Immunology 197937453
Kinetics of phagocytosis of Staphylococcus aureus and Escherichia coli by human granulocytes
P. C. J. LEIJH, MARIA TH. VAN DEN BARSELAAR, THEDA L. VAN ZWET, IVONNE DUBBELDEMAN-REMPT & R. VAN FURTH Department of Infectious Diseases, University Hospital, Leiden, The Netherlands
Received 16 October 1978; acceptedfor publication 20 November 1978
Summary. Although phagocytosis of micro-organisms by granulocytes is one of the most important defence mechanisms against infection, little is known about the kinetics of this process. The present study showed that the rate of ingestion of Staphylococcus aureus and Escherichia coli depends on the concentrations of the granulocytes and bacteria. Phagocytosis of bacteria at a bacteria-to-cell ratio in the range between 100:1 and 1:10 showed an exponential course during the first 30 min. At a bacteria-to-cell ratio of 1: 1, application of a correction for the outgrowth of extracellular bacteria gave an exponential course of ingestion over the first 90-min period. Since it was found that the phagocytosis of bacteria by granulocytes at various bacteriato-cell ratios can be described with Michaelis-Menten kinetics, we studied the kinetics of phagocytosis on the basis of the initial rate for the first 30-min period. The rate of phagocytosis and the maximal degree of ingestion of bacteria by granulocytes proved to be related to the concentration of serum used in the assay. The minimal serum concentration required for maximal ingestion was 2 5% for Staphylococcus aureus and 5% for Escherichia coli. When bacteria were preopsonized, the duration of pre-opsonization proved to be limiting for the rate of phagocytosis in dependence
on the serum concentration. The effect of temperature on the phagocytosis of micro-organisms proved to be two-fold. First, at temperatures between 4 and 330 a decrease in the functioning of the cells leads to a
decrease in the rate of phagocytosis. Above 420, the temperature affects mainly the opsonization of the micro-organisms and has only a slight influence on the ingestion process. From the data obtained in this study, maximal rates of 6-3 x 106 Staphylococcus aureusi5 x 106 granulocytes/min and of 7-1 x 106 Escherichia colil5 x 106 granulocytes/min were calculated for phagocytosis at a bacteria-to-cell ratio of 100:1 at 370, i.e. on average about one bacterium per granulocyte per min. The maximum calculated number of bacteria ingested by one granulocyte lies between 40 and 50.
INTRODUCTION Phagocytosis of micro-organisms by granulocytes and monocytes is one of the most important of the body's defence mechanisms against infection, since it is the only mechanism by which micro-organisms which have invaded the tissues can be eliminated. Information about this process has been obtained with both in vivo and in vitro techniques. The former, which are based on measurement of the clearance of substances injected into an animal, have the disadvantage of being influenced by many unknown variables, such as
Correspondence. Professor R. van Furth, Department of Internal Medicine & Infectious Diseases, University Hospital, Leiden, The Netherlands. 00 19-2805/79/0600-0453$02.00 C) 1979 Blackwell Scientific Publications
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P. C. J. Leijh et al.
humoral factors, the blood flow, the concentration of particles, the number and functional state of the phagocytic cells. In vivo techniques are also limited by the fact that the measurements concern mainly the functioning of the Kupffer cells and to a lesser extent that of the spleen macrophages (Normann, 1973; Stiffel, Mouton & Biozzi, 1970; Bouveng, Schildt & Sjogvist, 1975). Studies carried out in vitro have the advantage that both homogeneous, well defined and characterized phagocytes and particles can be used. Phagocytosis experiments can be performed with cells in different functional states and under various experimental conditions, for instance attached to glass or plastic surfaces (Michell, Pancake, Noseworthy & Karnovsky, 1969) or in suspension (Cohn & Morse, 1959; van Furth, van Zwet & Leijh, 1978; Stossel, Mason, Hartwig & Vaughan, 1972). Serum factors, such as immunoglobulins, complement components, or other proteins, that are necessary for the opsonization of the particles (Stossel, Alper & Rosen, 1973; Johnston, Klemperer, Alper & Rosen 1969; Griffin, Griffin, Leider & Silverstein 1975; Michl, Ohlbaum & Silverstein, 1976; Rabinovitch, 1968), can also be kept under control in in vitro experiments. On this basis, techniques of several kinds have been developed to measure phagocytosis in vitro. For instance, the number of particles (micro-organisms, yeast cells, latex particles, or oil droplets) ingested per cell can be determined microscopically (Capo, Bongrand, Benoliel & Depieds, 1974; Weisman & Korn, 1967). The number of ingested micro-organisms can be determined with the use of radiolabelled dead or living organisms after the removal of the extracellular population (Peterson, Verhoef & Quie, 1977). The ingestion of viable microorganisms can also be measured microbiologically as a function of their extracellular disappearance (Cohn et al., 1959; Solberg & Hellum, 1973; Quie, White, Holmes & Good, 1967). In our opinion, this last approach gives the best approximation of the situations occurring at the site of an inflammation in vivo. Although this method has been used by many investigators, quantitative information about the rate of phagocytosis by normal human granulocytes in various bacteria-to-cell ratios and about the opsonization of bacteria is not available. Such information is indispensable for the interpretation of the results obtained using granulocytes and serum opsonins from patients. The present study was therefore undertaken to investigate the kinetics of opsonization and phagocytosis of live Staphylococcus
aureus and Escherichia coli by normal human granulocytes as determined in vitro by a microbiological method. MATERIALS AND METHODS Reagents A stock solution of 300 ug/ml lysostaphin (Sigma Chemical Co., St Louis, Mo.) was prepared in phosphate-buffered saline and stored in l-ml aliquots at -20°. A stock solution of 02 M sodium fluoride (Merck, Darmstadt, Germany) was prepared in Hanks's balanced salt solution and stored at 4°.
Granulocytes Blood was obtained from healthy donors by venepuncture. Granulocytes were collected by sedimentation of erythrocytes with a 5% solution of dextran in buffered saline (mol. wt 2000,000; 3 ml solution to 10 ml blood). The leucocyte-rich layer was washed twice with phosphate-buffered saline containing 0-5 u/ml heparin, after which a cell suspension of 1-2 x 107 granulocytes/ml in Hanks's salt solution with 0.1% (w/v) gelatin was prepared. Serum In all experiments, serum prepared from blood of healthy AB blood donors was used. The blood was clotted for I h at room temperature, centrifuged for 20 min at 1100 g, and stored until use in aliquots of 2 ml for up to 2 months at -20°. Micro-organisms The Staphylococcus aureus (type 42D) and Escherichia coli (O 54) used in all experiments, were stored on agar slants at 4° and transferred every 14 days. The microorganisms were cultured overnight in Nutrient Broth no. 2 (Oxoid Ltd, London), harvested by centrifugation at 1500 g for 10 min, and washed twice with phosphate-buffered saline (pH 7 2) after which a suspension containing 107 bacteria per ml in Hanks's balanced salt solution with 01% (w/v) gelatin was prepared. Pre-opsonization of bacteria Pre-opsonized micro-organisms were obtained by incubation of suspensions (5 x 106 bacteria/ml) with normal AB serum for 25 min at 370 under slow rotation (4 r.p.m.) followed by centrifugation at 1 5OOg for 10 min and two washings in ice-cold gelatin-
455
Kinetics ofphagocytosis by human granulocytes
Hanks's solution. The bacteria then were suspended to a concentration of approximately I07 organisms/ml in gelatin-Hanks's solution.
the percentage decrease of the initial number of viable extracellular bacteria according to the formula No- N. ~ x 100, R No in which Ft) is the phagocytosis index at time t, No is the number of viable extracellular bacteria at time t=0, and N. is the number of viable extracellular bacteria at time t = t. The initial rate of phagocytosis (V0) was calculated according to the formula Vo =k No, in which k is the initial rate constant. This rate constant was calculated according to the formula lnN0 - lnN, =
Phagocytosis Phagocytosis was measured as described elsewhere (van Furth et al., 1978). In short, for a standard assay a suspension of 107 granulocytes per ml was incubated with an equal volume of 107 bacteria per ml with 10% (v/v) serum at 370 under slow rotation (4 r.p.m.). At various time points, a 0-5 ml sample of the suspension was added to 1 5 ml ice-cold gelatin-Hanks's salt solution to stop phagocytosis. After 4 min of centrifugation at 110 g to spin down the granulocytes, the number of viable micro-organisms in the supernatant was determined by a microbiological plate method. At this sedimentation gravity, 99% of the Staphylococcus aureus and Escherichia coli remain in suspension (van Furth et al., 1978). In this study other bacteria-togranulocyte ratios were also used.
Calculations Phagocytosis at a given time point was expressed as
RESULTS
Phagocytosis of Staphylococcus aureus and Escherichia coi Incubation of Staphylococcus aureus or Escherichia coli with granulocytes, both in final concentration of
Escherichia coli
Staphylococcus aureus
bacteria serum
c
standing
100
control
._.
i
10-
a!
