transient calcium elevation in polymorphonuclear leukocytes triggered by thrombin-activated...

Transient calcium elevation in polymorphonuclear leukocytes triggered by thrombin-activated platelets

Takahashi A, Yamamoto K, Okuma M, Sasada M. Transient calcium elevation in polymorphonuclear leukocytes triggered by thrombin-activated

A. Takahashi ’, K. Yamamoto ’, M, Okuma ’ and M, Sasada ‘2

platelets. Eur J Haematol 1992: 48: 196-201.

Abstract: The effect of thrombin-activated platelets and their release products on the intracellular free calcium concentration ([Ca” ]i) of human polymorphonuclear leukocytes (PMNs) was studied by loading PMNs with a fluorescent indicator of calcium, fura-2. [Ca2+]i of PMNs was transiently elevated by thrombin-activated platelets. The supernatant of thrombin-activated platelets also elicited a transient elevation of [Ca” li in PMNs. Pretreatment of the supernatant with hexokinase caused a decrease in the transient [ Ca2 + ]i elevation of PMNs, while hexokinase abrogated the [Ca” ]i elevation of PMNs elicited by 80 pmol/l adenosine triphosphate (ATP). Pretreatment of the supernatant with trypsin also decreased the magnitude of the elevation, while trypsin had no effect on the response to ATP. These findings suggest that thrombin-activated platelets induce a transient [ Ca’ + ]i elevation in PMNs by releasing ATP and some trypsin-sensitive factor(s).

Introduction

Platelets and their release products such as adenosine thriphosphate (ATP), platelet-derived growth factor, platelet factor 4, and 12-hydroxy- eicosatetraenoic acid (12-HETE) have been shown to modulate functions of polymorphonuclear leukocytes (PMNs) in v i m , including synthesis of leukotrienes (1-3), lipoxins (4) and platelet-activating factor (PAF) ( 5 ) , chemotaxis (649 , phagocytosis (9, lo), degranulation (1 1- 13), and superoxide release (10, 12, 14-17).

It has been reported that ATP is released by activated platelets and that exogenously added ATP induces a transient elevation of intracellular free calcium concentration (a Ca2+ transient) in PMNs (14, 18-20). However, there has been no direct demon- stration of a Ca” transient in PMNs induced by activated platelets. Furthermore, no conclusion has yet been drawn as to whether ATP is solely respon- sible for the Ca2+ transient evoked by activated platelets. Therefore, we studied the Ca2+ transient in PMNs induced by thrombin-activated platelets and characterized the platelet release product(s) that triggered the Ca2+ transient in PMNs.

’ The First Division, Department of Internal Medicine, Faculty of Medicine, and * College of Medical Technology, Kyoto University, Kyoto, Jaoan

Key words: platelet-neutrophil interactions - intracellular calcium - thrombin - ATP - hexokinase - trypsin

Correspondence: Atsushi Takahashi, The First Division, Department of Internal Medicine, Faculty of Medicine, Kyoto University, 54 Shogoin-Kawaramachi, Sakyo-ku, Kyoto 606, Japan

Accepted for publication 29 January 1992

Material and methods Preparation of PMNs

Peripheral blood of healthy volunteers anticoagu- lated with 1/10 volume of 3.8% citrate was centrifuged at 200g for 15 minutes at 25°C to obtain platelet-rich plasma. The pellet was resuspended in calcium- and magnesium-free phosphate-buffered saline (PBS) containing 1% dextran (high molecular) and allowed to stand for 30min at room temperature for sedimentation of erythrocytes. Then, PMNs were isolated between discontinuous Percoll (Pharmacia, Uppsala, Sweden) gradients of 1.085 and 1.103 by a modification of our previously described method (21). The cells were washed with PB S, resuspended in phenol red-free Hank’s bal- anced salt solution (HBSS), and stored on ice until use.

Preparation of platelets

Platelet-rich plasma was centrifuged at 200 g for 15 min at 25°C to remove contaminating erythrocytes and leukocytes. The supernatant was supplemented with 1/10 volume of 77 mmol/l ethylenedia-

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mine tetraacetic acid (EDTA) (pH 7.4) and centrifuged at 1150 g for 10 rnin at 4" C. The pellet was washed with calcium-free Tyrode's buffer (containing 1 mg/ml glucose and adjusted to pH 7.30) (22) supplemented with 7 mmol/l EDTA, resuspended in the same buffer without EDTA, and stored at room temperature until use. PMNs and platelets isolated from the same blood specimen were used for each experiment.

