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In vitro assessment of phagocytosis: Interspecies comparison of chemiluminescence response
Toxic. in Vitro Vol. 7, No. 4, pp. 317-320, 1993 Printed in Great Britain.All rights reserved
0887-2333/93 $6.00+ 0.00 Copyright© 1993Pergamon Press Ltd
I N VITRO ASSESSMENT OF PHAGOCYTOSIS: INTERSPECIES COMPARISON OF CHEMILUMINESCENCE RESPONSE F. VERDIER*t,F. CONDEVAUX*,R. TEDONEt, M. VIRAT* and J. DESCOTESt *Pharmakon Europe, BP 0118, 69593 L'Arbresle Cedex and tLaboratoire d'Immunotoxicologie Fondamentale et Clinique, INSERM U80, Facult6 de M6decine A. Carrel, 69008 Lyon, France Abstraet--Phagocytosis is a major component of the host's defences against pathogens. Particulate or soluble stimuli trigger the intracellular respiratory burst in activated phagocytes which can be measured in vitro by the luminol-enhanced chemiluminescent response. In this study, the phagocytic capacity of peripheral blood leucocytes was assayed using chemiluminescence and preliminary in vitro activation by phorbol myristate acetate, opsonized zymosan or latex beads. Leucocytes from rats, mini-pigs, dogs, monkeys and humans were preincubated for 2 hr with either doxycycline (0, 2 and 50 #g/ml) (n = 5) or lead acetate (0, 2 and 20 pg/ml) (n = 5), both compounds impairing phagocytosis. Whatever the species and the activator used, a similar dose-dependent decrease in chemiluminescence response was observed with either doxycycline or lead acetate, showing that the results can be extrapolated between species and to humans. The chemiluminescence assay is proposed as a tool for assessment of the safety of drugs and chemicals.
Introduction
Materials and Methods
The immune system is a potential target organ for the toxicity of xenobiotics. Non-specific immunity plays a major part in the host's defence mechanisms against infections and tumours; indeed, impairment of non-specific immune function is associated with serious health problems (Peterson, 1984). Phagocytosis is the primary non-specific defence mechanism against pathogens resulting in the activation of phagocytes: the respiratory oxidative burst (Sbarra and Karnofsky, 1959) is a series of changes in the oxidative metabolism of phagocytes, including increased production of hydrogen peroxide (H202), production of superoxide anion (O~-) and generation of photons or chemiluminescence (CL). The emission of CL by phagocytes is associated with the initial biochemical mechanisms of uptake, killing and digestion of micro-organisms. Measurement of the CL response has been proposed as a means of assessment of the phagocytic capacity of polymorphonuclear neutrophils and monocytes (Descamps-Latscha et al., 1982; Steele, 1991). Various stimuli, either particulate or soluble, can be used to activate phagocytes. The aim of this study was to compare the CL response of peripheral blood leucocytes from five different species (humans, monkey, dog, mini-pig and rat) using three different stimuli, namely opsonized zymosan, latex beads and phorbol myristate acetate (PMA), and following incubation with doxycycline and lead acetate,
Reagents. Zymosan, PMA, latex beads (0.83/tm diameter) and luminol (5-amino-2,3-dihydro-l,4phthalazinedione) were obtained from Sigma (St Louis, MO, USA). Phosphate buffered saline (PBS) was used as the cell medium. The red blood cell lysing solution was prepared with 8.23 g NH4CI, 37mg ethylenediaminetetraacetic acid and 1 g KHCO3 in 100ml sterile water. Doxycycline and lead acetate (supplied by Sigma) were suspended in saline. Blood sampling. Heparinized blood samples (5 ml) were collected from healthy human donors, purposebred male cynomolgus (Macaca fascicularis) monkeys (Charles River, St-Aubin-16s-Elbeuf, France), male beagle dogs (Hazleton RP, Cumberland, VA, USA), Yucatan and G6ttingen mini-pigs (Charles River and Genipig, Marseille, France, respectively) and male Sprague--Dawley rats ( I F F A CREDO, l'Arbresle, France). Animals were housed in airconditioned facilities with a 12-hr light/dark cycle. Unanaesthetized animals [with the exception of rats (ether anaesthesia)] were sampled between 8.00 and 9.00 a.m. Cell preparation. Red blood cells were lysed twice by the addition of 10 ml of the lysing solution to each blood sample. White blood cells were washed twice and resuspended in 5 ml PBS. Polymorphonuclear neutrophils and monocytes were counted. In vitro treatment. White blood cells were incubated for 2 h r at 37°C with either 0, 2 or 50/tg doxycycline/ml or 0, 2 or 20/~g lead acetate/ml. The viability of treated cells, assessed by the trypan blue exclusion method, was more than 90% at the end of the incubation period.