._.
a
1-
'IOU
bacteria
!0.1 serum ~ ~ ~~'vcorrected
0.
\
85granulocytes bacteria
0 v
0
W0
120
0
60
corrected
120
9;
minutes
Figure 1. The kinetics of phagocytosis of Staphylococcus aureus and Escherichia coli by human granulocytes. 5 x 106 granulocytes/ml were incubated with 5 x 106 bacteria/ml and 10% serum at 370 under rotation (4 r.p.m.). The number of viable extracellular bacteria determined at 5 min (x) or 15 min (o) intervals. Incubation without rotation (standing control 4), or without granulocytes (-) Corrected (v) is curve obtained after correction for bacterial growth.
P. C. J. Leijh et al.
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5 x 106/ml, in the presence of 10% serum under-slow rotation (4 r.p.m.), showed that phagocytosis-of these micro-organisms was a rapid process (Fig. 1). Incubation of either strain with granulocytes in a ratio of 1:1 at 370 showed an exponential decrease in the number of viable extracellular micro-organisms-determined at intervals of 5 min-during the first 20-30 min. During this period more than 80% of the initial number of micro-organisms were ingested, whereas during the remaining 90 min this percentage rose to 99 5 for Staphylococcus aureus and 99 8 for Escherichia coli. When, instead of 5 min, intervals of 15 or 30 min were used to measure the number of viable extracellular micro-organisms, the same curve was obtained (Fig. 1). Thus, the same information about the rate of phagocytosis is obtained with the use of longer intervals. Calculation of the rate of phagocytosis for periods of 30 and 90 min showed a marked decrease for the longer compared with the shorter period (Table 1). Table 1. Effect of correction for bacterial growth on the initial rate of phagocytosis Initial rate
(bacteria/min/5 x 106 granulocytes) Interval
S. aureus
E. coli
30 min, direct experiment 90 min, direct experiment 90 min, after growth correction
5-1 x 105 2 8 x 105 4 8 x 105
4 6 x 105 2 7 x 105 4 3 x 105
Since multiplication of micro-organisms in the presence of serum tends to start during the second hour of incubation, a correction must be made for this growth in the calculation of the rate of phagocytosis. Under the assumption that the growth of micro-organisms in a bacteria-granulocyte suspension equals that in a suspension of bacteria only, the corrected number of extracellular bacteria can be calculated according to the formula Bo
NC, = N,B., in which NC, is the corrected number of extracellular bacteria at time t, N, the measured number of extracellular bacteria at time t, Bo the initial number of bacteria in the control suspension containing only bacteria, and B. the number of bacteria in the control
at time t. After this correction, the results showed an exponential decrease in the number of viable extracellular bacteria over a period of 90 min (Fig. 1). The rate constant of this decrease calculated for this interval did not differ from that obtained for the first 30 min without correction (Table 1). From these results we may conclude that the difference between uncorrected rates of phagocytosis for incubation periods of 30 and 90 min is due solely to the multiplication of extracellular micro-organisms, and that the information on the rate of phagocytosis obtained during the first 30 min is representative for the entire experimental period. Control studies to exclude extracellular killing of microorganisms The decrease in the number of viable extracellular bacteria was not due to extracellular killing of microorganisms, because: (1) incubation of a bacterial suspension without granulocytes in the presence of serum showed no decrease but an increase in the number of micro-organisms, which indicates that there was no bactericidal activity in the serum used for both bacteria species (Fig. 1); (2) incubation of the microorganisms with granulocytes in the presence of 10% serum at 37° without rotation (i.e. the standing control, Fig. 1) showed no decrease in the number of extracellular micro-organisms, thus indicating that no phagocytosis occurs without contact between cells and bacteria and that no extracellular factors reduced the number of viable micro-organisms; (3) supernatants from granulocytes and bacteria prepared after 0, 60, or 120 min of phagocytosis showed no bactericidal activity for either Staphylococcus aureus and Escherichia coli when incubated with bacteria for 2 h at 37°. Control studies to demonstrate the ingestion of microorganisms To find out whether the decrease in the number of extracellular micro-organisms was due to attachment of bacteria to granulocytes or to ingestion of the bacteria by these cells, the number of cell-associated bacteria in the cell pellet after 0 or 60 min of incubation was determined before and after the removal of the attached bacteria. First, the effect of washing of the granulocyte pellet with Hanks's solution was investigated. After washing, there was a decrease of 7.3% (range 4.3-10.3% and 114% (range 6-8-15l1%) for Staphylococcus aureus and Escherichia coli, respectively, in the total number of viable bacteria in the cell
457
Kinetics ofphagocytosis by human granulocytes
pellet determined after disruption of the granulocytes with distilled water with 00 00% (w/v) human albumin. Second, the cell pellet was incubated for 5 min at 370 in the presence of lysostaphin (10 pg/ml) to lyse extracellular Staphylococcus aureus (Tan, Watanakunakorn & Phair, 1971), which reduced the total number ofviable bacteria by 13.4% (range 6-4-21-3%) as compared with incubation of the cell pellet for 5 min at 370 in medium only. (Incubation of 5 x 106 Staphylococcus aureus with 10 Mg lysostaphin/ml for 5 min at 370 gave a reduction of more than 99 9% of the viable bacteria.) Third, the phagocytic assay was performed in the presence of sodium fluoride, which is known to inhibit ingestion (Karnovsky, Simmons, Glass, Shafer & D'Arcy Hart, 1970). Incubation with 0 2 M sodium fluoride for 60 min gave a phagocytic index of 45% for Staphylococcus aureus and 38% for Escherichia coli. From the findings in these three kinds of control experiments it may be concluded that in the assay used in this study, the decrease in the number of viable extracellular bacteria mainly represents phagocytosis of these micro-organisms. Effect of various bacteria-to-cell ratioson the kinetics of phagocytosis Incubation of 5 x 106 granulocytes/ml with various concentrations of micro-organisms (range 5 x 104 to 5 x 109/ml) in the presence of serum showed that at bacteria-to-granulocyte ratios higher than 1:1 the phagocytosis of both Staphylococcus aureus and Escherichia coli was limited (Fig. 2). Incubation of granulocytes and bacteria at bacteria-to-granulocyte ratios of 100:1 and 1000:1 only gave reliable information about the kinetics of phagocytosis for periods of 90 and 60 min, respectively, because after these intervals the granulocytes were saturated with bacteria and became disrupted. Bacteria concentrations of 1010/ml or more were not used because of bacterial clumping, which hampers accurate counting of the micro-organisms. The values obtained in these experiments were used to calculate the rate of phagocytosis during the initial 30 min of the assay for the various bacteria-to-granulocyte ratios. The results (see Fig. 3) for the incubation of 5 x 106 granulocytes/ml showed a proportional relationship between the logarithm of the initial rate of phagocytosis and the logarithm of the initial number of bacteria at concentrations of 5 x 104 to 4 x I07 bacteria/ml, whereas at higher concentrations a maximal rate of ingestion was reached, amounting to 6-3 x 106 bacteria/5 x 106 cells/min for Staphylococcus aureus
Staphylococcus aureus
Escherichia coli
bacteria to
cell ratio
bacteria to cell ratio
0 a)
n c ._
a)
-o 0
D 0-
Time(min)
Figure 2. The kinetics of phagocytosis at various bacteria-tocell ratios. 5 x 106 granulocytes/ml were incubated with various concentrations of bacteria at 37°.
Granulocyte
concentration (cells/ml)
5x107 _n
5x 106
0
0 en
.x 0 0 0
5x105
OL 0 c .'
0 -J
Log initial concentration of bacteria
Figure 3. Effect of the concentrations of Staphylococcus aureus (&) and Escherichia coli (a) on the initial rate of phagocytosis (first 30 min period) at various granulocyte concentrations. The data for 5 x 106 granulocytes/ml are taken from Fig. 2.
P. C. J.
458
and of 7-1 x 106 bacteria/5 x 106 cells/min for Escherichia coli. Next, the effect of various granulocyte concentrations on the kinetics of phagocytosis was studied at concentrations ranging from 5 x 104 to 5 x 107 cells per ml. Incubation of 5 x 104 granulocytes per ml with bacteria suspensions of 104 to 106/ml at 370 under rotation (4 r.p.m.) did not lead to any detectable decrease in the number of extracellular micro-organisms. Phagocytosis during incubation of 5 x 105 to 5 x 107 granulocytes per ml with various concentrations of bacteria was characterized by an initial rate depending on the bacteria-to-cell ratio and the number of granulocytes. Figure 3 shows the relationship between the logarithm of the initial rate of phagocytosis and the logarithm of the initial number of micro-organisms as calculated for the various experiments with 104-109 micro-organisms and 5 x 105 to 5 x 107 granulocytes/ml. A log-log plot based on these data (Fig. 4) shows that there is a relationship between the intitial rate of phagocytosis and the granulocyte concentration. From these experiments it may be concluded that the rate of phagocytosis in vitro depends not only on the bacteria-to-cell ratio but also on the concentration of both the bacteria and the granulocytes.
