JOURNAL OF BIOLUMINESCENCE AND CHEMILUMINESCENCE VOL 5 31-36
(1990)
Magnesium-dependent Induction of Phagocytosis-associated Chemiluminescence of Adherent Human Polymorphonuclear Leukocytes by Non-opsonized Zymosan Paul Roschger,'* Wolfgang Graninger2 and Herbert Klirna' 'Atorninstitut der Oesterreichischen Universitaten, Schuttelstrasse 115, A-1020 Vienna, Austria 'Universitatsklinik fur Chemotherapie, Vienna, Austria
A severe dysfunction in the cellular response of human polymorphonuclear leukocytes (PMNL) t o non-opsonized zymosan was observed under a deficiency of extracellular Mg2+. The phagocytosis-association native (luminol-independent) luminescence (NL), as well as luminol-dependent luminescence (LDL) (detected simultaneously and discriminated by spectral methods), was strongely inhibited. Apart from a general decrease of total light production, a Mg2+-concentration-dependent delay of the maximum of NL and LDL was observed. A disorder in recruitment of activated membrane-bound NADPH-oxidase of PMNL is suggested. The presence of extracellular Ca2+ did not compensate for the Mg2+ deficit. In the presence of Mg2+ only a slight Ca2+-dependent reduction of NL was obtained, but Ca2+ seemed t o selectively promote LDL. This may indicate a positive influence of Ca2+ on the myeloperoxidase release from the cells. Experiments with the metalions-chelating agents EDTA and EGTA, which complex Mg2+ t o differing extents, confirmed the important role of Mg2+ in PMNL-activation by non-opsonized zyrnosan. Keywords: Luminol-dependent luminescence; native luminescence; polymorphonuclear leukocytes; Mg2+;Ca2+
INTRODUCTION Polymorphonuclear leukocytes (PMNL) have a mechanism for non-opsonic phagocytosis, i.e. a phagocytosis which is not mediated by serum components like C3b and IgG coating of the particles (Rodriguez-Ortega et al., 1987; Morikawa et al., 1986). Surface glycoproteins on *Author for correspondence
0884-3996/90/010031-06$05 .OO 0 1990 by John Wiley & Sons, Ltd
PMNL membranes were identified as receptors for phagocytosis. In the case of zymosan, a glucan of a defined chemical structure seems to be responsible for the ligand-receptor interaction on PMNL (Wilkinson, 1975). Divalent cations are important in the mechanisms of activation and execution of the cellular functions observed in PMNL, particularly Ca2+
32 which is involved in transmembrane signalling, and acts as an intracellular messenger and an effector molecule (Berridge and Irvine, 1984; Boxer et al., 1985; DeChatelet and Shirley, 1982; Hoffstein, 1979; Lew et al., 1985; Murata et al., 1987; Snyderman and Verghese, 1987). Mg2+ is known to play an important role in various ATP-dependent reactions, there are reports describing the requirement of extracellular Mg2+ for optimal function of PMNL in chemotaxis, adherence and phagocytosis (Stossel, 1973; Williams et d., 1986). In this study the dependence of extracellular contents of Ca2+ and Mg2+ on the cellular response of PMNL to non-opsonized zymosan was investigated. Phagocytosis-associated native (luminol-independent) luminescence (NL) and luminol-dependent luminescence (LDL) were evaluated simultaneously. Since NL is related to an 02-mediated reaction (Rosen and Klebanoff, 1976), it reflects the formation of OF, which is catalysed through an activation of a membranebound NADPH-dependent oxidoreductase by the process of phagocytosis (Goldstein et al., 1977). In contrast to NL, LDL seems to be linked to an MPO-mediated reaction (DeChatelet et al., 1982) and may therefore reflect the release of the granular enzyme, MPO.
MATERIALS AND METHODS Polymorphonuclear leukocytes
PMNL were prepared from fresh human blood. Blood was collected in citrate anticoagulant. Leukocyte-enriched plasma was obtained- by l g sedimentation in presence of 1% (w/v) dextran (M.W. 250,000). The residual erythrocytes were lysed by 0.83% NH4Cl solution at 0°C. The PMNL were separated by subsequent centrifugation on a density gradient (Lymphoprep, Nyegaard & Co. AIS, Norway) and suspended in phosphate-buffered-saline (PBS) without Ca2+ and Mg2+ (Dulbecco A , Oxoid Ltd, England) containing 10 mmol/l glucose, 0.05% bovine serum albumin (BSA) (Sigma Chemical Co.) and 16 nmol/l luminol (Lumanol-100, LUMAC System A G , Switzerland).