Abbreviations: CL = chemiluminescence; PBS = phosphate
buffered saline; PMA = phorbol myristate acetate,
317
318
F. VERDIERet al.
Chemiluminescence assay. Aliquots of P M A suspended in 1 0 - 2 m o l dimethyl sulphoxide/litre were diluted 100 times in PBS before use. Opsonized zymosan was prepared by 2 hr incubation of l0 mg zymosan with 750/~l autologous plasma mixed with 250/al PBS at 37°C and centrifugation for 5 min at 1800rpm. The final pellet was washed twice and resuspended in 1 ml PBS. Latex beads were diluted two-fold (1:2) in PBS before use (solid content, 5%). The C L response was measured in a LKB 1251 luminometer (Wailac, Finland) immediately after mixing 2 x l0 5 M-luminol, 500/~l of the cell suspension, 100/zl of the stimulus solution and 200/~1 PBS in 4 ml polystyrene cuvettes. The reaction was recorded every 30see for at least 15min at 37°C with continuous mixing. The peak values (mV) per thousand phagocytes/mm a and the time of the peak were noted. Statistical analysis. Results were compared using the non-parametric Friedman test for multiple group comparisons and Wilcoxon test for pairwise comparisons, Results
Baseline CL response Whatever the species used, the peak values and the kinetics of the CL response were similarly dependent on the stimulating agent, but showed a different trend (Table 1). Peak values ranked zymosan > latex beads > P M A , whereas the time of the peak ranked latex beads > zymosan > PMA. Effects o f doxycycline Whatever the species used, a dose-dependent decrease in the C L reponse was induced by doxycycline with the three different stimuli. However, only the higher dose of doxycycline induced a consistent statistically significant decrease in peak values (Fig. 1). Similarly, a dose-dependent delay in the CL response was noted (data not shown). Effects o f lead acetate With the exception of mini-pigs (zymosan stimulus) and dogs (latex beads), the higher dose of lead acetate induced a statistically significant decrease in peak values (Fig. 1). However, no clear dosedependent trend was noted,
Discussion
A variety of processes are known to be involved in the emission of light by activated phagocytes. Opsonized zymosan has been shown to interact with the phagocyte membrane by means of complement receptors (i.e. CRI, CR3) and glucan- and mannanspecific receptors (Lindena and Burkhardt, 1987; Williams et al., 1986) whereas latex beads develop hydrophobic interactions with the cell surface (Nguyen et al., 1983). By contrast, the soluble stimulus P M A results in the direct activation of the respiratory burst by protein kinase C stimulation (Babior, 1984; Johansson and Dahlgren, 1989). Whatever the initial signal for phagocyte activation, the production of free radicals, oxidative metabolites (e.g. 0 2 and H202) and photons is a c o m m o n endpoint. The whole blood technique allowed the use of smaller samples than are required for density gradient separation. The combination of red blood cell lysis and centrifugation was used to avoid inhibition of light emission by plasma and red blood cells (Descamps-Latscha et al., 1982; Faden and Maciejewski, 1989). In addition, the use of ammonium chloride as a lysing solution has been shown to optimize the C L response (Halstensen, 1986). In all the species studied, the curve profiles of CL response were similar, but were clearly stimulusdependent, with latex beads inducing a quicker peak response. As previously observed by Lindena and Burkhardt 0988), the CL response of rats was lower. In addition, inter-animal variations were greater in rats than in the other species studied. Nevertheless, this study provided evidence that the CL responses can be readily investigated in all five species and gave similar results. This in vitro CL assay was validated by testing two compounds known to impair phagocyte runetion. The influence of antimicrobial agents on the C L response has been extensively studied in vitro (Bribeim and Dahlgren, 1987; Dumas et al., 1990; Duncker and Ullmann, 1986) and doxycycline was shown to inhibit the CL response of human granulocytes at concentrations above or equal to 50 mg/litre (Duncker and Ullmann, 1986). The study reported here confirmed these results. In addition, a doserelated trend was observed in all species and with all stimuli. The results obtained with lead acetate