Measurement of Ca2+ transients in PMNs

PMNs was loaded with a calcium indicator fura-2 (23) by incubating a cell suspension (1 x 107/ml) with 1 pmol/l fura-2/AM (Molecular Probes, Eugene, OR) for 30min at 37°C. The cells were washed twice and resuspended at 5 x 106/ml with HBSS. Fura-2-loaded PMNs with or without platelets were allowed to equilibrate while stirring in a thermostat- ted cuvette at 37°C for 2 min. Then a stimulus was added and fluorescence emission was monitored at 510 nm with excitation at 340 nni in a Hitachi F- 3000 fluorescence spectrophotometer. The intracel- Mar free calcium concentration ([ Ca2 + ]i) was cal- culated from the fluorescence intensities (F) using the equation: [ Ca2 + ]i = Kd(F - Fn,in)/(F,,,,, - F). The calcium-dissociation constant of fura-2 in the cytoplasm of PMNs (Kd) was assumed to be 224nmol/l at 37°C (23). F,,, was determined by lysing the PMNs with 0.2% Triton-X. Fmin was determined by adding 5 mmol/l ethylene glycol-bis-(P- amhoethylether)-N,N,N' ,N ' -tetraacetic acid (EGTA) (pH 7.4) to the PMN lysate.

Preparation and treatment of platelet release products

Platelet suspension (1 x 109/ml) was stimulated with bovine thrombin (1 unit/ml) (Sigma, St. Luis, MO) for 10 niin at 37°C in a water bath incubator. The supernatant was obtained after centrifugation at 1600g for 10 rnin at 4 ° C and divided into aliquots, each of which was treated as follows: 1) kept on ice until use; treated for 30 min at 37 " C in a water bath incubator with 2) hexokinase (5 units/ml) (from bak- ers yeast, type 111, Sigma), 3) trypsin (0.1 %) (type I11 from bovine pancreas, Sigma), 4) trypsin (0.1 %) and soybean trypsin inhibitor (1 mg/ml) (Type I-S, Sigma), 5) trypsin (0.1 %) and hexokinase ( 5 units/ ml). In 3) and 5), soybean trypsin inhibitor (1 mg/ml) was added after the treatment to stop digestion by trypsin.

When the supernatant was used as a stimulus to induce a Ca2' transient in PMNs, 1/10 volume of the reaction mixture was added so that the platelet release products equivalent to that released by 1 x lO'/ml (1 x 10' platelet equivalents/ml) would be present.

Other reagents

Arg-Gly-Asp-Ser (RGDS) was obtained from Pep- tide Institute, Osaka, Japan. Phorbol myristate acetate (PMA) obtained from Consolidated Midland, Brewster, NY was dissolved in dimethylsulfoxide, stored at - 20"C, thawed and diluted with HBSS immediately before use.

Statistical analysis

Statistical analysis was performed with the Wilcoxon signed-rank test.

Results Ca2+ transients in PMNs triggered by thrombin-activated platelets

The addition of thrombin to the mixture of fura-2- loaded PMNs and platelets caused a transient increase in fura-2 fluorescence with excitation at 340nm (Fig. 1,a). It reached a peak in about 30 seconds and returned to the baseline, which was slightly elevated, in about 2 min. When the fluorescence emission was monitored with excitation at 360nm, a point where fura-2 fluorescence is not affected by calcium concentration (23), only a grad- ual rise in fluorescence that reached a plateau in about 2 min was observed without the peak near

S W

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200

150-

100-

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50 Fig. 1. Effect ofthrombin-activated platelets on [ Ca2 + ]i of PMNs. PMNs ( 5 x 106/ml) loaded with fura-2 are preincubated with platelets (1 x 108/ml) for 2 rnin at 37°C with stirring before addition of thrombin at 1 unit/ml (indicated by an arrow). Fura-2 fluorescence is monitored in the absence (a) or presence (b) of EGTA (5 mmol/l, added 30 sec before thrombin). Essentially identical results were obtained in 16 (a) and 4 (b) separate experiments. The tracings were obtained with cells from the same batch of fura-2-loaded PMNs.

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30 s which was observed with excitation at 340 nm (data not shown). Therefore, the peak in fura-2 fluorescence was considered to reflect a transient elevation of [Ca2+ ]i in PMNs. While thrombin can affect functions of PMNs (24,25), thrombin itself did not induce an elevation of [Ca” ]i in PMNs in the absence of platelets (data not shown). Thus the Ca2+ transient in PMNs was considered to be mediated by platelets activated by thrombin.

