Thrombin enhances opsonized zymosan-induced chemiluminescence of neutrophils

TISSUE & CELL 1988 20 (1) &17 0 1988 Longman Group UK Ltd

KIN-LUN

CHAN, RENA BIZIOS”

and ASRAR B. MALlKt

THROMBIN ENHANCES OPSONIZED ZYMOSANINDUCED CHEMILUMINESCENCE OF NEUTROPHILS Keyword\: Thrombin. ncutrophils. C3h receptors. phagocytovs

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ABSTRACT. We examined the eftccts of n-thrombm (the native cnrymr:) on ncutrnphll activation as assessed by the measurement of chcm~lummcscencc a-Thrombm m phyvologul concentrations (lO~y-lO~” M) did not Induce neutrophil chemilummcscence Howcvcr. \*hcn neutrophils were coincubated wth opsonized zymown and cl-thrombin. the chem~lumincvxnce rwponse to opsonizcd zymoaan was enhanced in a concrntration-dependent manner. The neutrophil chemilumincsccnce responses to opsonized rymosan and to opsonized zymoun plw a-thrombin were dependent on the generation of oxygen-dewed free raduls wee the chemilumincacencc was inhibited by auperoxidc dlsmutase. The rewlts indxate that thromhln pu se does not induce neutrophil chemilumincscence. Howcwr. thromhm cnhancc\ the chcm~lumineacence response to opsonircd zymoaan suggatmg an interactwn hetwccn thrombm and complement receptors in inducing neutrophil activation. The chemilumincwx~ce rcqnc to thromhin and opsonizcd rymosan is the result of oxygen-derived free radicals

Introduction

by the measurement of chemiluminescence of these cells (Cheson et cd., 1976; Nelson CI al.. 1977). We also assessed the effect of thrombin on neutrophil chemiluminescencc induced by opsonized zymosan since thrombin may enhance the neutrophil activation response during phagocytosis.

Thrombin-induced pulmonary intravascular coagulation in experimental animals results in lung vascular injury and pulmonary edema (Malik, 1983). Neutrophil activation with the release of superoxide anion subsequent to the pulmonary neutrophil sequestration induced by thrombin has been implicated in mediating the vascular injury (Malik, 1983; Tahamont and Malik. 1983). Thrombin-induced intravascular coagulation may generate chemotaxins such as CT:, (Malik. 1983; Zimmerman et ul.. 1977) and lipoxygenase products of arachidonic acid (Malik, 1983). We have shown that thrombin also induces irl vitro neutrophil chemotaxis and aggregation (Bizios et al.. 1985, 1986). suggesting a direct effect of thrombin on neutrophil sequestration in the lungs. In the present study, we examined the possibility of a direct effect of thrombin on neutrophil activation as assessed

Materials and Methods Isolation of’ nrutrophils Sheep neutrophils were isolated from whole blood using the Percoll-isotonic saline gradient centrifugation technique as described by Cooper et al. (1984). The neutrophils were suspended in 1 X Williams’ Hepes buffer and cell viability was checked by Trypan Blue dye exclusion. Reagents Luminol (Sigma) was obtained in stock solutions of 10 mg/ml in dimethyl hulfoxide and was diluted in phosphate buffered saline (PBS) immediately before use. Purified human cl-thrombin (the native enzyme) was prepared as described by Fenton (1981) (gift of John Fenton, New State Department of Health, Albany, New York.)

Department of Physiology. Albany Medical College of Umon University, New York 12208. ‘Department of Biomedical Engineermg, Rensselaer Polytechmc Institute. Troy. New York 12180-3590. +To whom offprint requests should he addressed. Received 17 July 1987. Revised 14 September 1987. 13

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CHAN

Opsonized zymosan

Control experiments

Zymosan (Sigma) particles were opsonized with sheep serum. In this procedure, the dry zymosan was boiled for an hour before the opsonization step. The opsonized particles were resuspended with 25 mM Hepes/HBSS to a final concentration of 5 mg/ml.

In the absence of opsonized zymosan, the neutrophil chemiluminescence in buffer and luminol was low (background level). Comparable low neutrophil chemiluminescence was obtained with varying concentrations of

Chemiluminescence

assay

Scintillation glass vials were used for the chemiluminescence assay. The assay medium contained 0.5 ml Hepes/HBSS (25 mM), 0.2 ml opsonized zymosan (5 mg/ml), and 0.2 ml luminol. The luminol solution was added just prior to the addition of neutrophils to minimize exposure of luminol to light. An aliquot (O-1 ml) of neutrophil suspension (lo7 cells/ml) was added to the chemiluminescence-assay medium, and the sample was thoroughly mixed and immediately counted on a scintillation counter for 0.2 min. The scintillation counter (model Isocap/300, Searle-Nuclear, Chicago, IL) was operated at room temperature and in the off-coincidence mode. We examined the effects of a-thrombin and opsonized zymosan in the presence and absence of superoxide dismutase on the neutrophil chemiluminescence. For this purpose, 0.1 ml of superoxide dismutase (200 pg/ml), 0.1 ml of opsonized zymosan (5 mg/ml), or 0.1 ml of a-thrombin (1O-s-1O-y M) were added to the chemiluminescenceassay medium and appropriate adjustments were made to the Hepes/HBSS volume to keep the sample volume to 1 ml. Control samples (i.e. without neutrophils or opsonized zymosan or with buffer alone) were run with every experimental set. Each experiment was run four times and ten replicates were obtained for each experiment. The data are expressed as mean +l SEM. Differences maximum between the chemiluminescence response were compared using the Student’s t-test.

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Fig. 1. Comparison of chemiluminescent activity of isolated neutrophils stimulated by opsonized zymosan to those without opsonized zymosan. Bars indicate mean fl SEM.