Table I. Baseline chemiluminescencemeasurement of white blood cells from various species in response to particulate and soluble stimuli Species Stimuli Human Monkey Dog Mini-pig Rat Zymosan P* 2424 + 232 733 + 81 1667 + 343 353 + 90 379 + 131 (1 mg/ml) T 324 + 34 132 + 7 300 + 9 192 + 30 102 ± 7 Latex beads P 1403 +_110 602 4- 55 883 _+211 291 + 59 134 _+54 (0.5% solid content) T 174 + 29 80 + 8 30 -+0 138 + 43 42 +_7 PMA P 1170_+ 119 255+26 544+89 96±28 91 _+30 (10 nmol/ml) T 480 + 9 204 _+17 486 + 104 282 _+7 138 _+ 12 *Peak values (P) of the chemiluminescenceresponse in mV/103/mm3phagocytes (polymorphonuclear neutrophils and monocytes) and times of the peak (T) in seconds for 10 samples/stimulus/species(mean _4-SE).
Chemiluminescence assessment of phagocytosis %
%
DOXYCYCLINE
120 . Z
.
.
100
80
80
60
60
A
40
N
20
* *
.
120. 100:
E
80,
X
40
E A
20
D
0
S
MO
DOG
M-P
RAT
I HU
I 1,40
I DOG
I M-P
" RAT
120 T
l
80
*
.
*
6O
. ,
4O
,
2 , HU
~ MO
, DOG
, M-P
RAT
120
HU
MO
DOG
M-P
RAT
M-P
PAT
120
p
A
** ~
•
60.
B
.
2
HU
A
**
40
0
L
LEAD ACETATE
120
100
M
319
80
40
~
.
*
.
**
MO
DOG
*
2 HU
MO
DOG
M-P
PAT
HU
Fig. 1. Effects of doxycycline and lead acetate on the chemiluminescence response of white blood cells from five species [human (HU), monkey (MO), dog, mini-pig (M-P) and rat]. Three stimuli were used [opsonized zymosan, latex beads and phorbol myristate acetate (PMA)]. White blood cells were incubated for 2 hr with either doxycycline [0 ( I ) , 2 (I-I), 50 (1~1)Fg/ml] or lead acetate [0 ( I ) , 2 ([:]), 20 (1~) Mg/ml]. Each bar represents the mean relative value of the peak (percentage/control), n = 5, and asterisks denote significant difference (Wilcoxon test) from corresponding control values.
are also in agreement with those of Governa et al. (1987), who found a marked reduction in the CL response of phagocytes in vitro at the concentration of20/~g/ml. On the basis of the results reported here, this technique appears to give an accurate assessment of phagocytosis in vitro. A similar trend was observed, whatever the species and the stimulating agent used, with two reference compounds, suggesting that results can be extrapolated from one species to another, and in particular from animals to humans, This assay is proposed as a tool for use in the assessment of the safety of drugs and chemicals, REFERENCES
Babior B. M. (1984) The respiratory burst of phagocytes, Journal of Clinical Investigation 73, 599-601. TIV 7/4~aC
Briheim G. and Dahlgren C. (1987) Influence of antibiotics on formyl-methionyl-leucyl-phenylalanine-inducedleukocyte chemiluminescence. Antimicrobial Agents and Chemotherapy31, 736-767. Descamps-Latscha B., Nguyen A. T., Golub R. M. and Feuillet-Fieux M. N. (1982) Chemiluminescence in microamounts of whole blood for investigation of the human phagocyte oxidative metabolism function. Annales de l'Institut Pasteur. Immunology 133e, 349-364. Dumas R., Brouland J. P., Tedone R. and Descotes J. (1990) Influence of macrolide antibiotics on the chemiluminescence of zymosan-activated human neutrophils. Chemotherapy 36, 381-384. Duncker D. and Ullmann U. (1986) Influence of various antimicrobial agents on the chemiluminescence of phagocytosing human granulocytes. Chemotherapy 32, 18-24. Faden H. and Maciejewski N. (1981) Whole blood luminoldependent chemiluminescence. Journal of the Reticuloendothelial Society 30, 219-226.