Leijh et al. Determination of the phagocytic capacity of granulocytes To determine the maximum number of microorganisms that can be ingested by granulocytes, experiments were designed for a 3 h period in which the initial bacteria-to-cell ratio was re-established every 30 min by the addition of fresh bacteria. As can be seen from Fig. 5, at a bacteria-to-cell ratio of 1:1 the rate of
4o9
(a
Baccteria to cet.1 ratio
bacteria added
i
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I
x
I
.0
I
106
I~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
o
A
z
Initial bacteria
105 0
(bacteria/ml) 8
t.
60
90
120
150
Figure 5. Effect on the kinetics of phagocytosis of re-establishment of the bacteria-to-cell ratio at 30 min intervals. Incubation of 5 x 106 granulocytes with 5 x 106 (ratio= 1:1),
5x 108
5x l0' (ratio=10:1) or 5x 108 (ratio=100:1) Staphylococcus aureus/ml and 10% serum at 37° under rotation (4
5x107
r.p.m.).
7
-I
30
minutes
concentration
0
06 o
5x 106
0
a
2o '5
5
0'
o -J
5x105
4
7 8 Log granulocyte concentration
6
Figure 4. Log-log plot prepared from the data of Fig. 3, showing a linear relationship between the granulocyte concentration and the initial rate of phagocytosis.
phagocytosis of Staphylococcus aureus during the first 30 min was similar to that in the interval from 120 to 150 min. At a bacteria-to-cell ratio of 10:1 there was a decrease in the rate of phagocytosis after 90 min and no phagocytosis at all at 120 min. At a ratio of 100:1, detectable phagocytic activity ceased after 30 min. During incubation, the number of granulocytes was counted every 30 min and re-adjusted for the next interval of incubation to 5 x 106f cells/ml by changing the volume of the medium; during these experiments the viability of the cells remained above 90%. The same results were obtained when Escherichia coli was used as test organism (not shown). These data were used to calculate the total number
Kinetics of phagocytosis by human granulocytes of bacteria ingested by 5 x 106 granulocytes during each interval over 150 min. Figure 6 shows that at a bacteria-to-cell ratio of 100:1 the maximal number of Staphylococcus aureus (1 2 x 108) or Escherichia coli (1-1 X 108) is ingested within the first 30 min, after which there is almost no phagocytosis. At lower bacteria-to-cell ratios the uptake is lower, and for a ratio of 1:1 follows a linear course. x108
100:1
6x1070 XI8
0
:3
7
8X
O= 5x107 1
/
0)
2x107 07
CD
x107 25x105
0 1061:
106
30
60
90
120
150
Time (min)
Figure 6. Calculation of the maximal number of bacteria ingested by 5 x 106 granulocytes, on the basis of the data in Fig. 5. Summation of the total number of Staphylococcus aureus (a) or Escherichia coli (o) ingested during each 30 min interval.
The effect of serum on the rate of phagocytosis Incubation of Staphylococcus aureus or Escherichia coli with granulocytes in the presence of various concentrations of serum at 370 showed that both the rate of phagocytosis and maximal phagocytosis depend on the serum concentration (Fig. 7). This dose-response experiment showed that at least 2.5% serum was necessary to obtain maximum phagocytosis of Staphylococcus aureus whereas for Escherichia coli the minimum necessary serum concentration was 5%.
459
To find out whether opsonization of microorganisms was rate-limiting in the phagocytic process, phagocytosis was performed with pre-opsonized bacteria. Bacteria were pre-opsonized with various concentrations of serum for various periods before use in the phagocytosis assay in medium without serum. As a control, the standard phagocytosis assay of granulocytes and non pre-opsonized bacteria in the continuous presence of various concentrations of serum was used. The results show that Staphylococcus aureus pre-opsonized with 1% serum for 15 min or longer gave a higher initial rate than phagocytosis in the continuous presence of 1% serum (Fig. 8). At higher serum concentrations, 5 min of pre-opsonization gave the same initial rate as phagocytosis in the presence of the corresponding serum concentrations (Fig. 8). Similar results were obtained for Escherichia coli (Fig. 8). Pre-opsonization with 1 and 2.5% serum for 15 min or longer gave higher initial rates of phagocytosis compared with the experiments in which the same concentration of serum was present. With 5% serum a 5 min period of pre-opsonization is sufficient for a maximal initial rate phagocytosis. When pre-opsonized Staphylococcus aureus or Escherichia coli were used, phagocytosis only continued for 30 and 60 min, respectively, after this period, the number of extracellular bacteria started to increase (Fig. 9). When fresh serum was added to the granulocyte-bacteria suspension after 30 or 60 min, however, phagocytosis resumed (Fig. 9). When pre-opsonized Staphylococcus aureus were recovered by differential centrifugation after 60 min of phagocytosis, concentrated to 107 per ml, and then incubated with 5 x 106 granulocytes in the absence of serum, no phagocytosis occurred. But when 2.5% serum was added, these bacteria were phagocytosed at a normal rate. For Escherichia coli, the same results were obtained when the bacteria were recovered after 30 min of phagocytosis. The results of these experiments indicated that the decrease in phagocytosis of pre-opsonized bacteria after a certain period of incubation was due to the absence of opsonins on the surface of the bacteria.
Effect of temperature on the kinetics of phagocytosis To study the effect of temperature on the kinetics of phagocytosis, a series of phagocytosis experiments was performed after pre-warming or cooling of the bacteria, granulocytes, and serum for 1 h at various temperatures. Phagocytosis proved to be a tempera-
460
P. C. J. Leijhet al. Escherichia coti
Staphylococcus aureus
serum concentration
serum concentration
0/0
%/
0.1 1.0
4-
cL c
2.5 L.
tA c
5.0 10.0
0
30
60
90
120
0
30
60
90
120
minutes
Figure 7. Effect of the serum concentrations on the rate of phagocytosis. The phagocytosis assay was performed with a bacteria-to-cell ratio of 1:1 at 37 in the presence of various serum concentrations.
Escherichia coli
Staphylococcus oureus
IE 91) 0 -0
5
c CP
A)
X
4)
a
3,
n 0
2> U
x
_-
-)
No
5
15
5 No 60 Time of pre-opsonizotion (m n)
30
15
30
60
Figure 8. Effect of the duration and serum concentration during pre-opsonization on the initial rate of phagocytosis. Pre-opsonization of Staphylococcus aureus and Escherichia coli was performed for 5, 15, 30, or 60, min with 1 -0 (filled column), 2-5 (hatched columns), 5-0 (open columns), and 10-0 (stippled columns) DO serum, and the initial rate of phagocytosis was compared with that in the experiments in which bacteria were not pre-opsonized (No) but serum was present during the first 30 min.
461
Kinetics ofphagocytosis by human granulocytes Escherichia coLi
Staphylococcus aureus
serum concentration %/
serum concentration %/
c
2.5
100
m ,
c1m
5.0 10.0
c In C
10
a ._
1.
o 0
'a
ee 0.1 0.1 0
30
60
90
120
minutes
Figure 9. Effect of pre-opsonization on the kinetics of phagocytosis. Granulocytes were first incubated with bacteria preopsonized with 0 1, 1 0 or 10-0% serum (serum concentration) and after 30 or 60 min 10% fresh serum (4) was added.
Staphylococcus
Escherichia coil
aureus
temperature
temperature
°C
°c 100
4
-4c
c
(0
45 ._
LCL
'U
45
42
42
10
D
(._
o .0
22
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22
.0
33 39 37
10 Go
cm
33 37
39
L.
a. 0.1 0
30
60
90
120
0
30
60
90
120
minutes
Figure 10. Effect of temperature on the kinetics of phagocytosis. Phagocytosis took place at a bacteria-to-cell ratio of 1:1 in the of 10% serum at various temperatures. Sub-optimal phagocytosis at 45° is restored by the addition of 10% fresh serum
presence (1).