P. ROSCHGER, W. GRANINGER AND H. KLIMA
Zymosan and calcium and magnesium ions
Boiled and washed zymosan (Zymosan A from S. cerevisiae yeast, Sigma Chemical Co.) was used at a particle to cell ratio of 100:1 as stimulus without prior opsonization by serum treatment. For the addition of defined amounts of Ca2+ and Mg2+ to the reaction medium, a 24 mmol/l stock solution of CaC12 and of MgC12 in 0.9% NaCl were prepared from Ca2+ respectively Mg2+-standard solutions (Titrisol, E . Merck, FRG). For the experiments with the metal-ionchelating agents a 84 mmol/l stock solution of EDTA and of EGTA (Titriplex I11 and Titriplex VI, E . Merck, FRG) in 0.9% NaCl were made. Chemiluminescence measurement
The experiments were performed at 37°C with 5 x lo6 cells on culture dishes (50mm diameter, Petriperm, Heraeus Christ, FRG) enclosed in a light-proof, temperature-controlled specimen chamber. Luminescence was recorded by a special device developed for the detection of ultra-weak light emission including a cooled photomultiplier (Type R 562, Hamamatsu), a single-photon-counting device and a filter wheel. NL and LDL were detected simultaneously and discriminated by spectral methods. One measuring cycle consisted of two phases. During the first phase, the integral photon count rate was recorded without using any filter. In the second phase the spectral photon count rate of a selected wavelength range was determined by inserting a special filter. In the experiments presented a 550 nm cutoff filter (Schott, FRG) was employed. The contributions of the NL and the LDL in each measuring cycle were calculated from the resulting data as described by Roschger et al. (1984).
RESULTS AND DISCUSSION Ca2+ and Mg2+ concentrations
Only a weak light emission by human PMNL took place when they were incubated in Ca2+- and Mg2+-free medium and stimulated by nonopsonized zymosan (NZ) (Fig. l(a)). The light
INFLUENCE OF M g O N PHAGOCYTOSIS -ASSOCIATED
33
CHEMILUMINESCENCE
2000
f
a
NL
CPT
A
f \
I
--E
a)
LDL
1500
A
"-
'"'y
free
0)
c 3
-
1000
c
II
a
500
0
10
20
mn1
20
30
40
[mlnl
Figure 1. The time traces of native (NL) and of luminol-dependent (LDL) luminescence of human PMNL (5 x 10" cells) stimulated by non-opsonized zymosan (NZ). (a) in presence and in absence of extracellular Caz+ (1 mmol/l) and M g z + (1 mmolil), (b) when Ca2+ and Mg2+ (1 mmol/l final concentration for each) were added subsequently
emission was mainly due to NL, when Caz+ and Mg2+ was absent in the medium, whereas in presence of Ca2+ and Mg2+ (1 mmol/l each) the intensity of LDL exceeded much that of NL. Also differences of photon-emission kinetics between NL and LDL were observed. There was a clear delay of LDL maximum with respect to NL maximum. The subsequent decrease of light emission was much faster for LDL than that for NL. In experiments in which PMNL were incubated with N Z in Ca2+- and Mg2+-free medium followed by the addition of Ca2+ (1 mmol/l) alone, a weak additional activation with respect to light emission was detected (Fig. l(b)). However when Mg2+ (1 mmol/l) was added, then the luminescence response was as observed at physiological Ca2+ and Mg2+ concentrations (Fig. l(a)). This demonstrated the requirement of Mg2+ in the extracellular space in the case of activation of PMNL by non-opsonized zymosan. The dependence of this cellular response on Ca2+ and Mg2+ was studied in greater detail by varying Ca2+ and Mg2+ concentration in the range &1 mmol/l. When the Mg2+ concentration was kept constant at lmmol/l and the Ca2+ concentration was reduced stepwise to 0 mmol/l (Fig. 2), the NL response (Fig. 2(a)) showed a slight reduction of intensity but only in the range from 0.25 mmol/l to 0.0 mmoVl Ca", while no reduction of NL was observed between 1 mmol/l
and 0.25 mmol/l Ca2+. At 0 mmol/l Ca2+ the amount of NL within a reaction time of 30 minutes was reduced by 18%. In contrast to that LDL showed a much greater sensitivity to the deprivation of Ca2+ from the medium (Fig. 2(b)). It decreased about 50% at 0 mmol/l Ca2+. In the experiments (Fig. 3), in which the Ca2+ concentration was kept constant at lmmol/l and the Mg2+ concentration decreased stepwise (1, 0.25, 0.1, 0 mmol/l), a stepwise diminution of NL and LDL was observed. In the absence of a Mg2+ decrease of NL of about 60% and of LDL of about 92% and an alteration in the kinetics of cell activation was observed. Apart from general decrease of total luminescence, an Mg2+dependent delay of the NL and the LDL maximum was detected. The results of these experiments indicated a severe dysfunction of PMNL, generated by a deficiency of extracellular Mg2+. Because of the close relationship between the amount of 0;production and NL intensity (Rosen and Klebanoff, 1976), the delay and overall decrease of NL generation suggests a reduction in the speed of recruiting and in the amount of activated NADPH-oxidase on the plasma membrane, which in return is coupled to the process of phagocytosis. Extracellular Ca2+ was less important for this kind of activation (Fig. 2(b)). The possibility of Ca2+ recruitment from intracellular stores cannot be excluded (Hoffstein, 1979;
34
P. ROSCHGER, W. GRANINGER AND H. KLIMA
Figure 2. The influence of the addition of different amounts of Ca2' together with a constant amount of Mg2 (1 mmolil final concentration) on phagocytosis-associated luminescence of PMNL (5 x lo6 cells) Non-opsonized zymosan (NZ) was added to the cells 20 minutes before Ca". together with Mg2+ was added The indicated amounts of Ca2+ belongs to the final concentration in the reaction medium (a) native luminescence (NL) (b) Simultaneously detected luminol-dependent luminescence (LDL), reduced by a factor of 0 4 +
B
0 1
.