320
F. VEROIER et al.
Governa M., Valentino M. and Visona I. (1987) In vitro impairment of human granulocyte functions by lead. Archives of Toxicology 59, 421~425. Halstensen A., Haneberg B., Glette J., Sandberg S. and Solberg C. O. (1986) Factors important for the measurement of chemiluminescence production by polymorphonuclear leukocytes. Journal of Immunological Methods 88, 121-128. Johansson A. and Dahlgren C. (1989) Characterization of the luminol-amplified light generating reaction induced in human monocytes. Journal of Leukocyte Biology 45, 444~451. Lindena J. and Burkhardt H. (1988) Separation and chemiluminescence properties of human, canine and rat polymorphonuclear cells. Journal of Immunological Methods 115, 141 147. Nguyen A. T., Golub R., Feuillet-Fieux M. N. and Descamps-Latscha B. (1983) Modulation of human
granulocyte and monocyte chemiluminescence response: evidence for distinct free radical generating systems. Journal of Clinical Laboratory Immunology 12, 47-55. Peterson P. K. (1984)Host defense abnormalities predisposing the patient to infection. American Journal of Medicine 76, 2-10. Sbarra J. and Karnofsky M. L. (1959) The biochemical basis of phagocytosis. I. Metabolic changes during the ingestion of particles by polymorphonuclear leukocytes. Journalof Biological Chemistry 234, 1355-1362. Steele R. W. (1991) Clinical applications of chemiluminescence of granulocytes. Reviews of Infectious Diseases 13, 918-925. Williams J. D., Topley N., Alobaidi H. M. and Harber M . J . (1986) Activation of human polymorphonuclear leucocytes by particulate zymosan is related to both its major carbohydrate components: glucan and mannan. ImmunologySg, 117-124.
0887-2333/93 $6.00+ 0.00 Copyright© 1993Pergamon Press Ltd
I N VITRO ASSESSMENT OF PHAGOCYTOSIS: INTERSPECIES COMPARISON OF CHEMILUMINESCENCE RESPONSE F. VERDIER*t,F. CONDEVAUX*,R. TEDONEt, M. VIRAT* and J. DESCOTESt *Pharmakon Europe, BP 0118, 69593 L'Arbresle Cedex and tLaboratoire d'Immunotoxicologie Fondamentale et Clinique, INSERM U80, Facult6 de M6decine A. Carrel, 69008 Lyon, France Abstraet--Phagocytosis is a major component of the host's defences against pathogens. Particulate or soluble stimuli trigger the intracellular respiratory burst in activated phagocytes which can be measured in vitro by the luminol-enhanced chemiluminescent response. In this study, the phagocytic capacity of peripheral blood leucocytes was assayed using chemiluminescence and preliminary in vitro activation by phorbol myristate acetate, opsonized zymosan or latex beads. Leucocytes from rats, mini-pigs, dogs, monkeys and humans were preincubated for 2 hr with either doxycycline (0, 2 and 50 #g/ml) (n = 5) or lead acetate (0, 2 and 20 pg/ml) (n = 5), both compounds impairing phagocytosis. Whatever the species and the activator used, a similar dose-dependent decrease in chemiluminescence response was observed with either doxycycline or lead acetate, showing that the results can be extrapolated between species and to humans. The chemiluminescence assay is proposed as a tool for assessment of the safety of drugs and chemicals.
