. Among the granular enzyme markers, lysozyme was released up to 40-50% of its total activity with the ionophore or during phagocytosis at optimal concentrations for ECF release, while with arachidonic acid as stimulus, only 20% of lysozyme appears in the supernatant. In the latter case, toxic concentrations of AA (more than 1-78 mM) increased the release of lysozyme but decreased the amount of ECF present in the supernatant. Additional enzymes of the specific granules, such as acid P-glycerophosphatase and P-glucuronidase appear to a lesser degree in the supernatant as compared to lysozyme. Only minor amounts of peroxidase (10-20%) which is present in the azurophilic granules can be detected, while almost no microsomal activity such as acid or alkaline PNP phosphatase, Mg2 + ATPase, alkaline phosphodiesterase was recovered. In the described experiments ECF activity is always obtained prior to the maximum of enzyme release and is not related to the amount of enzymes present in the supernatant. It has been described that the granular associated tetrapeptides ECF-A and the eosionophilotactic oligopeptides are preformed within mast cells and can be totally recovered by cell sonication or cell lysis (Goetzl, 1976). In contrast, the PMN derived ECF is not preformed and no ECF activity can be obtained from the sonicated cells (Konig et al., 1978). In addition, the release of a preformed mediator from
Eosinophilotactic activity from human polymorphnuclear neutrophils
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Figure 1 (a) Kinetics of ionophore induced ECF-release. PMNs (1 x 107/ml) were incubated with the calcium ionophore. At various times, the mixture was centrifuged and the supernatant (200 pl) assayed for chemotactic activity. The precision coefficient of the chemotactic assay varied between 8-12% (Eos/5 HPF, eosinophils per five high power fields). Lower part of the figure represents the pattern of enzyme release. The mean variation coefficient of the enzymes was calculated from the mean SD values obtained from eight separate experiments, which were performed in quadriplicate. Peroxidase, 4-8%; lysozyme, 4-2%; f-glucuronidase, 3-2%; acid f-glycerophosphatase, 7 8%; acid PNP phosphatase, 5 7%; alkaline PNP phosphatase, 5-8%; Mg ATPase, 8 8%; LDH, 4-0%. (b) Release pattern of ECF and enzymes from human PMNs on stimulation with opsonized zymosan. Conditions for the chemotactic assay were the same as described in a (c) Kinetics of ECF and enzyme release from human PMNs by arachidonic acid. Chemotactic assay was carried out as described in a. Lysozyme; o, f-glycerophosphatase; a, f3-glucuronidase; *, peroxidase; hatched area, acid p-NP-Ph, alkalinep-NP-Ph, ATPase, alkaline phosphodiesterase, LDH. In c hatched area includes peroxidase. e,
mast cells such as histamine, parallels that of the lyso-
somal enzyme marker /3-glucuronidase suggesting a similar release process for the enzyme and the mediator. Since our results indicated a different release pattern of ECF activity and granular enzymes after noncytotoxic stimulation, experiments were carried out to study the hypotonic exposure of human neutrophils. Human PMNs were incubated in a hypotonic Tris-HCI medium (0 01 M, pH 7-5) at 37°. It is apparent from Fig. 2a that under hypotonic conditions LDH and acid PNP phosphatase are released up to 100% into the supernatant after 3-4 min of incubation, while 55-65% of lysozyme, P-glycerophosphatase, fi-glucuronidase and total protein can be recovered. Alkaline PNP phosphatase and alkaline phosphodiesterase I are present up to 15-30% of their total while less than 10% of the plasma membrane marker Mg 2 + ATPase and of the azurophilic granular enzyme marker peroxidase can be obtained from the supernatant. As is apparent from the DNA measurement, hypotonic treatment does not lead to a destruction of the cell nucleus. ECF activity appears in a fluctuating pattern at 60 and 110 min of incubation (Fig. 2b) after the maximum of enzyme release. In a more hypotonic