P. C. J. Leijh et al.
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Table 2 Effect of temperature on the opsonization of micro-organisms Degree of phagocytosis Temperature during Temperature during Staphylococcus aureus* Escherichia colit
pre-opsonization (0) 4 22 37 37 37 37 42 45
phagocytosis (0)
(%)
(%)
37 37 37 4 42 45 37 37
95.5 97.7
73.4
97 6 70 97 1 923 96 1 650
75 1 70 3
5*1
732 67-1 71 3 600
* Determined after 45 min of incubation. t Determined after 30 min of incubation.
ture-dependent process, with no detectable phagocytosis at 40 and maximal ingestion at 33-39° (Fig. 10). At higher temperatures (420 and 450) there was a remarkable decrease in the rate of phagocytosis. To find out whether the influence of temperature on the rate of phagocytosis was due to an effect on opsonization or ingestion, micro-organisms were pre-opsonized at various temperatures. After preopsonization at 420 or lower, phagocytosis was normal at 37°. When the bacteria were pre-opsonized at 450, phagocytosis was sub-optimal at 370 (Table 2), which indicates that opsonization was insufficient at this temperature. After pre-opsonization at 37°, temperature-dependent ingestion was observed, with no phagocytosis at 40, maximum ingestion at 370, and near maximum ingestion at 450 (Table 2). Since both opsonization and ingestion were affected at 450, phagocytosis was performed in the presence of 10% serum at 450 and fresh serum was added after 30 and 60 min of incubation. The results show that addition of fresh serum results in continuation of phagocytosis (Fig. 10), which indicates that at this temperature the decrease in the ingestion of bacteria is caused by inadequate opsonization presumably due to inactivation of opsonins. From these results it may be concluded that at temperatures lower than 330, decreased phagocytosis is caused by reduced functioning of the granulocytes, whereas at temperatures above 420 the effect of the temperature is mainly on opsonization. DISCUSSION Phagocytosis of micro-organisms (both Staphylo-
coccus aureus and Escherichia coli) by human granulocytes proved to be a rapid process with an exponential course. At a bacteria-to-cell ratio of 1: 1, less than 1% of the initial number of bacteria remained extracellular after 2 h of phagocytosis. Calculation showed that under these conditions one granulocyte can ingest a maximum of 40-50 bacteria. The rate of phagocytosis in vitro proved to be dependent on the concentration of both micro-organisms and granulocytes, whereas the concentration of serum was rate-limiting when the concentration was too low to provide optimum opsonization. A maximum rate of phagocytosis was reached at temperatures between 33 and 390. The temperature affects the ingestion process above 390, because inactivation of heat-labile opsonins results in a lower rate of ingestion. At lower temperatures the temperature effect proved to be on the ingestion itself, since opsonization was normal. The conclusion that the phagocytosis of microorganisms by granulocytes follows an exponential course could only be reached after a correction had been made. Without this correction the exponential decrease in the number of viable extracellular bacteria seems to be limited to the first 30 min of the assay. The calculated rate of phagocytosis for the initial 30 min does not differ from that calculated for 90 min of phagocytosis if a correction is made for the multiplication of extracellular bacteria. This means that for the study of the kinetics of phagocytosis the initial period of 30 min is adequate. To make certain that the decrease in the number of viable extracellular bacteria is not due to extracellular killing of bacteria and truly represents ingestion of the micro-organisms, a number of control experiments
Kinetics ofphagocytosis by human granulocytes were performed. A bactericidal activity of the medium could be ruled out, because incubation of the investigated strains of Staphylococcus aureus and Escherichia coli with serum alone or with supernatants prepared after various periods of phagocytosis, showed no decrease, and even led to an increase in the number of viable bacteria. Furthermore, no decrease in the number of extracellular bacteria was found when the phagocytosis test was performed in the presence of sodium fluoride, a known inhibitor of ingestion (Karnovsky et al., 1970), and lysostaphin treatment of the granulocytes after phagocytosis of Staphylococcus aureus showed that only 13.4% of the total number of cell-associated bacteria were attached to the cell surface. From these control experiments it could be concluded that the decrease in the number of viable extracellular bacteria during incubation of granulocytes and bacteria under rotation, really represents the ingestion of micro-organisms. To describe the kinetics of phagocytosis in relation to the number of particles and phagocytic cells on the basis of enzyme kinetics, Stossel et al. (1972) used the uptake of oil droplets by granulocytes, Weisman et al. (1967) the phagocytosis of latex particles by Acanthamoeba, Michell et al. (1969) the uptake of starch particles or polystyrene spherules by monolayers of granulocytes, and Magnusson et al. (1977) the ingestion of heat-killed Saccharomyces cerevisiae and granulocytes. This type of approach implies that the rate of phagocytosis is proportional to both the logarithm of the concentration of particles and the logarithm of the concentration of phagocytic cells. Since the present study has shown that that rate of phagocytosis calculated for the initial 30 min represents the real rate of phagocytosis during the entire period of the assay, the initial rate was used throughout our calculations. Incubation of a known number of granulocytes with various concentrations of bacteria showed that the initial rate is proportional to the concentration of bacteria until a maximum is reached. Saturation kinetics for the micro-organisms could not be studied, however, because such high concentrations of bacteria were required that the decrease in the number of viable extracellular bacteria due to phagocytosis could not be measured accrurately. Furthermore, the use of higher concentrations than 1010 bacteria/ml leads to clumping of the micro-organisms, which also hampers accurate counting. With respect to the granulocyte concentration, the rate of phagocytosis is directly proportional to the number of cells above a certain minimum level. Con-
463
centrations of more than 108 granulocytes/ml cannot be used, because agglutination of the cells occurs. From the results of the calculations it may be concluded that it is also valid to apply enzyme kinetics to the phagocytosis of live bacteria by granulocytes, which is the best model for the situation in vivo at the site of an infection. The ingestion of almost all species of live bacteria is dependent on opsonization (Rabinovitch, 1968; Stossel et al., 1973; Griffin, Griffin & Silverstein, 1976; Michl et al., 1976). Therefore, for the study of the kinetics of phagocytosis, experiments must be performed in such a way that opsonization is not ratelimiting. In this respect two aspects have to be considered, namely the duration of pre-opsonization and the serum concentration for opsonization. The results of the present study show that regardless of the serum concentration, at least 15 min of pre-opsonization is required, because otherwise opsonization will be ratelimiting for the ingestion process. The concentration of the serum used for pre-opsonization or present during the entire period of phagocytosis proved to determine the maximum degree of ingestion of bacteria. Maximum ingestion of Staphylococcus aureus is only reached with a serum concentration of at least 2.5% and for Escherichia coli with a serum concentration of 5% or higher. These observations were made in sera from normal donors and granulocytes from healthy individuals, which means that when the phagocytosis-promoting activity of an unknown serum is investigated, a conclusion can only be drawn if the assay performed with bacteria (pre)opsonized with the same minimal serum concntration as that required for normal donor serum to obtain the maximum rate and degree of ingestion. Otherwise, a (partial) deficiency of opsonins may be obscured. It is clear from all this that if some other strain or species of bacteria is used to determine the opsonizing activity of an unknown serum, the minimum concentration required to obtain the maximum rate and degree of ingestion with normal donor serum must be determined first to serve as reference. The effect of temperature on the kinetics of phagocytosis was two-fold: first, an effect was observed on the ingestion process at temperatures from 40 to 330 and maximum ingestion in the temperature range of 33-39°, and second an effect on the opsonization of the micro-organisms was found at higher temperatures. The effect at 420 and higher was mainly due to inactivation of the heat-labile opsonins. Restoration of this activity gave a normal rate of phagocytosis.
464
P. C. J. Leijh et al.
The fact that other investigators (Mandell, 1975; Craig & Suter, 1966) were unable to detect an effect of temperature on the phagocytosis of micro-organisms might be due to the fact that they did not pre-incubate the cells and bacteria at various temperatures. The present results are in conflict with the findings of Peterson et al. (1977), who found abnormal opsonization at 40, whereas attachment of the bacteria to the cells was mainly affected at 410. Previous reports on the phagocytosis of microorganisms based on microbiological assays (Cohn et al., 1959; Li, Mudd & Kapral, 1963; Quie et al., 1967; Solberg et al., 1973) do not deal with many of the interactions studied in the present investigation and show only the curve of the decrease in the number of viable extracellular bacteria, or describe the maximum degree of phagocytosis at various time points, or concern an over-all bactericidal index that includes both ingestion and intracellular killing. In general, if we calculate the initial rate of phagocytosis with their data and taking into account the granulocyte and bacteria concentrations they used, the results are in agreement with ours, but the present study provided more detailed information about the process ofphagocytosis over a wider range of bacteria and granulocyte concentrations. The present method of assessing the phagocytosis of micro-organisms made it possible to calculate the maximum number of bacteria ingested by a single cell. Since 5 x 106 granulocytes can phagocytose a maximum of 1 6 x 108 colony-forming units of Staphylococcus aureus and a colony-forming unit represents on average 1[47 bacteria-counted microscopically and microbiologically (unpublished observation)-the calculated average uptake is 47-3 staphylococci per granulocyte. The corresponding uptake of Escherichia coli, for which one colony-forming unit represents 1-54 bacteria, is 43-0 bacteria per cell. These data are close to those obtained by Bjorksten (1977), who used radiolabelled bacteria and found an uptake of 35-40 bacteria for Escherichia coli and 40 for Streptococcus group B per granulocyte. The uptake of 230 Staphylococcus aureus per granulocyte as calculated by Verbrugh, Peters, Perterson & Verhoef (1978) seems to us too high; this discrepancy is probably attributable to differences in the methods used. The values we obtained can be used to calculate the number of granuloctyes necessary to eliminate the bacteria from the site of an infection. If an inflammatory exudate contains 109 bacteria/ml (in an optimal medium, bacteria reach a concentration of
109-1010/ml), 2 x 107 granulocytes are needed to ingest the bacteria in I ml exudate if one cell ingests 50 micro-organisms. If one granulocyte ingests only 25 bacteria, this number is 4x 107. Since I ml blood contains on average 5 x 106 granulocytes, elimination of the bacteria in I ml exudate would require the migration of the granulocytes in 4 (or 8) ml blood to the site of the inflammation.