e
0.0
0.0
"
20
.B"%, .
30
40
mn1
20
30
40
Figure 3. The influence of the addition of different amounts of Mg2+,together with a constant amount of Ca2+ (1 mmol/l final concentration), on phagocytosis-associated luminescence of PMNL (5 x 1O6 cells) Non-opsonized zymosan (NZ) was added to the cells 20 minutes before Mg*+, together with Ca2+ as injected The indicated amounts of Mg'+ belongs to the final concentration in the reaction medium (a) Native luminescence (NL) (b) Simultaneously detected luminol-dependent luminescence [LDL), reduced by a factor of 0 4
Murata et al., 1987). It has still to be elucidated if extracellular Mg2' is required directly for this kind of stimulation or if it is only necessary to maintain a certain intracellular level of Mg2+. The observed differences between NL and LDL behaviour may be explained by a dissociation of 02-production and of the release of the granular enzyme MPO, because LDL is dependent on an MPO-mediated reaction. For that
reason the much greater sensitivity of LDL on Ca2+ concentration in the experiments shown in Fig. 2 , may indicate, that the enzyme release is promoted by the presence of extracellular Ca2+. EDTA and EGTA treatment
To confirm the results demonstrating the Mg*+dependence on phagocytosis-associated lumines-
INFLUENCE OF M g ON PHAGOCYTOSIS -ASSOCIATED
CHEMILUMINESCENCE
35
be expected. Fig. 4 shows the results of EDTA (0.7 mmol/l) and of EGTA (0.7 mmol/l) pretreatment of PMNL incubated together with N Z in Ca2+- and Mg2+-free medium. In both cases the NL was strongly compared to the control (Fig. 4(a)). A subsequent addition of Ca2+ (1.5 mmoli 1) together with Mg2+ (1.5 mmol/l) did not restore normal NL response in the case of EDTA treatment, whereas for the EGTA NL it achieved
cence, experiments were performed with the metal-ion-chelating agents, EDTA and EGTA. Both agents have similar stability constants (log K values) to form Ca2+ complexes, EDTA 10.7 and EGTA 11, but they are quite different for the formation of Mg2+ complexes, EDTA with a log K for Mg2+ of 8.7 and EGTA only of 5.2 (West, 1969). Therefore different influences of these agents on the cellular response to zymosan could
Fiaure 4. The influence of Dretreatment of the PMNL with the metal-ion-chelatina aaents. EDTA ( 0 7 rnrnol/ll on pt&ocytosis-associated luminescence Non-opsonized zyrnosan was added toget h& w;th EDTA. respectively EGTA (0 7 rnmolll final concentration). 20 minutes before Ca2+and Mg2+ (each 1 5 rnmolll final concentration) were injected In the case of 'controls' no EDTA or EGTA pretreatment occurred and the amount of injected Ca2+ and Mg2+ was 1 rnmol/l (final concentration) for each (a) Native luminescence (NL) (b) Simultaneously detected lurninol-dependent luminescence (LDL) reduced by a factor of 0 4
-a
800 NL :
A
i
I
--m
2
a)
600
a,
EDTA
LDL'
b)
A
EDTA
x
B
I
EGTA
c 3
-
400
0 c
c a
)a+Mg
200
t\
d
Ca+Mg
\ 0
20
30
40
[min]
Figure 5. The effects of t h e metal-ion-chelating agents, EDTA and EGTA (each 2 rnrnolll final concentration), on phagocytosis-associated luminescence of PMNL (5 x 10% when EDTA or EGTA was added during the cellular response to non-opsonized ryrnosan in presence of Ca2' and Mg2+ (each 1 rnrnoVI final concentration) (a) Native luminescence (NL) (b) Simultaneously detected luminol-dependent luminescence (LDL) reduced by a factor of 0 4
P. ROSCHGER, W. GRANINGER AND H. KLIMA
36