medium (0-005 M Tris-HCl), a high amount of ECF activity appears even after 10 min of incubation showing repetitive fluctuation over time with sharp peaks and steep fall-offs (Fig. 3). An almost similar pattern of enzyme release as is apparent from Fig. 2a was observed when human PMNs were exposed to a hypotonic medium at 00 (data not shown). As compared to the previous experiments (Fig. 2a), the maximum of enzyme release occurred slightly later, i.e. after 6 min of incubation. In repeated experiments, however, no ECF activity could be recovered from the supernatant over time suggesting that the generation and release of ECF is temperature dependent. When human PMNs were exposed to a hypertonic medium (two-fold concentrated TCM buffer at 26 mS), LDH increased up to 30% of its total after 60 min of incubation, while no ECF activity appeared in the supernatant as was determined over a period of 2 h incubation. As was mentioned previously, ECF activity unlike ECF-A in mast cells is not preformed within human PMNs. Sonicated PMNs did not reveal eosinophilotactic activity and furthermore incubation of a standard ECF with sonicated but not intact PMNs reduced its activity suggesting a cellular inactivator
W. Konig, N. Frickhofen & H. Tesch
738
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Figure 2. (a) Enzyme release from human PMNs after their exposure to hypotonic buffer. Human PMNs (1 x 107/ml) were exposed to Tris-HCI buffer (0 1 M, pH 7-5) at 37°. After various times, cells were centrifuged and the supernatant were assayed for enzyme and ECF activity (Fig. 2b). LDH; v, acid p-NP-phosphatase; o, lysozyme; o, protein; +, DNA; *, ,-glucuronidase; ,B-glycerophosphatase; alkaline phosphodiesterase I; alkaline p-NP-phosphatase; +, Mg2 + -ATPase; x, peroxidase. (b) Kinetics of ECF release during hypotonic exposure of PMNs. For chemotaxis, 200 pl of the supernatant restored to isotonicity (see Materials and Methods) were assayed. .,
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(Konig et al., 1976; Czarnetzki et al., 1978). Our recent data show that the inactivator co-elutes with the peroxidase positive azurophilic granules of human PMNs after subcellular fractionation and equilibrium density gradient centrifugation (Frickhofen & Konig, 1978). One could assume that ECF might already be present as a preformed mediator within human PMNs but biologically inactive, in the presence of the inactivator. Since hypotonic exposure leads to a rapid and high release of granular enzymes, it might then be possible to detect ECF activity in the cell button prior to its
release into the supernatant. Therefore, human PMNs (1 x 107/ml) were exposed to hypotonic medium (0-005 M Tris-HCl, pH 7 5). After various lengths of time, the cells were centrifuged and the supernatants were obtained. The cell pellets were washed in TCM buffer at 40 and the sonicated. Both, the supernatant and the sonicated cell pellets were then assayed for ECF activity. It could be demonstrated that ECF activity increased after 15-25 min of incubation; the major amount of enzyme release had already occurred after 4 min of incubation. ECF activity in the cell pellet was negligible. Only a slight amount of activity was recovered after 15 min of incubation as compared to the amount of ECF released into the supernatants. These data indicate that ECF is rapidly secreted into the supernatant as has been previously shown for the ionophore and phagocytosis induced ECF. Since the ECFs induced by various stimuli have been described as low molecular weight mediators, experiments were carried out to analyse the eosinophil chemotactic factor generated by hypotonic lysis. Human PMNs (5 x 107 cells/2 ml) were incubated with the calcium ionophore for 15 min at 370 or exposed to hypotonic buffer (0-01 M Tris-HCI, pH 7-5 for 50 min at 37°. After incubation, aliquots of the supernatant (500 pl) were obtained and applied to gel filtration on Sephadex G-25. As can be seen, the elution profile of the ECF induced by hypotonic exposure parallels the elution profile of the ionophore induced ECF on
Eosinophilotactic activity from human polymorphnuclear neutrophils Dextron blue (2 x 106)
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Figure 4. Elution pattern of the ionoph ore (e) and lysis (o) induced ECF on sephadex G25 (1-6 x 254cm). Fractions of 1-5 ml were collected. The column was ecjuilibrated in TCM buffer. As molecular weight markers, clextran blue 2 x 106 and vitamin B12 (1355) were used.