ACKNOWLEDGMENTS This study was partially supported by the Foundation for Medical Research Fungo, which is subsidized by The Netherlands Organization for the Advancement of Pure Research (ZWO) and by the J. A. Cohen Institute of Radiopathology and Radiation Protection.
REFERENCES Bj6RKSTEN B., PETERSON P.K., VERHOEF J. & QUIE P.G. (1977) Limiting factors in bacterial phagocytosis by human polymorphonuclear leukocytes. Acta pathol. microbiol. scand. Sect. S, 85, 345. BOUVENG R., SCHILDT B. & SJOGVIST J. (1975) Estimation of RES phagocytosis and catabolism in man by the use of I251-labeled micro-aggregates of human serum albumin. J. reticuloendothel. Soc. 18, 151. CAPO C., BONGRAND P., BENOLIEL A.M. & DEPIEDS R. (1974) Phagocytosis. J. theor. Biol. 47, 177. COHN Z.A. & MORSE S.I. (1959) Interactions between rabbit polymorphonuclear leucocytes and staphylococci. J. exp Med. 110, 419. CRAIG C.P. & SUTER E. (1966) Extracellular factors influencing staphylocidal capacity of human polymorphonuclear leukocytes. J. Immunol. 97,287. FURTH R. VAN, VAN ZWET TH.L. & LEIJH P.C.J. (1978) In vitro determination of phagocytosis and intracellular killing by polymorphonuclear and mononuclear phagocytes. in: Handbook ofExperimental Immunology, 3rd edn. (Ed. by D. M. Weir), Ch. 32. Blackwell Scientific Publications, Oxford. GRIFFIN F.M., GRIFFIN J.A., LEIDER J. & SILVERSTEIN S.C. (1975) Studies on the mechanism of phagocytosis I. Requirements for circumferential attachment of particlebound ligands to specific receptors on the macrophage plasma membrane. J. exp Med. 142, 1263. GRIFFIN F.M., GRIFFIN J.A. & SILVERSTEIN S.C. (1976) Studies on the mechanism of phagocytosis. II. The interaction of macrophages with anti-immunoglobulin IgGcoated bone marrow derived lymphocytes. J. exp Med. 144, 788. JOHNSTON R.B., KLEMPERER, M.R., ALPER C.A. & ROSEN F.S. (1969) The enhancement of bacterial phagocytosis by serum: the role of complement components and two cofactors. J. exp Med. 124. 1275
Kinetics ofphagocytosis by human granulocytes KARNOVSKY M.L., SIMMONS S., GLASS E.A., SHAFER A.W. & D'ARCY HART P. (1970) Metabolism of macrophages. In: Mononuclear Phagocytes (Ed. by R. van Furth), p. 103. Blackwell Scientific Publications, Oxford. Li I.W., MUDD S. & KAPRAL F.A. (1963) Dissociation of phagocytosis and intracellular killing of Staphylococcus aureus by human blood leukocytes. J. Immunol. 90, 804. MALE 0. (1946) On the Relation Between Alexin and
Opsonin. Munksgaard, Copenhagen. MAGNUSSON K. E., DAHLGREN C., STENDAHL 0. & SUNDQVIST T. (1977) Characteristics of the phagocytic process assessed by coulter counter. Acta pathol. microbiol. scand. Sect. C, 85, 215. MANDELL M. L. (1975) Effect of temperature on phagocytosis by human polymorphonuclear neutrophils. Infect. Immun. 12, 221. MICHELL R.H., PANCAKE S.J., NOSEWORTHY J. & KARNOVSKY M.L. (1969) Measurement of rate of phagocytosis: The use of cellular monolayers. J. cell. Biol. 40,216. MICHL J., OHLBAUM D.I. & SILVERSTEIN S.C. (1976) 2Deoxyglucose selectively inhibits Fc and complement receptor-mediated phagocytosis in mouse peritoneal macrophages. description of the inhibiting effect. J. exp Med. 144, 1465. NORMANN S.J. (1973) The kinetics of phagocytosis. J. reticuloendothel. Soc. 14, 587. PETERSON P.K., VERHOEF J. & QUIE P.G. (1977 Influence of temperature on opsonization and phagocytosis of staphylococci. Infect. Immun. 15. 175. QUIE P.G., WHITE J.G., HOLMES B. & GOOD R.A. (1967) In
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vitro bactericidal capacity of human polymorphonuclear leucocytes: diminished activity in chronic granulomatous disease of childhood. J. clin. Invest. 46, 668. RABINOVITCH M. (1968) Phagocytosis: the engulfment stage. Semin. Haematol. 5,134. SOLBERG C.O. & HELLUM K.B. (1973) Influence of serum on the bactericidal activity of neutrophil granulocytes. Acta pathol. microbiol. scand. Sect. B, 81, 621. STIFFEL C., MOUTON D. & Biozzi G. (1970) Kinetics of the phagocytic function of reticuloendothelial macrophages in vivo. In: Mononuclear Phagocytes (Ed. by R. van Furth), p. 335. Blackwell Scientific Publications, Oxford. STOSSEL T.P., MASON R.J., HARTWIG J. & VAUGHAN M. (1972) Quantitative studies of phagocytosis by polymorphonuclear leukocytes: use of emulsions to measure the initial rate of phagocytosis. J. clin. Invest. 51, 615. STOSSEL T.P., ALPER C.A. & ROSEN F.S. (1973) Serum-dependent phagocytosis of paraffin oil emulsified with bacterial lipopolysaccharide. J. exp Med. 137,690. TAN J.S., WATANAKUNAKORN C. & PHAIR J.P. (1971) A modified assay of neutrophil function: use of lysostaphin to differentiate defective phagocytosis from impaired intracellular killing. J. lab. clin. Med. 78, 316. VERBRUGH H.A., PETERS R., PETERSON P.K. & VERHOEF J. (1978) Phagocytosis and killing of staphylococci by human polymorphonuclear and mononuclear leucocytes. J. clin. Pathol. 31, 539. WEISMAN R.A. & KORN E.D. (1967) Phagocytosis of latex beads by Acanthamoeba. I. Biochemical properties. Biochemistry 6, 485.
Kinetics of phagocytosis of Staphylococcus aureus and Escherichia coli by human granulocytes
P. C. J. LEIJH, MARIA TH. VAN DEN BARSELAAR, THEDA L. VAN ZWET, IVONNE DUBBELDEMAN-REMPT & R. VAN FURTH Department of Infectious Diseases, University Hospital, Leiden, The Netherlands
Received 16 October 1978; acceptedfor publication 20 November 1978
Summary. Although phagocytosis of micro-organisms by granulocytes is one of the most important defence mechanisms against infection, little is known about the kinetics of this process. The present study showed that the rate of ingestion of Staphylococcus aureus and Escherichia coli depends on the concentrations of the granulocytes and bacteria. Phagocytosis of bacteria at a bacteria-to-cell ratio in the range between 100:1 and 1:10 showed an exponential course during the first 30 min. At a bacteria-to-cell ratio of 1: 1, application of a correction for the outgrowth of extracellular bacteria gave an exponential course of ingestion over the first 90-min period. Since it was found that the phagocytosis of bacteria by granulocytes at various bacteriato-cell ratios can be described with Michaelis-Menten kinetics, we studied the kinetics of phagocytosis on the basis of the initial rate for the first 30-min period. The rate of phagocytosis and the maximal degree of ingestion of bacteria by granulocytes proved to be related to the concentration of serum used in the assay. The minimal serum concentration required for maximal ingestion was 2 5% for Staphylococcus aureus and 5% for Escherichia coli. When bacteria were preopsonized, the duration of pre-opsonization proved to be limiting for the rate of phagocytosis in dependence
on the serum concentration. The effect of temperature on the phagocytosis of micro-organisms proved to be two-fold. First, at temperatures between 4 and 330 a decrease in the functioning of the cells leads to a
decrease in the rate of phagocytosis. Above 420, the temperature affects mainly the opsonization of the micro-organisms and has only a slight influence on the ingestion process. From the data obtained in this study, maximal rates of 6-3 x 106 Staphylococcus aureusi5 x 106 granulocytes/min and of 7-1 x 106 Escherichia colil5 x 106 granulocytes/min were calculated for phagocytosis at a bacteria-to-cell ratio of 100:1 at 370, i.e. on average about one bacterium per granulocyte per min. The maximum calculated number of bacteria ingested by one granulocyte lies between 40 and 50.