Introduction
Materials and Methods
The immune system is a potential target organ for the toxicity of xenobiotics. Non-specific immunity plays a major part in the host's defence mechanisms against infections and tumours; indeed, impairment of non-specific immune function is associated with serious health problems (Peterson, 1984). Phagocytosis is the primary non-specific defence mechanism against pathogens resulting in the activation of phagocytes: the respiratory oxidative burst (Sbarra and Karnofsky, 1959) is a series of changes in the oxidative metabolism of phagocytes, including increased production of hydrogen peroxide (H202), production of superoxide anion (O~-) and generation of photons or chemiluminescence (CL). The emission of CL by phagocytes is associated with the initial biochemical mechanisms of uptake, killing and digestion of micro-organisms. Measurement of the CL response has been proposed as a means of assessment of the phagocytic capacity of polymorphonuclear neutrophils and monocytes (Descamps-Latscha et al., 1982; Steele, 1991). Various stimuli, either particulate or soluble, can be used to activate phagocytes. The aim of this study was to compare the CL response of peripheral blood leucocytes from five different species (humans, monkey, dog, mini-pig and rat) using three different stimuli, namely opsonized zymosan, latex beads and phorbol myristate acetate (PMA), and following incubation with doxycycline and lead acetate,
Reagents. Zymosan, PMA, latex beads (0.83/tm diameter) and luminol (5-amino-2,3-dihydro-l,4phthalazinedione) were obtained from Sigma (St Louis, MO, USA). Phosphate buffered saline (PBS) was used as the cell medium. The red blood cell lysing solution was prepared with 8.23 g NH4CI, 37mg ethylenediaminetetraacetic acid and 1 g KHCO3 in 100ml sterile water. Doxycycline and lead acetate (supplied by Sigma) were suspended in saline. Blood sampling. Heparinized blood samples (5 ml) were collected from healthy human donors, purposebred male cynomolgus (Macaca fascicularis) monkeys (Charles River, St-Aubin-16s-Elbeuf, France), male beagle dogs (Hazleton RP, Cumberland, VA, USA), Yucatan and G6ttingen mini-pigs (Charles River and Genipig, Marseille, France, respectively) and male Sprague--Dawley rats ( I F F A CREDO, l'Arbresle, France). Animals were housed in airconditioned facilities with a 12-hr light/dark cycle. Unanaesthetized animals [with the exception of rats (ether anaesthesia)] were sampled between 8.00 and 9.00 a.m. Cell preparation. Red blood cells were lysed twice by the addition of 10 ml of the lysing solution to each blood sample. White blood cells were washed twice and resuspended in 5 ml PBS. Polymorphonuclear neutrophils and monocytes were counted. In vitro treatment. White blood cells were incubated for 2 h r at 37°C with either 0, 2 or 50/tg doxycycline/ml or 0, 2 or 20/~g lead acetate/ml. The viability of treated cells, assessed by the trypan blue exclusion method, was more than 90% at the end of the incubation period.
Abbreviations: CL = chemiluminescence; PBS = phosphate
buffered saline; PMA = phorbol myristate acetate,
317
318
F. VERDIERet al.
Chemiluminescence assay. Aliquots of P M A suspended in 1 0 - 2 m o l dimethyl sulphoxide/litre were diluted 100 times in PBS before use. Opsonized zymosan was prepared by 2 hr incubation of l0 mg zymosan with 750/~l autologous plasma mixed with 250/al PBS at 37°C and centrifugation for 5 min at 1800rpm. The final pellet was washed twice and resuspended in 1 ml PBS. Latex beads were diluted two-fold (1:2) in PBS before use (solid content, 5%). The C L response was measured in a LKB 1251 luminometer (Wailac, Finland) immediately after mixing 2 x l0 5 M-luminol, 500/~l of the cell suspension, 100/zl of the stimulus solution and 200/~1 PBS in 4 ml polystyrene cuvettes. The reaction was recorded every 30see for at least 15min at 37°C with continuous mixing. The peak values (mV) per thousand phagocytes/mm a and the time of the peak were noted. Statistical analysis. Results were compared using the non-parametric Friedman test for multiple group comparisons and Wilcoxon test for pairwise comparisons, Results