Sephadex G-25 (Fig. 4). With all EC]Fs, peak activity is eluted after vitamin B1 2 indicating
Generation and secretion of eosinophilotactic activity from human polymorphonuclear neutrophils by various mechanisms of cell activation
W. KONIG*, N. FRICKHOFEN & H. TESCH Institute of Medical Microbiology Johannes Gutenberg Universitdt 65 Mainz, Hochhaus Augustusplatz, FRG
Received 8 June 1978; acceptedfor publication 17 August 1978
Summary. An eosinophil chemotactic factor(s) (ECF) can be generated from human polymorphonuclear neutrophils by the calcium ionophore, phagocytosis, arachidonic acid and hypotonic lysis. In kinetic studies it is observed that peak ECF activity is released prior to the maximum of lysosomal enzyme release with the calcium ionophore, phagocytosis and arachidonic acid, while under conditions of hypotonic exposure ECF activity appears after the maximum of enzyme release. The ECF obtained by hypotonic exposure shows a fluctuating pattern with sharp peaks and steep fall-offs in activity. The ECF-release for each stimulus is temperature dependent; extracellular calcium is required when the ionophore or phagocytosis are used as stimuli, while with arachidonic acid and hypotonic exposure no extracellular calcium is necessary for ECF-release. On Sephadex G-25 each preparation of ECF eluted in the low molecular weight range at 500
daltons. Eosinophils can be deactivated and crossdeactivated with the various ECF-preparations indicating either a molecular identity or a common mode of action on eosinophils.
INTRODUCTION Recent interest has focused on the immunobiological role of the eosinophil leucocyte (Parish, 1970; Kay, 1970; Greene & Colley, 1974). Since eosinophils have been recognized to exert specific killer functions on parasites (Butterworth, Sturrock, Houba & Rees, 1975; Mahmoud, Warren & Peters, 1975) and to be prominent participants at sites of inflammation (Goetzl, 1976), the mechanisms of their chemo-attraction are of major importance. It has been shown that with their enzymes, the eosinophil leucocytes are able to counteract the mediators of inflammation such as histamine (Zeiger, Twang & Colten, 1976), the slowreacting substance of anaphylaxis (Wasserman, Goetzl & Austen, 1975) and the platelet aggregating factor (Kater, Goetzl & Austen, 1976). In recent years, several eosinophilotactic factors have been described which are mast cell (Clark, Gallin & Kaplan, 1975; Kay, Stechschulte & Austen, 1971; Goetzl & Austen, 1975) and serum derived products (Ward, Cochrane & Muller-Eberhard, 1976). Among these, ECF-A has been studied best with regard to its
-
Abbreviations: AA, arachidonic acid; ECF, eosinophil chemotactic factor; Ion, ionophore; Zx, opsonized zymosan; Eos, eosinophils; HETE, 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid; HHT, 12-L-hydroxy-5,8,10-heptadecatrienoic acid; HPF, high power field; PMN, polymorphonuclear neutrophil; SRS, slow reacting substance; TACM, T: tris hydroxyaminomethan; A: albumin; SDS, sodium dodecylsulphate. *Correspondence: Dr W. Konig, Institute for Medical Microbiology, 65 Mainz, Augustusplatz, FRG 0079-2805/79/0400-0733$02.00 ©0 1979 Blackwell Scientific Publications
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structure and its eosinophilotactic properties (Goetzl & Austen, 1975). ECF-A is preformed within mast cells and consists of two tetrapeptides. On sonication or lysis of the cells, the total eosinophilotactic activity can be recovered from the supernatant. In addition to the tetrapeptides, oligopeptides have been shown with eosinophilotactic activity which are released from mast cells during the allergic reaction (Boswell, Austen & Goetzl, 1977). The eosinophilotactic peptides account in part for the eosinophilotactic activity of stimulated mast cells. Furthermore, lipid products such as HETE and HHT, derived from arachidonic acid after incubation with the microsomal fraction of platelets have been shown to attract eosinophils and neutrophils by chemokinesis and chemotaxis (Goetzl, Woods & Forman, 1977; Goetzl & Gorman, 1978). In the past, we demonstrated that human neutrophils generate and release an eosinophil chemotactic factor after stimulation with the calcium ionophore A 23187 (Czarnetzki, Konig & Lichtenstein, 1976) and during phagocytosis