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Results

The neutrophil preparation contained 86% neutrophils, 10% eosinophils, 4% lymphocytes, and less than 1% basophils. Platelets comprised less than 1% of the cells. Over 98% of the neutrophils excluded Trypan Blue dye.

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Fig. 2. Chemiluminescence of isolated sheep neutrophils activated by the different thrombin concentrations. Bars indicate +l SEM.

THROMBIN-INDUCED

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CHEMILUMINESCENCE

a-thrombin (Fig. 1). However, in the presence of opsonized zymosan, neutrophil chemiluminescence increased 218% from baseline in a time-dependent manner (Fig. 1). This response was greater than the chemiluminescence at all concentrations of thrombin. Effects of a-thrombin with opsonized zymosan

a-Thrombin enhanced the opsonized zymosan-induced chemiluminescence of neutrophils in a concentration-related manner (Fig, 2). a-Thrombin concentrations of 1 x 10mx,5 x lo-” and 1 x lOmyM were found to increase (RO.05) the chemiluminescent activity by 25, 23 and 12%, respectively, above the response obtained with opsonized zymosan alone (Fig. 2). Effects of sodium azide and of superoxide dismutase

Sodium azide (0.1 M) abolished the chemiluminescent activity of all samples assayed (Fig. 3). Superoxide dismutase (200 ,ug/ml) reduced the chemiluminescence activity obtained with opsonized zymosan alone and with opsonized zymosan plus SX 10e9 M a-thrombin (Fig. 3).

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sodium indicate

Effects of superoxide dismutase (SOD) and aide on neutrophil chemiluminescence. Bars tl SEM.

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Discussion Oxygen-free radicals are generated by neutrophils during phagocytosis of opsonized microorganisms and zymosan particles (Cheson et al., 1976; Nelson et al., 1977; Klebanoff, 1975; Webb et al., 1974). Oxygen radicals are also generated through the interaction of neutrophils with soluble ligands such as CS, and LTBl (Newman and Johnston, 1979). The opsonized zymosan particles serve as a ligand for C3b receptors on the neutrophil cell membrane (Fearon. 1980; Mentovani, 197.5; Wright and Griffin, 1985). The opsonic fragment of the complement component C3 is Fragment C3b, which mediates the binding of the particle to the cell surface of neutrophils (Fearon. 1980; Mentovani, 1975; Wright and Griffin, 1985). The interaction of C3b receptors with the zymosan particles may lead to the generation of oxygen-free radicals without phagocytosis (Wright and Griffin, 1985). Luminol-dependent chemiluminescence qualitatively measures the production of oxygen-derived metabolites by activated neutrophils (Nelson et al., 1977; Webb et al.. 1974; Babior et al., 1977). A variety of oxygen-derived metabolites including superoxide anion, hydrogen peroxide, and singlet chemiluminescence oxygen, can induce (Cheson et al., 1976; Nelson et al., 1977: Webb et al., 1974; Babior et al., 1977). The measurement of chemiluminescence in the presence of superoxide dismutase provides an indication of the dependence of the response on the presence of superoxide anions. In the present study, thrombin did not elicit neutrophil chemiluminescence. However, opsonized zymosan proved to be a necessary prerequisite for neutrophil chemiluminescence with thrombin since the chemiluminescence response was only seen in experiments with the opsonized zymosan particles. The finding that thrombin did not directly induce neutrophil activation as assessed by chemiluminescence agrees with our observation that thrombin, which is known to induce neutrophil chemotaxis and aggregation (Bizios et al., 1986; Baehner, 1975), did not cause the generation of superoxide anions from isolated neutrophils (Malik, unpublished observation). The chemiluminescence response of neutrophils to opsonized zymosan was

16

CHAN

enhanced in a concentration-dependent manner by the addition of a-thrombin. Therefore, a cellular interaction may occur between a-thrombin and the C3b membrane receptors, thereby exposing more of the C3b receptors for opsonized zymosan. This may involve thrombin-induced ‘unmasking’ of C3b receptors due to the proteolytic activity of thrombin (Speer et al., 1984). Thrombin may prime neutrophils for enhanced release of oxygen metabolites as has been reported for lipopolysaccharide (Guthrie et al., 1984). Another explanation for the increased chemiluminescence is that a-thrombin alters the complement fragments on the zymosan particles such that the C3b receptors become more receptive to the altered zymosan particles (Wright and Griffin, 1985), thus enhancphagocytosis and the ing neutrophil generation of reactive oxygen-derived metabolites. The interaction between opsonized zymosan and thrombin may have implications in the neutrophil-dependent lung vascular injury. Complement activation is associated with activation of the clotting cascades during which thrombin is generated (Zimmerman et al., 1977); therefore, both processes may act synergistically to enhance

ETAL.

the degree of neutrophil activation, and thus induce lung vascular injury. Most of the chemiluminescence response of neutrophils appeared to be the result of generation of superoxide anion since superoxide dismutase (an enzyme that removes the generated superoxide anions) greatly diminished the chemiluminescence. However, hydroxyl radicals may also contribute to the response since the superoxide anion is the required substrate for generation of hydroxyl radical via the Haber-Weiss reaction. Neutrophil chemiluminescence was dependent on oxidative metabolism since the chemiluminescence responsible was abolished with sodium azide (Sagone etul., 1977). In summary, the results indicate that neutrophil chemiluminescence induced by opsonized zymosan is enhanced by the addition of physiological concentrations of athrombin. The response is the result of the generation of oxygen-derived free radicals. Acknowledgements

We wish to thank Linda Lai for technical assistance and Lynn McCarthy for typing the manuscript. Supported by Grant HL-32418 from the National Institutes of Health.

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