INTRODUCTION Phagocytosis of micro-organisms by granulocytes and monocytes is one of the most important of the body's defence mechanisms against infection, since it is the only mechanism by which micro-organisms which have invaded the tissues can be eliminated. Information about this process has been obtained with both in vivo and in vitro techniques. The former, which are based on measurement of the clearance of substances injected into an animal, have the disadvantage of being influenced by many unknown variables, such as
Correspondence. Professor R. van Furth, Department of Internal Medicine & Infectious Diseases, University Hospital, Leiden, The Netherlands. 00 19-2805/79/0600-0453$02.00 C) 1979 Blackwell Scientific Publications
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P. C. J. Leijh et al.
humoral factors, the blood flow, the concentration of particles, the number and functional state of the phagocytic cells. In vivo techniques are also limited by the fact that the measurements concern mainly the functioning of the Kupffer cells and to a lesser extent that of the spleen macrophages (Normann, 1973; Stiffel, Mouton & Biozzi, 1970; Bouveng, Schildt & Sjogvist, 1975). Studies carried out in vitro have the advantage that both homogeneous, well defined and characterized phagocytes and particles can be used. Phagocytosis experiments can be performed with cells in different functional states and under various experimental conditions, for instance attached to glass or plastic surfaces (Michell, Pancake, Noseworthy & Karnovsky, 1969) or in suspension (Cohn & Morse, 1959; van Furth, van Zwet & Leijh, 1978; Stossel, Mason, Hartwig & Vaughan, 1972). Serum factors, such as immunoglobulins, complement components, or other proteins, that are necessary for the opsonization of the particles (Stossel, Alper & Rosen, 1973; Johnston, Klemperer, Alper & Rosen 1969; Griffin, Griffin, Leider & Silverstein 1975; Michl, Ohlbaum & Silverstein, 1976; Rabinovitch, 1968), can also be kept under control in in vitro experiments. On this basis, techniques of several kinds have been developed to measure phagocytosis in vitro. For instance, the number of particles (micro-organisms, yeast cells, latex particles, or oil droplets) ingested per cell can be determined microscopically (Capo, Bongrand, Benoliel & Depieds, 1974; Weisman & Korn, 1967). The number of ingested micro-organisms can be determined with the use of radiolabelled dead or living organisms after the removal of the extracellular population (Peterson, Verhoef & Quie, 1977). The ingestion of viable microorganisms can also be measured microbiologically as a function of their extracellular disappearance (Cohn et al., 1959; Solberg & Hellum, 1973; Quie, White, Holmes & Good, 1967). In our opinion, this last approach gives the best approximation of the situations occurring at the site of an inflammation in vivo. Although this method has been used by many investigators, quantitative information about the rate of phagocytosis by normal human granulocytes in various bacteria-to-cell ratios and about the opsonization of bacteria is not available. Such information is indispensable for the interpretation of the results obtained using granulocytes and serum opsonins from patients. The present study was therefore undertaken to investigate the kinetics of opsonization and phagocytosis of live Staphylococcus
aureus and Escherichia coli by normal human granulocytes as determined in vitro by a microbiological method. MATERIALS AND METHODS Reagents A stock solution of 300 ug/ml lysostaphin (Sigma Chemical Co., St Louis, Mo.) was prepared in phosphate-buffered saline and stored in l-ml aliquots at -20°. A stock solution of 02 M sodium fluoride (Merck, Darmstadt, Germany) was prepared in Hanks's balanced salt solution and stored at 4°.
Granulocytes Blood was obtained from healthy donors by venepuncture. Granulocytes were collected by sedimentation of erythrocytes with a 5% solution of dextran in buffered saline (mol. wt 2000,000; 3 ml solution to 10 ml blood). The leucocyte-rich layer was washed twice with phosphate-buffered saline containing 0-5 u/ml heparin, after which a cell suspension of 1-2 x 107 granulocytes/ml in Hanks's salt solution with 0.1% (w/v) gelatin was prepared. Serum In all experiments, serum prepared from blood of healthy AB blood donors was used. The blood was clotted for I h at room temperature, centrifuged for 20 min at 1100 g, and stored until use in aliquots of 2 ml for up to 2 months at -20°. Micro-organisms The Staphylococcus aureus (type 42D) and Escherichia coli (O 54) used in all experiments, were stored on agar slants at 4° and transferred every 14 days. The microorganisms were cultured overnight in Nutrient Broth no. 2 (Oxoid Ltd, London), harvested by centrifugation at 1500 g for 10 min, and washed twice with phosphate-buffered saline (pH 7 2) after which a suspension containing 107 bacteria per ml in Hanks's balanced salt solution with 01% (w/v) gelatin was prepared. Pre-opsonization of bacteria Pre-opsonized micro-organisms were obtained by incubation of suspensions (5 x 106 bacteria/ml) with normal AB serum for 25 min at 370 under slow rotation (4 r.p.m.) followed by centrifugation at 1 5OOg for 10 min and two washings in ice-cold gelatin-
455
Kinetics ofphagocytosis by human granulocytes
Hanks's solution. The bacteria then were suspended to a concentration of approximately I07 organisms/ml in gelatin-Hanks's solution.
the percentage decrease of the initial number of viable extracellular bacteria according to the formula No- N. ~ x 100, R No in which Ft) is the phagocytosis index at time t, No is the number of viable extracellular bacteria at time t=0, and N. is the number of viable extracellular bacteria at time t = t. The initial rate of phagocytosis (V0) was calculated according to the formula Vo =k No, in which k is the initial rate constant. This rate constant was calculated according to the formula lnN0 - lnN, =
Phagocytosis Phagocytosis was measured as described elsewhere (van Furth et al., 1978). In short, for a standard assay a suspension of 107 granulocytes per ml was incubated with an equal volume of 107 bacteria per ml with 10% (v/v) serum at 370 under slow rotation (4 r.p.m.). At various time points, a 0-5 ml sample of the suspension was added to 1 5 ml ice-cold gelatin-Hanks's salt solution to stop phagocytosis. After 4 min of centrifugation at 110 g to spin down the granulocytes, the number of viable micro-organisms in the supernatant was determined by a microbiological plate method. At this sedimentation gravity, 99% of the Staphylococcus aureus and Escherichia coli remain in suspension (van Furth et al., 1978). In this study other bacteria-togranulocyte ratios were also used.
Calculations Phagocytosis at a given time point was expressed as
RESULTS
Phagocytosis of Staphylococcus aureus and Escherichia coi Incubation of Staphylococcus aureus or Escherichia coli with granulocytes, both in final concentration of
Escherichia coli
Staphylococcus aureus
bacteria serum
c
standing
100
control
._.
i
10-
a!
._.
a
1-
'IOU
bacteria
!0.1 serum ~ ~ ~~'vcorrected
0.
\
85granulocytes bacteria
0 v
0
W0
120
0
60
corrected
120
9;
minutes
Figure 1. The kinetics of phagocytosis of Staphylococcus aureus and Escherichia coli by human granulocytes. 5 x 106 granulocytes/ml were incubated with 5 x 106 bacteria/ml and 10% serum at 370 under rotation (4 r.p.m.). The number of viable extracellular bacteria determined at 5 min (x) or 15 min (o) intervals. Incubation without rotation (standing control 4), or without granulocytes (-) Corrected (v) is curve obtained after correction for bacterial growth.