of opsonized zymosan (Konig Czarnetzki & Lichtenstein, 1976). Quite recently, we observed that human PMNs but not human lymphocytes generate and release an eosinophilotactic activity on stimulation with arachidonic acid (Konig, Tesch & Frickhofen, 1978), which is indistinguishable from the ionophore or phagocytosis induced ECF. With each stimulus, the ECFs proved to be of low molecular weight (500 daltons) and highly specific for guinea-pig and human eosinophils; in contrast to HETE and HHT, the ECFs do not attract guinea-pig or human neutrophils. Furthermore, ECF is not preformed within human PMNs, and no ECF activity can be obtained from sonicated cells (Konig, Czarnetzki & Lichtenstein, 1978). We now, however, present evidence, that potent ECF activity can be recovered from human PMNs during their exposure to hypotonic buffer. An intact cell membrane is required for each stimulus (ionophore, phagocytosis, arachidonic acid, hypotonic lysis) to generate and release ECF. The kinetics of ECF release with each stimulus appear to be similar in that after an early rise in ECF activity a steep fall off occurs at later times of secretion suggesting a mechanism of inactivation. Quite recently, we obtained evidence by subcellular fractionation studies of unstimulated PMNs that a cell derived inactivator is present within the azurophilic granules of human PMNs which most likely inactivates ECF by binding (Frickhofen & Konig, 1978). In addition, we
demonstrated by equilibrium gradient centrifugation that ECF activity in stimulated PMNs cosediments
with the microsomal fractions and most likely with the plasma membrane. The purpose of this paper is to analyse carefully the ECF release pattern from human PMNs as compared to the release pattern of granular and microsomal (acid, alkaline PNP phosphatase, Mg2 + ATPase, alkaline phosphodiesterase I) enzymes. These studies will help to understand the molecular basis of activation for human PMNs and to clarify the origin and the release process of ECF induced by various stimuli. It will be demonstrated that eosinophilotactic activity can be recovered prior to the release of granular enzymes with the calcium ionophore, phagocytosis and arachidonic acid, while under conditions of hypotonic exposure, ECF activity appears after the release of the granular enzyme markers.
MATERIALS AND METHODS
Commercial source of reagents Glycogen, phenol reagent, H202, casein, vitamin B12, glucose (E. Merck, Darmstadt, W. Germany). Heparin grade II, horseradish peroxidase type II, zymosan A, f-glycerophosphate, phenolphthalein glucuronic acid, phenolphthalein, lysozyme, Micrococcus lysdeikticus, p-nitrophenol, adenosine 5'-triphosphate, thymidine 5'-monophospho-p-nitrophenylester, pyruvic acid, calf thymus DNA type I (Sigma, Munchen, W. Germany)' Ficoll 400, Sephadex G-25, blue dextran 2000 (Pharmacia, Uppsala, Sweden). o-tolidine, triton X-100 (Serva, Heidelberg, W. Germany); dextran = Macrodex 6% (Knoll, Ludwigshafen, W. Germany); sodium metrizoate 75% (Nyegaard & Co., Oslo, Norway); sterile glucose 5% (Fresenius, Bad-Homburg, W. Germany); ethyl acetate (Riedel de Haen Hannover, W. Germany);
p-nitrophenyl-phosphate (Boehringer, Mannheim, W. Germany); [C14]-histamine (Amersham Buchler, W. Germany); human serum albumin (Behringwerke, Marburg, W. Germany). Reagents not further listed were purchased from E. Merck, Darmstadt, W. Germany. Preparations of cells Human leucocytes were obtained from heparinized blood of healthy donors and separated on a Ficoll metrizoate gradient followed by dextran sedimentation (Boyum, 1968). This method has been previously described and leads to more than 95% pure PMNs. As target cells for the chemotactic assay,
Eosinophilotactic activity from human polymorphnuclear neutrophils guinea-pig peritoneal exudates rich in eosinophils (35-70%) were obtained by injecting human serum intraperitoneally at weekly intervals (Czarnetzki et al., 1976; Konig et al., 1976). Neutrophil-rich exudates (more than 95%) were obtained from guinea-pigs 15-18 h after injection of 0 1% glycogen (Ward et al., 1976). Two to three hours after intraperitoneal injection of sterile 5% glucose, the cells were harvested from the peritoneal cavity. The total yield ranged between 1-3 x 108 neutrophils per guinea-pig.