control level again (Fig. 4(a)). This effect is in Goldstein, I. M., Cerqueira, M., Lind, S. and Kaplan, H. B. (1977). Evidence that the superoxide-generating system of accordance with the above-observed dependency human leukocytes is associated with the cell surface. J . on availability of Mg2+ for normal cellular Clin. Invest., 59, 249-245. response. Green, T. R., Wu, D . E . and Wirtz, M. K. (1983). The 0;-generating oxidoreductase of human neutrophils: The influence of deprivation of free Ca2+ Evidence of an obligatory requirement for calcium and (1mmol/l) and Mg2+ (1mmol/l) during the magnesium for expression of catalytic activity. Biochem. cellular response to zymosan upon cell activity Biophys. Res. Commun., 110, 973-978. was tested by adding ETDA (2 mmol/l) or EGTA Hoffstein, S. T. (1979). Ultrastructural demonstration of calcium loss from local regions of the plasma membrane of (2mmol/l) 15 minutes after Ca2+ and Mg2+ surface stimulated human granulocytes. J . Immunol., 123, addition (Fig. 5 ) . In the case of EDTA an immediate decrease (65%) of NL within 10 Law,1359-1402. D. P., A n d e r s o n , T . , Di Virgilio, F., Pozann, T. and minutes was observed. In contrast, EGTA Stendahl, 0. (1985). Caz+-dependent and Cazfaddition caused only a slight depression of NL independent phagocytosis in human neutrophils. Nature, 315, 50P-511. (29%), which exhibited a delay of about 4 minutes (Fig. 5(a)). The LDL responses to the Morikawa, K., Noguchi, T., Yamazaki, M. and Mizuno, D. (1986). Calcium-dependent and -independent tumoricidal addition of both EDTA and EGTA were activities of polyrnorphonuclear leukocytes induced by a identical. The LDL decreased to 68% of the linear P-l,3-~-glucan and phorbol myristrate acetate in mice. Cancer Res., 46, 6670. control within 10 minutes (Fig. 5(b)). These results may indicate that the availability of Mg2+ Murata, T., Sullivan, J . A , , Sawyer, D. W. and Mandell, G . L. (1987). Influence of type and opsonization on ingested also has an influence on the activity of the particle on intracellular free calcium distribution and NADPH-oxidase. This assumption is confirmed superoxide production by human neutrophils. Infect. by experiments of Green et al., (1983) who Immun., 55, 17841791. worked with a cell-free preparation of NADPH- Rodriguez,-Ortega, M . , Ofek, I. and Sharon, N. (1987). Membrane glycoproteins of Human Polymorphonuclear oxidase.
Acknowledgement This work was supported by the ‘Fonds zur Foerderung der wissenschaftlichen Forschung P 5878 M’.
REFERENCES Berridge, M. J. and Irvine, R . F. (1984). Inositol trisphosphate, a novel second messenger in cellular signal transduction. Nature, 312, 315-321. Boxer, G. J . , Curnutte, J . T. and Boxer, L. A . (1985). Polymorphonuclear leukocyte function. Hosp. Pract., 15 March, 69-90. DeChatelet, L. R. and Shirley, P. S. (1982). Chemiluminescence of human neutrophils induced by soluble stimuli. Infect. Immun., 25, 206212. DeChatelet, L. R., Long, G . D . , Shirley, P. S . , Bass, D . A . , Thomas, M. J., Henderson, F. W . and Cohen, M. S. (1982). Mechanism of the luminol-dependent chemiluminescence of human neutrophils. J . Immunol., 129, 1589-1593.
leukocytes that act as receptors for mannose-specific Escherichia coli. Infect. Immun., 55, 968-973. Roschger, P., Graninger, W. and Klima, H . (1984). Simultaneous detection of native and luminol-dependent luminescence of stimulated human polymorphonuclear leukocytes. Biochem. Biophys. Res. Commun., 123, 1047-1053. Rosen, H. and Klebanoff, S. J. (1976). Chemiluminescence and superoxide production by myelperoxidase-deficient leukocytes. J . Clin. Invest., 58, 50-60. Snyderman, R . and Verghese, M. W. (1987). Leukocyte activation by chemoattractant receptors: roles of a guanine nucleotide regulatory protein and polyphosphoinositide metabolism. Rev. Infect. Dis., 9, Supp. 5 , S5624568. Stossel, T. P. (1973). Quantitative studies of phagocytosis: kinetic effects of cations and heat-labile opsonin. J . Cell Biol., 58, 346356. West, T. S. (1969) Complexometry with E D T A and related reagents. B D H Chemicals Ltd, Poole. Wilkinson, P. C. (1975) Leukocyte locomotion and chemotaxis: the influence of divalent cations and cation ionophores. Exp. Cell Res., 93, 42&426. Williams, J. D., Topley, H. M., Alobaidi, H. M. and Harber, M. J. (1986) Activation of human polymorphonuclear leukocytes by particulate zymosan is related to both its major carbohydrate components: glucan and mannan. Immunology, 58, 117-124.