P. C. J. Leijh et al.
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5 x 106/ml, in the presence of 10% serum under-slow rotation (4 r.p.m.), showed that phagocytosis-of these micro-organisms was a rapid process (Fig. 1). Incubation of either strain with granulocytes in a ratio of 1:1 at 370 showed an exponential decrease in the number of viable extracellular micro-organisms-determined at intervals of 5 min-during the first 20-30 min. During this period more than 80% of the initial number of micro-organisms were ingested, whereas during the remaining 90 min this percentage rose to 99 5 for Staphylococcus aureus and 99 8 for Escherichia coli. When, instead of 5 min, intervals of 15 or 30 min were used to measure the number of viable extracellular micro-organisms, the same curve was obtained (Fig. 1). Thus, the same information about the rate of phagocytosis is obtained with the use of longer intervals. Calculation of the rate of phagocytosis for periods of 30 and 90 min showed a marked decrease for the longer compared with the shorter period (Table 1). Table 1. Effect of correction for bacterial growth on the initial rate of phagocytosis Initial rate
(bacteria/min/5 x 106 granulocytes) Interval
S. aureus
E. coli
30 min, direct experiment 90 min, direct experiment 90 min, after growth correction
5-1 x 105 2 8 x 105 4 8 x 105
4 6 x 105 2 7 x 105 4 3 x 105
Since multiplication of micro-organisms in the presence of serum tends to start during the second hour of incubation, a correction must be made for this growth in the calculation of the rate of phagocytosis. Under the assumption that the growth of micro-organisms in a bacteria-granulocyte suspension equals that in a suspension of bacteria only, the corrected number of extracellular bacteria can be calculated according to the formula Bo
NC, = N,B., in which NC, is the corrected number of extracellular bacteria at time t, N, the measured number of extracellular bacteria at time t, Bo the initial number of bacteria in the control suspension containing only bacteria, and B. the number of bacteria in the control
at time t. After this correction, the results showed an exponential decrease in the number of viable extracellular bacteria over a period of 90 min (Fig. 1). The rate constant of this decrease calculated for this interval did not differ from that obtained for the first 30 min without correction (Table 1). From these results we may conclude that the difference between uncorrected rates of phagocytosis for incubation periods of 30 and 90 min is due solely to the multiplication of extracellular micro-organisms, and that the information on the rate of phagocytosis obtained during the first 30 min is representative for the entire experimental period. Control studies to exclude extracellular killing of microorganisms The decrease in the number of viable extracellular bacteria was not due to extracellular killing of microorganisms, because: (1) incubation of a bacterial suspension without granulocytes in the presence of serum showed no decrease but an increase in the number of micro-organisms, which indicates that there was no bactericidal activity in the serum used for both bacteria species (Fig. 1); (2) incubation of the microorganisms with granulocytes in the presence of 10% serum at 37° without rotation (i.e. the standing control, Fig. 1) showed no decrease in the number of extracellular micro-organisms, thus indicating that no phagocytosis occurs without contact between cells and bacteria and that no extracellular factors reduced the number of viable micro-organisms; (3) supernatants from granulocytes and bacteria prepared after 0, 60, or 120 min of phagocytosis showed no bactericidal activity for either Staphylococcus aureus and Escherichia coli when incubated with bacteria for 2 h at 37°. Control studies to demonstrate the ingestion of microorganisms To find out whether the decrease in the number of extracellular micro-organisms was due to attachment of bacteria to granulocytes or to ingestion of the bacteria by these cells, the number of cell-associated bacteria in the cell pellet after 0 or 60 min of incubation was determined before and after the removal of the attached bacteria. First, the effect of washing of the granulocyte pellet with Hanks's solution was investigated. After washing, there was a decrease of 7.3% (range 4.3-10.3% and 114% (range 6-8-15l1%) for Staphylococcus aureus and Escherichia coli, respectively, in the total number of viable bacteria in the cell
457
Kinetics ofphagocytosis by human granulocytes
pellet determined after disruption of the granulocytes with distilled water with 00 00% (w/v) human albumin. Second, the cell pellet was incubated for 5 min at 370 in the presence of lysostaphin (10 pg/ml) to lyse extracellular Staphylococcus aureus (Tan, Watanakunakorn & Phair, 1971), which reduced the total number ofviable bacteria by 13.4% (range 6-4-21-3%) as compared with incubation of the cell pellet for 5 min at 370 in medium only. (Incubation of 5 x 106 Staphylococcus aureus with 10 Mg lysostaphin/ml for 5 min at 370 gave a reduction of more than 99 9% of the viable bacteria.) Third, the phagocytic assay was performed in the presence of sodium fluoride, which is known to inhibit ingestion (Karnovsky, Simmons, Glass, Shafer & D'Arcy Hart, 1970). Incubation with 0 2 M sodium fluoride for 60 min gave a phagocytic index of 45% for Staphylococcus aureus and 38% for Escherichia coli. From the findings in these three kinds of control experiments it may be concluded that in the assay used in this study, the decrease in the number of viable extracellular bacteria mainly represents phagocytosis of these micro-organisms. Effect of various bacteria-to-cell ratioson the kinetics of phagocytosis Incubation of 5 x 106 granulocytes/ml with various concentrations of micro-organisms (range 5 x 104 to 5 x 109/ml) in the presence of serum showed that at bacteria-to-granulocyte ratios higher than 1:1 the phagocytosis of both Staphylococcus aureus and Escherichia coli was limited (Fig. 2). Incubation of granulocytes and bacteria at bacteria-to-granulocyte ratios of 100:1 and 1000:1 only gave reliable information about the kinetics of phagocytosis for periods of 90 and 60 min, respectively, because after these intervals the granulocytes were saturated with bacteria and became disrupted. Bacteria concentrations of 1010/ml or more were not used because of bacterial clumping, which hampers accurate counting of the micro-organisms. The values obtained in these experiments were used to calculate the rate of phagocytosis during the initial 30 min of the assay for the various bacteria-to-granulocyte ratios. The results (see Fig. 3) for the incubation of 5 x 106 granulocytes/ml showed a proportional relationship between the logarithm of the initial rate of phagocytosis and the logarithm of the initial number of bacteria at concentrations of 5 x 104 to 4 x I07 bacteria/ml, whereas at higher concentrations a maximal rate of ingestion was reached, amounting to 6-3 x 106 bacteria/5 x 106 cells/min for Staphylococcus aureus
Staphylococcus aureus
Escherichia coli
bacteria to
cell ratio
bacteria to cell ratio
0 a)
n c ._
a)
-o 0
D 0-
Time(min)
Figure 2. The kinetics of phagocytosis at various bacteria-tocell ratios. 5 x 106 granulocytes/ml were incubated with various concentrations of bacteria at 37°.
Granulocyte
concentration (cells/ml)
5x107 _n
5x 106
0
0 en
.x 0 0 0
5x105
OL 0 c .'
0 -J
Log initial concentration of bacteria
Figure 3. Effect of the concentrations of Staphylococcus aureus (&) and Escherichia coli (a) on the initial rate of phagocytosis (first 30 min period) at various granulocyte concentrations. The data for 5 x 106 granulocytes/ml are taken from Fig. 2.
P. C. J.
458
and of 7-1 x 106 bacteria/5 x 106 cells/min for Escherichia coli. Next, the effect of various granulocyte concentrations on the kinetics of phagocytosis was studied at concentrations ranging from 5 x 104 to 5 x 107 cells per ml. Incubation of 5 x 104 granulocytes per ml with bacteria suspensions of 104 to 106/ml at 370 under rotation (4 r.p.m.) did not lead to any detectable decrease in the number of extracellular micro-organisms. Phagocytosis during incubation of 5 x 105 to 5 x 107 granulocytes per ml with various concentrations of bacteria was characterized by an initial rate depending on the bacteria-to-cell ratio and the number of granulocytes. Figure 3 shows the relationship between the logarithm of the initial rate of phagocytosis and the logarithm of the initial number of micro-organisms as calculated for the various experiments with 104-109 micro-organisms and 5 x 105 to 5 x 107 granulocytes/ml. A log-log plot based on these data (Fig. 4) shows that there is a relationship between the intitial rate of phagocytosis and the granulocyte concentration. From these experiments it may be concluded that the rate of phagocytosis in vitro depends not only on the bacteria-to-cell ratio but also on the concentration of both the bacteria and the granulocytes.
Leijh et al. Determination of the phagocytic capacity of granulocytes To determine the maximum number of microorganisms that can be ingested by granulocytes, experiments were designed for a 3 h period in which the initial bacteria-to-cell ratio was re-established every 30 min by the addition of fresh bacteria. As can be seen from Fig. 5, at a bacteria-to-cell ratio of 1:1 the rate of
4o9
(a
Baccteria to cet.1 ratio
bacteria added
i
100:1 CL
10o
II
I
10:1 to -O.
A
-, Zs
I
L-
1:1
I
x
I
.0
I
106
I~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
o
A
z
Initial bacteria
105 0
(bacteria/ml) 8
t.
60
90
120
150
Figure 5. Effect on the kinetics of phagocytosis of re-establishment of the bacteria-to-cell ratio at 30 min intervals. Incubation of 5 x 106 granulocytes with 5 x 106 (ratio= 1:1),
5x 108
5x l0' (ratio=10:1) or 5x 108 (ratio=100:1) Staphylococcus aureus/ml and 10% serum at 37° under rotation (4
5x107
r.p.m.).
7
-I
30
minutes
concentration
0
06 o
5x 106
0
a
2o '5
5
0'
o -J
5x105
4
7 8 Log granulocyte concentration
6
Figure 4. Log-log plot prepared from the data of Fig. 3, showing a linear relationship between the granulocyte concentration and the initial rate of phagocytosis.
phagocytosis of Staphylococcus aureus during the first 30 min was similar to that in the interval from 120 to 150 min. At a bacteria-to-cell ratio of 10:1 there was a decrease in the rate of phagocytosis after 90 min and no phagocytosis at all at 120 min. At a ratio of 100:1, detectable phagocytic activity ceased after 30 min. During incubation, the number of granulocytes was counted every 30 min and re-adjusted for the next interval of incubation to 5 x 106f cells/ml by changing the volume of the medium; during these experiments the viability of the cells remained above 90%. The same results were obtained when Escherichia coli was used as test organism (not shown). These data were used to calculate the total number
Kinetics of phagocytosis by human granulocytes of bacteria ingested by 5 x 106 granulocytes during each interval over 150 min. Figure 6 shows that at a bacteria-to-cell ratio of 100:1 the maximal number of Staphylococcus aureus (1 2 x 108) or Escherichia coli (1-1 X 108) is ingested within the first 30 min, after which there is almost no phagocytosis. At lower bacteria-to-cell ratios the uptake is lower, and for a ratio of 1:1 follows a linear course. x108
100:1
6x1070 XI8
0
:3
7
8X
O= 5x107 1
/
0)
2x107 07
CD
x107 25x105
0 1061:
106
30
60
90
120
150
Time (min)
Figure 6. Calculation of the maximal number of bacteria ingested by 5 x 106 granulocytes, on the basis of the data in Fig. 5. Summation of the total number of Staphylococcus aureus (a) or Escherichia coli (o) ingested during each 30 min interval.