Buffers The medium used for washing the cells and for mediator release unless stated otherwise was a Tris buffer (pH 7 35, 0-025 M) with NaCl (120 mM), KCI (4mM) CaCl2 (0-6 mM) and Mg chloride (1 -0 mM). This buffer is referred to as TCM buffer; when human serum albumin (0 3 mg/ml) was added, we refer to it as TACM buffer. Preparation of ECF Methods for the release of ECF induced by phagocytosis, by the calcium ionophore or by arachidonic acid have been described previously (Konig et al., 1976; Czarnetzki et al., 1976; Konig et al., 1978). Briefly, I x 107 PMNs/ml were incubated with the calcium ionophore (a gift of Dr Hamill of the Eli Lilly Research Laboratories, Indianapolis, Indiana, USA) at 5 x 10-6M, with zymosan (Nutritional Biochemical Corporation, Cleveland, Ohio) coated with complement (Zx) or with arachidonic acid (Sigma GmbH., Munich, West Germany) at a concentration of 0-35 mM. Zymosan was coated with complement (Zx) as has been described. Briefly, zymosan (200 mg) was suspended in 3 ml human serum and incubated for 30 min at 37°. The particles were then washed in TCM buffer and used at 3 mg/ml final concentration. To study the kinetics of ECF release, human PMNs (I x 107/ml) were incubated with different stimuli (calcium ionophore, Zx or arachidonic acid) in TACM buffer. After various times, cells were centrifuged and the supernatant assayed for ECF activity. The generation and release of ECF in hypotonic buffer was carried out by using either a 001 M or 0005 M Tris-HCl buffer at pH 7 5 without the addition of Ca2 + chloride and Mg chloride. The conductivity of these buffers was 0 4 mS (0-005 M) and 0-7 mS (0 01 M). After the incubation, I ml of TCM buffer two-fold more concentrated than listed previously (26 inS) was added to restore isotonicity. In case, ECF activity was analysed within the cell, the cell pellets were washed,
735
resuspended in isotonic TCM buffer (1 ml) and then sonicated for 1 min at 4° (Mullard Equipment, Ltd, England). The conductivity of the buffers was measured using the Radiometer, Copenhagen instrument.
Chemotaxis The method for eosinophil chemotaxis has been described in detail (Czarnetzki et al., 1976). Briefly, 2-5 x 106 guinea-pig peritoneal cells containing 35-70% eosinophils were placed above a nitrocellulose filter (Sartorius Membranfilter, GmbH., Gottingen, West Germany, 8 gm pore size, 13 mm diameter) in a modified Boyden Chamber. The chemotactic factor was placed below the filter. After 3 h of incubation, eosinophils that had migrated through the filter were counted at 100 x magnification. Five highpower fields (HPF) were evaluated. Buffer control in each experiment never exceeded five to ten eosinophils per 5 HPF. Neutrophil chemotaxis was performed using 3 gm filter and casein (1-10 mg/ml) as a positive control (Ward et al., 1976). Assay conditions were the same as described for eosinophil chemotaxis. The precision coefficient of the chemotactic assay varied between 8-12%. The chemokinetic properties of the chemotactic factors was assayed by placing the factor either above the filter or above and below the filter. Random migration was then assayed after 3 h of incubation. Deactivation of cells The experiments follow those described by Wasserman et al. (Wasserman, Whitmer, Goetzl & Austen, 1975). Guinea-pig peritoneal cells containing 50% eosinophils were incubated with either the phagocytosis, ionophore, arachidonic acid or hypotonic lysis induced ECF for 15 min at 37°. After incubation, the cells were washed repeatedly and then exposed to phagocytosis, ionophore, arachidonic acid or hypotonic lysis induced ECF to measure their chemotactic response towards the various ECFs. In each experiment, an aliquot of cells was suspended in TACM buffer instead of ECF and served as a positive control.