Baseline CL response Whatever the species used, the peak values and the kinetics of the CL response were similarly dependent on the stimulating agent, but showed a different trend (Table 1). Peak values ranked zymosan > latex beads > P M A , whereas the time of the peak ranked latex beads > zymosan > PMA. Effects o f doxycycline Whatever the species used, a dose-dependent decrease in the C L reponse was induced by doxycycline with the three different stimuli. However, only the higher dose of doxycycline induced a consistent statistically significant decrease in peak values (Fig. 1). Similarly, a dose-dependent delay in the CL response was noted (data not shown). Effects o f lead acetate With the exception of mini-pigs (zymosan stimulus) and dogs (latex beads), the higher dose of lead acetate induced a statistically significant decrease in peak values (Fig. 1). However, no clear dosedependent trend was noted,
Discussion
A variety of processes are known to be involved in the emission of light by activated phagocytes. Opsonized zymosan has been shown to interact with the phagocyte membrane by means of complement receptors (i.e. CRI, CR3) and glucan- and mannanspecific receptors (Lindena and Burkhardt, 1987; Williams et al., 1986) whereas latex beads develop hydrophobic interactions with the cell surface (Nguyen et al., 1983). By contrast, the soluble stimulus P M A results in the direct activation of the respiratory burst by protein kinase C stimulation (Babior, 1984; Johansson and Dahlgren, 1989). Whatever the initial signal for phagocyte activation, the production of free radicals, oxidative metabolites (e.g. 0 2 and H202) and photons is a c o m m o n endpoint. The whole blood technique allowed the use of smaller samples than are required for density gradient separation. The combination of red blood cell lysis and centrifugation was used to avoid inhibition of light emission by plasma and red blood cells (Descamps-Latscha et al., 1982; Faden and Maciejewski, 1989). In addition, the use of ammonium chloride as a lysing solution has been shown to optimize the C L response (Halstensen, 1986). In all the species studied, the curve profiles of CL response were similar, but were clearly stimulusdependent, with latex beads inducing a quicker peak response. As previously observed by Lindena and Burkhardt 0988), the CL response of rats was lower. In addition, inter-animal variations were greater in rats than in the other species studied. Nevertheless, this study provided evidence that the CL responses can be readily investigated in all five species and gave similar results. This in vitro CL assay was validated by testing two compounds known to impair phagocyte runetion. The influence of antimicrobial agents on the C L response has been extensively studied in vitro (Bribeim and Dahlgren, 1987; Dumas et al., 1990; Duncker and Ullmann, 1986) and doxycycline was shown to inhibit the CL response of human granulocytes at concentrations above or equal to 50 mg/litre (Duncker and Ullmann, 1986). The study reported here confirmed these results. In addition, a doserelated trend was observed in all species and with all stimuli. The results obtained with lead acetate
Table I. Baseline chemiluminescencemeasurement of white blood cells from various species in response to particulate and soluble stimuli Species Stimuli Human Monkey Dog Mini-pig Rat Zymosan P* 2424 + 232 733 + 81 1667 + 343 353 + 90 379 + 131 (1 mg/ml) T 324 + 34 132 + 7 300 + 9 192 + 30 102 ± 7 Latex beads P 1403 +_110 602 4- 55 883 _+211 291 + 59 134 _+54 (0.5% solid content) T 174 + 29 80 + 8 30 -+0 138 + 43 42 +_7 PMA P 1170_+ 119 255+26 544+89 96±28 91 _+30 (10 nmol/ml) T 480 + 9 204 _+17 486 + 104 282 _+7 138 _+ 12 *Peak values (P) of the chemiluminescenceresponse in mV/103/mm3phagocytes (polymorphonuclear neutrophils and monocytes) and times of the peak (T) in seconds for 10 samples/stimulus/species(mean _4-SE).
Chemiluminescence assessment of phagocytosis %
%
DOXYCYCLINE
120 . Z
.
.
100
80
80
60
60
A
40
N
20
* *
.
120. 100:
E
80,
X
40
E A
20
D
0
S
MO
DOG
M-P
RAT
I HU
I 1,40
I DOG
I M-P
" RAT
120 T
l
80
*
.
*
6O
. ,
4O
,
2 , HU
~ MO
, DOG
, M-P
RAT
120
HU
MO
DOG
M-P
RAT
M-P
PAT
120
p
A
** ~
•
60.
B
.
2
HU
A
**
40
0
L
LEAD ACETATE
120
100
M
319
80
40
~
.
*
.