(1990)
Magnesium-dependent Induction of Phagocytosis-associated Chemiluminescence of Adherent Human Polymorphonuclear Leukocytes by Non-opsonized Zymosan Paul Roschger,'* Wolfgang Graninger2 and Herbert Klirna' 'Atorninstitut der Oesterreichischen Universitaten, Schuttelstrasse 115, A-1020 Vienna, Austria 'Universitatsklinik fur Chemotherapie, Vienna, Austria
A severe dysfunction in the cellular response of human polymorphonuclear leukocytes (PMNL) t o non-opsonized zymosan was observed under a deficiency of extracellular Mg2+. The phagocytosis-association native (luminol-independent) luminescence (NL), as well as luminol-dependent luminescence (LDL) (detected simultaneously and discriminated by spectral methods), was strongely inhibited. Apart from a general decrease of total light production, a Mg2+-concentration-dependent delay of the maximum of NL and LDL was observed. A disorder in recruitment of activated membrane-bound NADPH-oxidase of PMNL is suggested. The presence of extracellular Ca2+ did not compensate for the Mg2+ deficit. In the presence of Mg2+ only a slight Ca2+-dependent reduction of NL was obtained, but Ca2+ seemed t o selectively promote LDL. This may indicate a positive influence of Ca2+ on the myeloperoxidase release from the cells. Experiments with the metalions-chelating agents EDTA and EGTA, which complex Mg2+ t o differing extents, confirmed the important role of Mg2+ in PMNL-activation by non-opsonized zyrnosan. Keywords: Luminol-dependent luminescence; native luminescence; polymorphonuclear leukocytes; Mg2+;Ca2+
INTRODUCTION Polymorphonuclear leukocytes (PMNL) have a mechanism for non-opsonic phagocytosis, i.e. a phagocytosis which is not mediated by serum components like C3b and IgG coating of the particles (Rodriguez-Ortega et al., 1987; Morikawa et al., 1986). Surface glycoproteins on *Author for correspondence
0884-3996/90/010031-06$05 .OO 0 1990 by John Wiley & Sons, Ltd
PMNL membranes were identified as receptors for phagocytosis. In the case of zymosan, a glucan of a defined chemical structure seems to be responsible for the ligand-receptor interaction on PMNL (Wilkinson, 1975). Divalent cations are important in the mechanisms of activation and execution of the cellular functions observed in PMNL, particularly Ca2+
32 which is involved in transmembrane signalling, and acts as an intracellular messenger and an effector molecule (Berridge and Irvine, 1984; Boxer et al., 1985; DeChatelet and Shirley, 1982; Hoffstein, 1979; Lew et al., 1985; Murata et al., 1987; Snyderman and Verghese, 1987). Mg2+ is known to play an important role in various ATP-dependent reactions, there are reports describing the requirement of extracellular Mg2+ for optimal function of PMNL in chemotaxis, adherence and phagocytosis (Stossel, 1973; Williams et d., 1986). In this study the dependence of extracellular contents of Ca2+ and Mg2+ on the cellular response of PMNL to non-opsonized zymosan was investigated. Phagocytosis-associated native (luminol-independent) luminescence (NL) and luminol-dependent luminescence (LDL) were evaluated simultaneously. Since NL is related to an 02-mediated reaction (Rosen and Klebanoff, 1976), it reflects the formation of OF, which is catalysed through an activation of a membranebound NADPH-dependent oxidoreductase by the process of phagocytosis (Goldstein et al., 1977). In contrast to NL, LDL seems to be linked to an MPO-mediated reaction (DeChatelet et al., 1982) and may therefore reflect the release of the granular enzyme, MPO.
MATERIALS AND METHODS Polymorphonuclear leukocytes
PMNL were prepared from fresh human blood. Blood was collected in citrate anticoagulant. Leukocyte-enriched plasma was obtained- by l g sedimentation in presence of 1% (w/v) dextran (M.W. 250,000). The residual erythrocytes were lysed by 0.83% NH4Cl solution at 0°C. The PMNL were separated by subsequent centrifugation on a density gradient (Lymphoprep, Nyegaard & Co. AIS, Norway) and suspended in phosphate-buffered-saline (PBS) without Ca2+ and Mg2+ (Dulbecco A , Oxoid Ltd, England) containing 10 mmol/l glucose, 0.05% bovine serum albumin (BSA) (Sigma Chemical Co.) and 16 nmol/l luminol (Lumanol-100, LUMAC System A G , Switzerland).