Chromatography Gel filtration analysis of eosinophilotactic acitivity was performed on a Sephadex G-25 column (1 6 x 25 cm). Elution was performed in TCM buffer and fractions of 1-5 ml each were collected and assayed for their eosinophilotactic activity. Biological activity was compared to the elution profile of molecular weight
736
W. Konig, N. Frickhofen & H. Tesch
markers such as blue dextran (2 x 106), vitamin B12 (1335) and ['4C]-histamine (111). Blue dextran was determined spectrophotometrically (260 nm) as well as vitamin B12 (360 nm). ['4C]-histamine was measured in a scintillation counter (Packard).
determination were added to one part of the DNA solution and incubated for 10-20 h at 37°. The absorbency was read at 535 nm; calf thymus DNA was used as standard.
RESULTS
Biochemical assays Protein was measured at 280 nm in a Beckman photometer or by the Folin Lowry method (Lowry, Rosebrough, Farr & Randall, 1951). Enzyme determinations were performed as has been described: 1.11.1.7 peroxidase (Baggiolini, Hirsch & de Duve, 1909); 3.1.3.2 acid P-glycerophosphatase (Chen, Toribara & Warner, 1956; Bowers, Finkenstaedt & de Duve, 1967); 3.2.1.31 ,B-glucuronidase (Avila & Convit, 1973); 3.1.1.17 lysozyme (Sigma technical bulletin); 3.1.3.1 alkaline p-nitrophenylphosphatase (Bergmeyer, 1970); 3.1.3.2 acid p-nitrophenylphosphatase (Dingle, 1972); 3.6.1.3. Mg2+ ATPase (Harlan, de Chatelet, Iversion & McCall, 1977); 3.1.4.1. Alkaline phosphodiesterase I (Beaufay, Amar-Costesec, Feytmans, Thisnes-Sempoux, Wibo, Robbi & Berthet, 1974); 1.1.1.27 lactate dehydrogenase (Bretz & Baggiolini, 1974). The incubation volume is always 1 ml except for the lysozyme assay in which 0 05 ml sample was added to 1 25 ml substrate solution. Enzyme activities are expressed as percentage of the total activity calculated from unstimulated cells. Total enzyme activity was recovered after solubilization of the cells in 1% SDS. A solution of Na2HPO4 was used as standard for acid f-glycerophosphatase and Mg ATPase, while paranitrophenol (PNP) served as standard for alkaline PNP phosphatase, acid PNP phosphatase and alkaline phosphodiesterase I; phenolphthalein was used as standard in the,B-glucuronidase assay; horse radish peroxidase served as standard for peroxidase. One unit of lysozyme is defined as the decrease in optical density at 450 nm of 10 0 per min as has been described by Spitznagel, Dalldorf, Leffel, Folds, Welsh, Cooney & Martin, 1974). In all phosphatase assays, the effect of sodium tartrate (10 mM) was investigated to calculate the interference of the lysosomal acid phosphatase. For all enzyme determinations, a Carl Zeiss M4 Q3/PMQ2 spectrophotometer was used. DNA was determined as has been described according to Burton (1956). Briefly, the cell fractions (200-500 pl) were precipitated by 12 ml of ice-cold TCA (10%). The precipitate was re-suspended in NaOH (1-0 N; 0-1 ml) and HCIO4 (5-0 N; 0-4 ml) were added before the determination. Two parts of the Dische reagent which was prepared just before the
Human PMNs were incubated with the calcium ionophore, opsonized zymosan, arachidonic acid or exposed to hypotonic medium. On stimulation with ionophore, opsonized zymosan or arachidonic acid, ECF acitivity rapidly appears in the supernatant in the first 8 min of incubation (Fig. la-c). It appears from our data that the kinetics of ECF release induced by AA are more rapid as compared to stimulation with the ionophore or during phagocytosis. In repeated experiments, however, it was observed that with a less potent AA concentration, ECF activity peaked after 5 min of incubation. With all stimuli, the eosinophil chemotactic activity decreases markedly at later times of secretion and shows a fluctuating pattern with a second peak of activity. With the three stimuli, the generation and release of ECF occurs under non-cytotoxic conditions, as was determined by the enzyme LDH. Among the granular enzyme markers, lysozyme was released up to 40-50% of its total activity with the ionophore or during phagocytosis at optimal concentrations for ECF release, while with arachidonic acid as stimulus, only 20% of lysozyme appears in the supernatant. In