The Ca2+ transient was abrogated by chelation of extracelIularcalcium with 5 mmol/l EGTA (Fig. 1,b). RGDS at 0.5 mmol/l, which inhibits aggregation of platelets (26), did not significantly affect the Ca2+ transient (data not shown). The magnitude of the Ca2 + transient was dependent on the concentration of platelets (Fig. 2) and thrombin (Fig. 3).

At the start of the preincubation of PMNs with platelets, addition of platelets induced a small transient elevation of [Ca” ]i in PMNs. The magnitude of Ca2+ transient elicited by 1 x 108/ml platelets was about one-third of that elicited by the same concentration of platelets and 1 unit/ml of thrombin. Most likely, platelets were activated to some degree during preparation and mixing with PMNs in the cuvette at 37°C.

The elevation of the baseline fluorescence after the Ca2 + transient subsided was probably due to aggregation of platelets because it was observed when the fluorescence was monitored with excitation at 360 nm and because inhibition of the platelet aggre- gate formation by reducing the concentration of platelets or thrombin and by the presence of RGDS decreased the magnitude of this baseline shift (data not shown).

Ca2+ transients in PMNs triggered by platelet release products

The supernatant obtained from the suspension thrombin-activated platelets induced a transient

of el-

150 -

100 -

50 -

I

10 20 40

platelet/PMN

Fig. 2. Effect of the platelet to PMN ratio on the magnitude of the CaZ + transient induced by thrombin-activated platelets. Fura-2- preloaded PMNs (5 x 106/ml) are preincubated with platelets at the ratio as indicated and thrombin (1 unit/ml) is added. The figure represents 1 of 2 separate experiments with similar results.

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I

2 0.01

Thrombin ( u n i t /m I)

Fig. 3. Effect of thrombin concentration on the magnitude of the Ca2 + transient induced by thrombin-activated platelets. Varying concentrations of thrombin are added to fura-2-preloaded PMNs ( 5 x 106/ml) preincubated with platelets (1 x 10X/ml). The figure represents 1 of 2 separate experiments with similar results.

evation of [Ca”]i in PMNs that reached a peak almost immediately and returned to the baseline in less than 1 min (Fig. 4,a). This indicated that substance(s) released by thrombin-activated platelets induced a Ca2 + transient in PMNs. In the presence of 5 mmol/l EGTA, the Ca2+ transient induced by the supernatant was markedly diminished but was not totally abrogated (Fig. 4,b). Pretreatment of PMNs with PMA (100 ng/ml)for 30 sec caused total abrogation of the Ca2+ transient (Fig. 4,c). The Ca2 + transient was dependent on the concentration of the platelet supernatant (Fig. 5). The supernatant of a platelet suspension incubated at 37 O C for 10 min in the absence of thrombin, or that of a platelet suspension left stand at room temperature induced a small transient elevation of [ Ca2+ ]i with magnitude less than one-sixth of the Ca2+ transient induced by that of thrombin-activated platelets.

- , mm

Fig. 4. Effect of the supernatant of thrombin-activated platelets on [Ca2+]i of PMNs as assessed by fura-2 fluorescence. (a)The supernatant of thrombin-activated platelets (1 x lo8 platelet equivalents/ml) is added (arrow) to fura-2-preloaded PMNs ( 5 x 106/ml) in the absence of 5 mmol/l EGTA or (b) in its presence. (c) PMA (100 ng/ml) is added (arrowhead) prior to the addition of the supernatant (arrow). Other experimental conditions are the same as described in (a). All tracings were obtained with cells from the same batch of fura-2-loaded PMNs.

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Neutrophil Caz+ response to activated platelets

1001

I I

20 40

Platelet equivalent/PMN

Fig. 5 . Dose response for the supernatant of thrombin-activated platelets. The supernatant prepared as described in Material and methods is diluted and added to PMNs ( 5 x 106/ml) at the concentration as indicated.

Characterization of platelet release products

To characterize the platelet release products that elicited the Ca" transient in PMNs, the supernatant of thrombin-activated platelets was pretreated with hexokinase and/or trypsin as described in Material and methods. When the supernatant was pretreated with hexokinase, the Ca2 + transient was depressed, although not abrogated (data not shown). This confirmed that ATP released from platelets induces a Ca2+ transient in PMNs as expected from previous reports (14, 18-20).