P. ROSCHGER, W. GRANINGER AND H. KLIMA
Zymosan and calcium and magnesium ions
Boiled and washed zymosan (Zymosan A from S. cerevisiae yeast, Sigma Chemical Co.) was used at a particle to cell ratio of 100:1 as stimulus without prior opsonization by serum treatment. For the addition of defined amounts of Ca2+ and Mg2+ to the reaction medium, a 24 mmol/l stock solution of CaC12 and of MgC12 in 0.9% NaCl were prepared from Ca2+ respectively Mg2+-standard solutions (Titrisol, E . Merck, FRG). For the experiments with the metal-ionchelating agents a 84 mmol/l stock solution of EDTA and of EGTA (Titriplex I11 and Titriplex VI, E . Merck, FRG) in 0.9% NaCl were made. Chemiluminescence measurement
The experiments were performed at 37°C with 5 x lo6 cells on culture dishes (50mm diameter, Petriperm, Heraeus Christ, FRG) enclosed in a light-proof, temperature-controlled specimen chamber. Luminescence was recorded by a special device developed for the detection of ultra-weak light emission including a cooled photomultiplier (Type R 562, Hamamatsu), a single-photon-counting device and a filter wheel. NL and LDL were detected simultaneously and discriminated by spectral methods. One measuring cycle consisted of two phases. During the first phase, the integral photon count rate was recorded without using any filter. In the second phase the spectral photon count rate of a selected wavelength range was determined by inserting a special filter. In the experiments presented a 550 nm cutoff filter (Schott, FRG) was employed. The contributions of the NL and the LDL in each measuring cycle were calculated from the resulting data as described by Roschger et al. (1984).
RESULTS AND DISCUSSION Ca2+ and Mg2+ concentrations
Only a weak light emission by human PMNL took place when they were incubated in Ca2+- and Mg2+-free medium and stimulated by nonopsonized zymosan (NZ) (Fig. l(a)). The light
INFLUENCE OF M g O N PHAGOCYTOSIS -ASSOCIATED
33
CHEMILUMINESCENCE
2000
f
a
NL
CPT
A
f \
I
--E
a)
LDL
1500
A
"-
'"'y
free
0)
c 3
-
1000
c
II
a
500
0
10
20
mn1
20
30
40
[mlnl
Figure 1. The time traces of native (NL) and of luminol-dependent (LDL) luminescence of human PMNL (5 x 10" cells) stimulated by non-opsonized zymosan (NZ). (a) in presence and in absence of extracellular Caz+ (1 mmol/l) and M g z + (1 mmolil), (b) when Ca2+ and Mg2+ (1 mmol/l final concentration for each) were added subsequently
emission was mainly due to NL, when Caz+ and Mg2+ was absent in the medium, whereas in presence of Ca2+ and Mg2+ (1 mmol/l each) the intensity of LDL exceeded much that of NL. Also differences of photon-emission kinetics between NL and LDL were observed. There was a clear delay of LDL maximum with respect to NL maximum. The subsequent decrease of light emission was much faster for LDL than that for NL. In experiments in which PMNL were incubated with N Z in Ca2+- and Mg2+-free medium followed by the addition of Ca2+ (1 mmol/l) alone, a weak additional activation with respect to light emission was detected (Fig. l(b)). However when Mg2+ (1 mmol/l) was added, then the luminescence response was as observed at physiological Ca2+ and Mg2+ concentrations (Fig. l(a)). This demonstrated the requirement of Mg2+ in the extracellular space in the case of activation of PMNL by non-opsonized zymosan. The dependence of this cellular response on Ca2+ and Mg2+ was studied in greater detail by varying Ca2+ and Mg2+ concentration in the range &1 mmol/l. When the Mg2+ concentration was kept constant at lmmol/l and the Ca2+ concentration was reduced stepwise to 0 mmol/l (Fig. 2), the NL response (Fig. 2(a)) showed a slight reduction of intensity but only in the range from 0.25 mmol/l to 0.0 mmoVl Ca", while no reduction of NL was observed between 1 mmol/l
and 0.25 mmol/l Ca2+. At 0 mmol/l Ca2+ the amount of NL within a reaction time of 30 minutes was reduced by 18%. In contrast to that LDL showed a much greater sensitivity to the deprivation of Ca2+ from the medium (Fig. 2(b)). It decreased about 50% at 0 mmol/l Ca2+. In the experiments (Fig. 3), in which the Ca2+ concentration was kept constant at lmmol/l and the Mg2+ concentration decreased stepwise (1, 0.25, 0.1, 0 mmol/l), a stepwise diminution of NL and LDL was observed. In the absence of a Mg2+ decrease of NL of about 60% and of LDL of about 92% and an alteration in the kinetics of cell activation was observed. Apart from general decrease of total luminescence, an Mg2+dependent delay of the NL and the LDL maximum was detected. The results of these experiments indicated a severe dysfunction of PMNL, generated by a deficiency of extracellular Mg2+. Because of the close relationship between the amount of 0;production and NL intensity (Rosen and Klebanoff, 1976), the delay and overall decrease of NL generation suggests a reduction in the speed of recruiting and in the amount of activated NADPH-oxidase on the plasma membrane, which in return is coupled to the process of phagocytosis. Extracellular Ca2+ was less important for this kind of activation (Fig. 2(b)). The possibility of Ca2+ recruitment from intracellular stores cannot be excluded (Hoffstein, 1979;
34
P. ROSCHGER, W. GRANINGER AND H. KLIMA
Figure 2. The influence of the addition of different amounts of Ca2' together with a constant amount of Mg2 (1 mmolil final concentration) on phagocytosis-associated luminescence of PMNL (5 x lo6 cells) Non-opsonized zymosan (NZ) was added to the cells 20 minutes before Ca". together with Mg2+ was added The indicated amounts of Ca2+ belongs to the final concentration in the reaction medium (a) native luminescence (NL) (b) Simultaneously detected luminol-dependent luminescence (LDL), reduced by a factor of 0 4 +
B
0 1
.