the latter case, toxic concentrations of AA (more than 1-78 mM) increased the release of lysozyme but decreased the amount of ECF present in the supernatant. Additional enzymes of the specific granules, such as acid P-glycerophosphatase and P-glucuronidase appear to a lesser degree in the supernatant as compared to lysozyme. Only minor amounts of peroxidase (10-20%) which is present in the azurophilic granules can be detected, while almost no microsomal activity such as acid or alkaline PNP phosphatase, Mg2 + ATPase, alkaline phosphodiesterase was recovered. In the described experiments ECF activity is always obtained prior to the maximum of enzyme release and is not related to the amount of enzymes present in the supernatant. It has been described that the granular associated tetrapeptides ECF-A and the eosionophilotactic oligopeptides are preformed within mast cells and can be totally recovered by cell sonication or cell lysis (Goetzl, 1976). In contrast, the PMN derived ECF is not preformed and no ECF activity can be obtained from the sonicated cells (Konig et al., 1978). In addition, the release of a preformed mediator from
Eosinophilotactic activity from human polymorphnuclear neutrophils
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Figure 1 (a) Kinetics of ionophore induced ECF-release. PMNs (1 x 107/ml) were incubated with the calcium ionophore. At various times, the mixture was centrifuged and the supernatant (200 pl) assayed for chemotactic activity. The precision coefficient of the chemotactic assay varied between 8-12% (Eos/5 HPF, eosinophils per five high power fields). Lower part of the figure represents the pattern of enzyme release. The mean variation coefficient of the enzymes was calculated from the mean SD values obtained from eight separate experiments, which were performed in quadriplicate. Peroxidase, 4-8%; lysozyme, 4-2%; f-glucuronidase, 3-2%; acid f-glycerophosphatase, 7 8%; acid PNP phosphatase, 5 7%; alkaline PNP phosphatase, 5-8%; Mg ATPase, 8 8%; LDH, 4-0%. (b) Release pattern of ECF and enzymes from human PMNs on stimulation with opsonized zymosan. Conditions for the chemotactic assay were the same as described in a (c) Kinetics of ECF and enzyme release from human PMNs by arachidonic acid. Chemotactic assay was carried out as described in a. Lysozyme; o, f-glycerophosphatase; a, f3-glucuronidase; *, peroxidase; hatched area, acid p-NP-Ph, alkalinep-NP-Ph, ATPase, alkaline phosphodiesterase, LDH. In c hatched area includes peroxidase. e,
mast cells such as histamine, parallels that of the lyso-
somal enzyme marker /3-glucuronidase suggesting a similar release process for the enzyme and the mediator. Since our results indicated a different release pattern of ECF activity and granular enzymes after noncytotoxic stimulation, experiments were carried out to study the hypotonic exposure of human neutrophils. Human PMNs were incubated in a hypotonic Tris-HCI medium (0 01 M, pH 7-5) at 37°. It is apparent from Fig. 2a that under hypotonic conditions LDH and acid PNP phosphatase are released up to 100% into the supernatant after 3-4 min of incubation, while 55-65% of lysozyme, P-glycerophosphatase, fi-glucuronidase and total protein can be recovered. Alkaline PNP phosphatase and alkaline phosphodiesterase I are present up to 15-30% of their total while less than 10% of the plasma membrane marker Mg 2 + ATPase and of the azurophilic granular enzyme marker peroxidase can be obtained from the supernatant. As is apparent from the DNA measurement, hypotonic treatment does not lead to a destruction of the cell nucleus. ECF activity appears in a fluctuating pattern at 60 and 110 min of incubation (Fig. 2b) after the maximum of enzyme release. In a more hypotonic