**
MO
DOG
*
2 HU
MO
DOG
M-P
PAT
HU
Fig. 1. Effects of doxycycline and lead acetate on the chemiluminescence response of white blood cells from five species [human (HU), monkey (MO), dog, mini-pig (M-P) and rat]. Three stimuli were used [opsonized zymosan, latex beads and phorbol myristate acetate (PMA)]. White blood cells were incubated for 2 hr with either doxycycline [0 ( I ) , 2 (I-I), 50 (1~1)Fg/ml] or lead acetate [0 ( I ) , 2 ([:]), 20 (1~) Mg/ml]. Each bar represents the mean relative value of the peak (percentage/control), n = 5, and asterisks denote significant difference (Wilcoxon test) from corresponding control values.
are also in agreement with those of Governa et al. (1987), who found a marked reduction in the CL response of phagocytes in vitro at the concentration of20/~g/ml. On the basis of the results reported here, this technique appears to give an accurate assessment of phagocytosis in vitro. A similar trend was observed, whatever the species and the stimulating agent used, with two reference compounds, suggesting that results can be extrapolated from one species to another, and in particular from animals to humans, This assay is proposed as a tool for use in the assessment of the safety of drugs and chemicals, REFERENCES
Babior B. M. (1984) The respiratory burst of phagocytes, Journal of Clinical Investigation 73, 599-601. TIV 7/4~aC
Briheim G. and Dahlgren C. (1987) Influence of antibiotics on formyl-methionyl-leucyl-phenylalanine-inducedleukocyte chemiluminescence. Antimicrobial Agents and Chemotherapy31, 736-767. Descamps-Latscha B., Nguyen A. T., Golub R. M. and Feuillet-Fieux M. N. (1982) Chemiluminescence in microamounts of whole blood for investigation of the human phagocyte oxidative metabolism function. Annales de l'Institut Pasteur. Immunology 133e, 349-364. Dumas R., Brouland J. P., Tedone R. and Descotes J. (1990) Influence of macrolide antibiotics on the chemiluminescence of zymosan-activated human neutrophils. Chemotherapy 36, 381-384. Duncker D. and Ullmann U. (1986) Influence of various antimicrobial agents on the chemiluminescence of phagocytosing human granulocytes. Chemotherapy 32, 18-24. Faden H. and Maciejewski N. (1981) Whole blood luminoldependent chemiluminescence. Journal of the Reticuloendothelial Society 30, 219-226.
320
F. VEROIER et al.
Governa M., Valentino M. and Visona I. (1987) In vitro impairment of human granulocyte functions by lead. Archives of Toxicology 59, 421~425. Halstensen A., Haneberg B., Glette J., Sandberg S. and Solberg C. O. (1986) Factors important for the measurement of chemiluminescence production by polymorphonuclear leukocytes. Journal of Immunological Methods 88, 121-128. Johansson A. and Dahlgren C. (1989) Characterization of the luminol-amplified light generating reaction induced in human monocytes. Journal of Leukocyte Biology 45, 444~451. Lindena J. and Burkhardt H. (1988) Separation and chemiluminescence properties of human, canine and rat polymorphonuclear cells. Journal of Immunological Methods 115, 141 147. Nguyen A. T., Golub R., Feuillet-Fieux M. N. and Descamps-Latscha B. (1983) Modulation of human
granulocyte and monocyte chemiluminescence response: evidence for distinct free radical generating systems. Journal of Clinical Laboratory Immunology 12, 47-55. Peterson P. K. (1984)Host defense abnormalities predisposing the patient to infection. American Journal of Medicine 76, 2-10. Sbarra J. and Karnofsky M. L. (1959) The biochemical basis of phagocytosis. I. Metabolic changes during the ingestion of particles by polymorphonuclear leukocytes. Journalof Biological Chemistry 234, 1355-1362. Steele R. W. (1991) Clinical applications of chemiluminescence of granulocytes. Reviews of Infectious Diseases 13, 918-925. Williams J. D., Topley N., Alobaidi H. M. and Harber M . J . (1986) Activation of human polymorphonuclear leucocytes by particulate zymosan is related to both its major carbohydrate components: glucan and mannan. ImmunologySg, 117-124.