e
0.0
0.0
"
20
.B"%, .
30
40
mn1
20
30
40
Figure 3. The influence of the addition of different amounts of Mg2+,together with a constant amount of Ca2+ (1 mmol/l final concentration), on phagocytosis-associated luminescence of PMNL (5 x 1O6 cells) Non-opsonized zymosan (NZ) was added to the cells 20 minutes before Mg*+, together with Ca2+ as injected The indicated amounts of Mg'+ belongs to the final concentration in the reaction medium (a) Native luminescence (NL) (b) Simultaneously detected luminol-dependent luminescence [LDL), reduced by a factor of 0 4
Murata et al., 1987). It has still to be elucidated if extracellular Mg2' is required directly for this kind of stimulation or if it is only necessary to maintain a certain intracellular level of Mg2+. The observed differences between NL and LDL behaviour may be explained by a dissociation of 02-production and of the release of the granular enzyme MPO, because LDL is dependent on an MPO-mediated reaction. For that
reason the much greater sensitivity of LDL on Ca2+ concentration in the experiments shown in Fig. 2 , may indicate, that the enzyme release is promoted by the presence of extracellular Ca2+. EDTA and EGTA treatment
To confirm the results demonstrating the Mg*+dependence on phagocytosis-associated lumines-
INFLUENCE OF M g ON PHAGOCYTOSIS -ASSOCIATED
CHEMILUMINESCENCE
35
be expected. Fig. 4 shows the results of EDTA (0.7 mmol/l) and of EGTA (0.7 mmol/l) pretreatment of PMNL incubated together with N Z in Ca2+- and Mg2+-free medium. In both cases the NL was strongly compared to the control (Fig. 4(a)). A subsequent addition of Ca2+ (1.5 mmoli 1) together with Mg2+ (1.5 mmol/l) did not restore normal NL response in the case of EDTA treatment, whereas for the EGTA NL it achieved
cence, experiments were performed with the metal-ion-chelating agents, EDTA and EGTA. Both agents have similar stability constants (log K values) to form Ca2+ complexes, EDTA 10.7 and EGTA 11, but they are quite different for the formation of Mg2+ complexes, EDTA with a log K for Mg2+ of 8.7 and EGTA only of 5.2 (West, 1969). Therefore different influences of these agents on the cellular response to zymosan could
Fiaure 4. The influence of Dretreatment of the PMNL with the metal-ion-chelatina aaents. EDTA ( 0 7 rnrnol/ll on pt&ocytosis-associated luminescence Non-opsonized zyrnosan was added toget h& w;th EDTA. respectively EGTA (0 7 rnmolll final concentration). 20 minutes before Ca2+and Mg2+ (each 1 5 rnmolll final concentration) were injected In the case of 'controls' no EDTA or EGTA pretreatment occurred and the amount of injected Ca2+ and Mg2+ was 1 rnmol/l (final concentration) for each (a) Native luminescence (NL) (b) Simultaneously detected lurninol-dependent luminescence (LDL) reduced by a factor of 0 4
-a
800 NL :
A
i
I
--m
2
a)
600
a,
EDTA
LDL'
b)
A
EDTA
x
B
I
EGTA
c 3
-
400
0 c
c a
)a+Mg
200
t\
d
Ca+Mg
\ 0
20
30
40
[min]
Figure 5. The effects of t h e metal-ion-chelating agents, EDTA and EGTA (each 2 rnrnolll final concentration), on phagocytosis-associated luminescence of PMNL (5 x 10% when EDTA or EGTA was added during the cellular response to non-opsonized ryrnosan in presence of Ca2' and Mg2+ (each 1 rnrnoVI final concentration) (a) Native luminescence (NL) (b) Simultaneously detected luminol-dependent luminescence (LDL) reduced by a factor of 0 4
P. ROSCHGER, W. GRANINGER AND H. KLIMA
36