medium (0-005 M Tris-HCl), a high amount of ECF activity appears even after 10 min of incubation showing repetitive fluctuation over time with sharp peaks and steep fall-offs (Fig. 3). An almost similar pattern of enzyme release as is apparent from Fig. 2a was observed when human PMNs were exposed to a hypotonic medium at 00 (data not shown). As compared to the previous experiments (Fig. 2a), the maximum of enzyme release occurred slightly later, i.e. after 6 min of incubation. In repeated experiments, however, no ECF activity could be recovered from the supernatant over time suggesting that the generation and release of ECF is temperature dependent. When human PMNs were exposed to a hypertonic medium (two-fold concentrated TCM buffer at 26 mS), LDH increased up to 30% of its total after 60 min of incubation, while no ECF activity appeared in the supernatant as was determined over a period of 2 h incubation. As was mentioned previously, ECF activity unlike ECF-A in mast cells is not preformed within human PMNs. Sonicated PMNs did not reveal eosinophilotactic activity and furthermore incubation of a standard ECF with sonicated but not intact PMNs reduced its activity suggesting a cellular inactivator
W. Konig, N. Frickhofen & H. Tesch
738
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Figure 2. (a) Enzyme release from human PMNs after their exposure to hypotonic buffer. Human PMNs (1 x 107/ml) were exposed to Tris-HCI buffer (0 1 M, pH 7-5) at 37°. After various times, cells were centrifuged and the supernatant were assayed for enzyme and ECF activity (Fig. 2b). LDH; v, acid p-NP-phosphatase; o, lysozyme; o, protein; +, DNA; *, ,-glucuronidase; ,B-glycerophosphatase; alkaline phosphodiesterase I; alkaline p-NP-phosphatase; +, Mg2 + -ATPase; x, peroxidase. (b) Kinetics of ECF release during hypotonic exposure of PMNs. For chemotaxis, 200 pl of the supernatant restored to isotonicity (see Materials and Methods) were assayed. .,
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(Konig et al., 1976; Czarnetzki et al., 1978). Our recent data show that the inactivator co-elutes with the peroxidase positive azurophilic granules of human PMNs after subcellular fractionation and equilibrium density gradient centrifugation (Frickhofen & Konig, 1978). One could assume that ECF might already be present as a preformed mediator within human PMNs but biologically inactive, in the presence of the inactivator. Since hypotonic exposure leads to a rapid and high release of granular enzymes, it might then be possible to detect ECF activity in the cell button prior to its
release into the supernatant. Therefore, human PMNs (1 x 107/ml) were exposed to hypotonic medium (0-005 M Tris-HCl, pH 7 5). After various lengths of time, the cells were centrifuged and the supernatants were obtained. The cell pellets were washed in TCM buffer at 40 and the sonicated. Both, the supernatant and the sonicated cell pellets were then assayed for ECF activity. It could be demonstrated that ECF activity increased after 15-25 min of incubation; the major amount of enzyme release had already occurred after 4 min of incubation. ECF activity in the cell pellet was negligible. Only a slight amount of activity was recovered after 15 min of incubation as compared to the amount of ECF released into the supernatants. These data indicate that ECF is rapidly secreted into the supernatant as has been previously shown for the ionophore and phagocytosis induced ECF. Since the ECFs induced by various stimuli have been described as low molecular weight mediators, experiments were carried out to analyse the eosinophil chemotactic factor generated by hypotonic lysis. Human PMNs (5 x 107 cells/2 ml) were incubated with the calcium ionophore for 15 min at 370 or exposed to hypotonic buffer (0-01 M Tris-HCI, pH 7-5 for 50 min at 37°. After incubation, aliquots of the supernatant (500 pl) were obtained and applied to gel filtration on Sephadex G-25. As can be seen, the elution profile of the ECF induced by hypotonic exposure parallels the elution profile of the ionophore induced ECF on
Eosinophilotactic activity from human polymorphnuclear neutrophils Dextron blue (2 x 106)
Vit B1l12 (1355'
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Figure 4. Elution pattern of the ionoph ore (e) and lysis (o) induced ECF on sephadex G25 (1-6 x 254cm). Fractions of 1-5 ml were collected. The column was ecjuilibrated in TCM buffer. As molecular weight markers, clextran blue 2 x 106 and vitamin B12 (1355) were used.
Sephadex G-25 (Fig. 4). With all EC]Fs, peak activity is eluted after vitamin B1 2 indicating