Pretreatment of the supernatant with trypsin decreased the magnitude of the Ca2+ transient (Ta- ble l), while incubation of the supernatant at 37°C without trypsin did not. The elevation of [ Ca2 + ]i induced by the supernatant preincubated with trypsin was significantly lower than the Ca2+ transient induced by that incubated at 37 O C without trypsin. When the soybean trypsin inhibitor was added at the same time as trypsin, the Ca2+ transient was not significantly affected (Table 1). In contrast, pretreatment of ATP (80 pmol/l) with trypsin had no influence on the Ca2+ transient triggered by ATP. Pretreatment of ATP with hexokinase abrogated the ATP-induced Ca2+ transient (data not shown). When the supernatant was pretreated

Table 1, Effect of various pretreatments of the supernatant of thrombin-activated platelets on the Ca2+ transient of PMNs

Pretreatment N [ca2+]i increase (nmolil)

Trypsin Buffer used (0.1 to %) dissolve trypsin

Trypsin (0.1 %) t soybean trypsin inhibitor

The results are expressed as the meankS.E.M. NS=not statistically different.

7 45.5k8.0 7 5 , 7 k 1 3 , 2 ] ;;<0.05

4 6 1 , l k 10.5

with both hexokinase and trypsin, an additive inhibition of the Ca2+ transient was observed (data not shown).

Discussion

Our results demonstrate that thrombin-activated platelets elicit a transient elevation of [ Ca2 + ]i in PMNs in v i m . The Ca2+ transient is completely dependent on extracellular calcium because it was abrogated with EGTA. Aggregation of platelets is not essential because the Ca2+ transient was not affected by the inhibition of platelet aggregation with RGDS. The magnitude of the Ca2+ transient is dependent on the concentration of platelets and thrombin.

The supernatant of the suspension of thrombin- activated platelets also induced a Ca2 + transient in PMNs. However, the Ca2+ transient elicited by the supernatant had shorter duration and was less dependent on extracellular calcium than that elicited by thrombin-activated platelets because it was not totally abrogated with EGTA. One possible explanation for the different requirement for extracellular calcium is that the release of the platelet release product(s) which trigger the Ca2+ transient is decreased in the absence of extracellular calcium. An- other explanation might be that extracellular calcium is necessary for a close platelet-to-PMN contact that allows the platelet release product(s) to concentrate around PMNs and to trigger the Ca" transient. Adhesion of platelets to PMNs mediated by CD62 (GMP- 140/PADGEM)on thrombin-activatedplate- lets and its carbohydrate ligands on PMNs is dependent on extracellular calcium (27, 28).

PMA abrogated the Ca2+ transient elicited by the supernatant of thrombin-activated platelets. PMA has been reported to inhibit the Ca2+ transient in PMNs elicited by formyl-methionyl-leucyl-phenylalanine (FMLP) (29, 30), leukotriene B4 (29), PAF (31), and ATP (19). PMA inhibits the formation by PMNs of inositol 1,4,5-triphosphate (IP,) (30), which releases Ca2 + from intracellular stores and stimulates, perhaps indirectly, Ca2 + influx through the plasma membrane (32). These studies suggest that Ca2+ transients elicited by the platelet supernatant and the agents described above are mediated at least in part by a common signal transduction mechanism, IP, formation.

We have confirmed that ATP released from platelets induces a Ca2+ transient in PMNs. However, the Ca2+ transient induced by the platelet supernatant was not abrogated by depletion of ATP with hexokinase. This indicates that the Ca2 + transient in PMNs evoked by platelets cannot be accounted for solely on the basis of ATP. Furthermore, pretreatment with trypsin inhibited the Ca2+ transient and

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pretreatment with both hexokinase and trypsin caused an additive inhibition. These findings suggest that there is at least another platelet release product which is sensitive to trypsin that induces or modu- lates the Ca2 + transient of PMNs.

Wright et al. reported a platelet release product with characters of a labile protein that primes PMNs for augmented FMLP-induced superoxide release (15). However, its effect on [Ca'+]i of PMNs was not shown in the study. Neutrophil-activating peptide-2, which could be generated from platelet basic protein and/or connective tissue-activating peptide I11 released from activated platelets, has been reported to induce a rise in [ Ca2+ ]i of PMNs (33). However, its sensitivity to trypsin was not described.

Further characterization of the platelet release products that can influence PMNs will provide us much information concerning the roles of platelet- PMN interactions at inflammatory sites such as in- fection, vasculitis, ischemia and adult respiratory distress syndrome (34, 35).

Acknowledgments

We thank Eiri Shibata for technical assistance; Junko Hirai and Akiko Kitagawa for secretarial assistance.

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