control level again (Fig. 4(a)). This effect is in Goldstein, I. M., Cerqueira, M., Lind, S. and Kaplan, H. B. (1977). Evidence that the superoxide-generating system of accordance with the above-observed dependency human leukocytes is associated with the cell surface. J . on availability of Mg2+ for normal cellular Clin. Invest., 59, 249-245. response. Green, T. R., Wu, D . E . and Wirtz, M. K. (1983). The 0;-generating oxidoreductase of human neutrophils: The influence of deprivation of free Ca2+ Evidence of an obligatory requirement for calcium and (1mmol/l) and Mg2+ (1mmol/l) during the magnesium for expression of catalytic activity. Biochem. cellular response to zymosan upon cell activity Biophys. Res. Commun., 110, 973-978. was tested by adding ETDA (2 mmol/l) or EGTA Hoffstein, S. T. (1979). Ultrastructural demonstration of calcium loss from local regions of the plasma membrane of (2mmol/l) 15 minutes after Ca2+ and Mg2+ surface stimulated human granulocytes. J . Immunol., 123, addition (Fig. 5 ) . In the case of EDTA an immediate decrease (65%) of NL within 10 Law,1359-1402. D. P., A n d e r s o n , T . , Di Virgilio, F., Pozann, T. and minutes was observed. In contrast, EGTA Stendahl, 0. (1985). Caz+-dependent and Cazfaddition caused only a slight depression of NL independent phagocytosis in human neutrophils. Nature, 315, 50P-511. (29%), which exhibited a delay of about 4 minutes (Fig. 5(a)). The LDL responses to the Morikawa, K., Noguchi, T., Yamazaki, M. and Mizuno, D. (1986). Calcium-dependent and -independent tumoricidal addition of both EDTA and EGTA were activities of polyrnorphonuclear leukocytes induced by a identical. The LDL decreased to 68% of the linear P-l,3-~-glucan and phorbol myristrate acetate in mice. Cancer Res., 46, 6670. control within 10 minutes (Fig. 5(b)). These results may indicate that the availability of Mg2+ Murata, T., Sullivan, J . A , , Sawyer, D. W. and Mandell, G . L. (1987). Influence of type and opsonization on ingested also has an influence on the activity of the particle on intracellular free calcium distribution and NADPH-oxidase. This assumption is confirmed superoxide production by human neutrophils. Infect. by experiments of Green et al., (1983) who Immun., 55, 17841791. worked with a cell-free preparation of NADPH- Rodriguez,-Ortega, M . , Ofek, I. and Sharon, N. (1987). Membrane glycoproteins of Human Polymorphonuclear oxidase.
Acknowledgement This work was supported by the ‘Fonds zur Foerderung der wissenschaftlichen Forschung P 5878 M’.
REFERENCES Berridge, M. J. and Irvine, R . F. (1984). Inositol trisphosphate, a novel second messenger in cellular signal transduction. Nature, 312, 315-321. Boxer, G. J . , Curnutte, J . T. and Boxer, L. A . (1985). Polymorphonuclear leukocyte function. Hosp. Pract., 15 March, 69-90. DeChatelet, L. R. and Shirley, P. S. (1982). Chemiluminescence of human neutrophils induced by soluble stimuli. Infect. Immun., 25, 206212. DeChatelet, L. R., Long, G . D . , Shirley, P. S . , Bass, D . A . , Thomas, M. J., Henderson, F. W . and Cohen, M. S. (1982). Mechanism of the luminol-dependent chemiluminescence of human neutrophils. J . Immunol., 129, 1589-1593.
leukocytes that act as receptors for mannose-specific Escherichia coli. Infect. Immun., 55, 968-973. Roschger, P., Graninger, W. and Klima, H . (1984). Simultaneous detection of native and luminol-dependent luminescence of stimulated human polymorphonuclear leukocytes. Biochem. Biophys. Res. Commun., 123, 1047-1053. Rosen, H. and Klebanoff, S. J. (1976). Chemiluminescence and superoxide production by myelperoxidase-deficient leukocytes. J . Clin. Invest., 58, 50-60. Snyderman, R . and Verghese, M. W. (1987). Leukocyte activation by chemoattractant receptors: roles of a guanine nucleotide regulatory protein and polyphosphoinositide metabolism. Rev. Infect. Dis., 9, Supp. 5 , S5624568. Stossel, T. P. (1973). Quantitative studies of phagocytosis: kinetic effects of cations and heat-labile opsonin. J . Cell Biol., 58, 346356. West, T. S. (1969) Complexometry with E D T A and related reagents. B D H Chemicals Ltd, Poole. Wilkinson, P. C. (1975) Leukocyte locomotion and chemotaxis: the influence of divalent cations and cation ionophores. Exp. Cell Res., 93, 42&426. Williams, J. D., Topley, H. M., Alobaidi, H. M. and Harber, M. J. (1986) Activation of human polymorphonuclear leukocytes by particulate zymosan is related to both its major carbohydrate components: glucan and mannan. Immunology, 58, 117-124.