The effect of serum on the rate of phagocytosis Incubation of Staphylococcus aureus or Escherichia coli with granulocytes in the presence of various concentrations of serum at 370 showed that both the rate of phagocytosis and maximal phagocytosis depend on the serum concentration (Fig. 7). This dose-response experiment showed that at least 2.5% serum was necessary to obtain maximum phagocytosis of Staphylococcus aureus whereas for Escherichia coli the minimum necessary serum concentration was 5%.
459
To find out whether opsonization of microorganisms was rate-limiting in the phagocytic process, phagocytosis was performed with pre-opsonized bacteria. Bacteria were pre-opsonized with various concentrations of serum for various periods before use in the phagocytosis assay in medium without serum. As a control, the standard phagocytosis assay of granulocytes and non pre-opsonized bacteria in the continuous presence of various concentrations of serum was used. The results show that Staphylococcus aureus pre-opsonized with 1% serum for 15 min or longer gave a higher initial rate than phagocytosis in the continuous presence of 1% serum (Fig. 8). At higher serum concentrations, 5 min of pre-opsonization gave the same initial rate as phagocytosis in the presence of the corresponding serum concentrations (Fig. 8). Similar results were obtained for Escherichia coli (Fig. 8). Pre-opsonization with 1 and 2.5% serum for 15 min or longer gave higher initial rates of phagocytosis compared with the experiments in which the same concentration of serum was present. With 5% serum a 5 min period of pre-opsonization is sufficient for a maximal initial rate phagocytosis. When pre-opsonized Staphylococcus aureus or Escherichia coli were used, phagocytosis only continued for 30 and 60 min, respectively, after this period, the number of extracellular bacteria started to increase (Fig. 9). When fresh serum was added to the granulocyte-bacteria suspension after 30 or 60 min, however, phagocytosis resumed (Fig. 9). When pre-opsonized Staphylococcus aureus were recovered by differential centrifugation after 60 min of phagocytosis, concentrated to 107 per ml, and then incubated with 5 x 106 granulocytes in the absence of serum, no phagocytosis occurred. But when 2.5% serum was added, these bacteria were phagocytosed at a normal rate. For Escherichia coli, the same results were obtained when the bacteria were recovered after 30 min of phagocytosis. The results of these experiments indicated that the decrease in phagocytosis of pre-opsonized bacteria after a certain period of incubation was due to the absence of opsonins on the surface of the bacteria.
Effect of temperature on the kinetics of phagocytosis To study the effect of temperature on the kinetics of phagocytosis, a series of phagocytosis experiments was performed after pre-warming or cooling of the bacteria, granulocytes, and serum for 1 h at various temperatures. Phagocytosis proved to be a tempera-
460
P. C. J. Leijhet al. Escherichia coti
Staphylococcus aureus
serum concentration
serum concentration
0/0
%/
0.1 1.0
4-
cL c
2.5 L.
tA c
5.0 10.0
0
30
60
90
120
0
30
60
90
120
minutes
Figure 7. Effect of the serum concentrations on the rate of phagocytosis. The phagocytosis assay was performed with a bacteria-to-cell ratio of 1:1 at 37 in the presence of various serum concentrations.
Escherichia coli
Staphylococcus oureus
IE 91) 0 -0
5
c CP
A)
X
4)
a
3,
n 0
2> U
x
_-
-)
No
5
15
5 No 60 Time of pre-opsonizotion (m n)
30
15
30
60
Figure 8. Effect of the duration and serum concentration during pre-opsonization on the initial rate of phagocytosis. Pre-opsonization of Staphylococcus aureus and Escherichia coli was performed for 5, 15, 30, or 60, min with 1 -0 (filled column), 2-5 (hatched columns), 5-0 (open columns), and 10-0 (stippled columns) DO serum, and the initial rate of phagocytosis was compared with that in the experiments in which bacteria were not pre-opsonized (No) but serum was present during the first 30 min.
461
Kinetics ofphagocytosis by human granulocytes Escherichia coLi
Staphylococcus aureus
serum concentration %/
serum concentration %/
c
2.5
100
m ,
c1m
5.0 10.0
c In C
10
a ._
1.
o 0
'a
ee 0.1 0.1 0
30
60
90
120
minutes
Figure 9. Effect of pre-opsonization on the kinetics of phagocytosis. Granulocytes were first incubated with bacteria preopsonized with 0 1, 1 0 or 10-0% serum (serum concentration) and after 30 or 60 min 10% fresh serum (4) was added.
Staphylococcus
Escherichia coil
aureus
temperature
temperature
°C
°c 100
4
-4c
c
(0
45 ._
LCL
'U
45
42
42
10
D
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.0
33 39 37
10 Go
cm
33 37
39
L.
a. 0.1 0
30
60
90
120
0
30
60
90
120
minutes
Figure 10. Effect of temperature on the kinetics of phagocytosis. Phagocytosis took place at a bacteria-to-cell ratio of 1:1 in the of 10% serum at various temperatures. Sub-optimal phagocytosis at 45° is restored by the addition of 10% fresh serum
presence (1).
P. C. J. Leijh et al.
462
Table 2 Effect of temperature on the opsonization of micro-organisms Degree of phagocytosis Temperature during Temperature during Staphylococcus aureus* Escherichia colit
pre-opsonization (0) 4 22 37 37 37 37 42 45
phagocytosis (0)
(%)
(%)
37 37 37 4 42 45 37 37
95.5 97.7
73.4
97 6 70 97 1 923 96 1 650
75 1 70 3
5*1
732 67-1 71 3 600
* Determined after 45 min of incubation. t Determined after 30 min of incubation.
ture-dependent process, with no detectable phagocytosis at 40 and maximal ingestion at 33-39° (Fig. 10). At higher temperatures (420 and 450) there was a remarkable decrease in the rate of phagocytosis. To find out whether the influence of temperature on the rate of phagocytosis was due to an effect on opsonization or ingestion, micro-organisms were pre-opsonized at various temperatures. After preopsonization at 420 or lower, phagocytosis was normal at 37°. When the bacteria were pre-opsonized at 450, phagocytosis was sub-optimal at 370 (Table 2), which indicates that opsonization was insufficient at this temperature. After pre-opsonization at 37°, temperature-dependent ingestion was observed, with no phagocytosis at 40, maximum ingestion at 370, and near maximum ingestion at 450 (Table 2). Since both opsonization and ingestion were affected at 450, phagocytosis was performed in the presence of 10% serum at 450 and fresh serum was added after 30 and 60 min of incubation. The results show that addition of fresh serum results in continuation of phagocytosis (Fig. 10), which indicates that at this temperature the decrease in the ingestion of bacteria is caused by inadequate opsonization presumably due to inactivation of opsonins. From these results it may be concluded that at temperatures lower than 330, decreased phagocytosis is caused by reduced functioning of the granulocytes, whereas at temperatures above 420 the effect of the temperature is mainly on opsonization. DISCUSSION Phagocytosis of micro-organisms (both Staphylo-
coccus aureus and Escherichia coli) by human granulocytes proved to be a rapid process with an exponential course. At a bacteria-to-cell ratio of 1: 1, less than 1% of the initial number of bacteria remained extracellular after 2 h of phagocytosis. Calculation showed that under these conditions one granulocyte can ingest a maximum of 40-50 bacteria. The rate of phagocytosis in vitro proved to be dependent on the concentration of both micro-organisms and granulocytes, whereas the concentration of serum was rate-limiting when the concentration was too low to provide optimum opsonization. A maximum rate of phagocytosis was reached at temperatures between 33 and 390. The temperature affects the ingestion process above 390, because inactivation of heat-labile opsonins results in a lower rate of ingestion. At lower temperatures the temperature effect proved to be on the ingestion itself, since opsonization was normal. The conclusion that the phagocytosis of microorganisms by granulocytes follows an exponential course could only be reached after a correction had been made. Without this correction the exponential decrease in the number of viable extracellular bacteria seems to be limited to the first 30 min of the assay. The calculated rate of phagocytosis for the initial 30 min does not differ from that calculated for 90 min of phagocytosis if a correction is made for the multiplication of extracellular bacteria. This means that for the study of the kinetics of phagocytosis the initial period of 30 min is adequate. To make certain that the decrease in the number of viable extracellular bacteria is not due to extracellular killing of bacteria and truly represents ingestion of the micro-organisms, a number of control experiments
Kinetics ofphagocytosis by human granulocytes were performed. A bactericidal activity of the medium could be ruled out, because incubation of the investigated strains of Staphylococcus aureus and Escherichia coli with serum alone or with supernatants prepared after various periods of phagocytosis, showed no decrease, and even led to an increase in the number of viable bacteria. Furthermore, no decrease in the number of extracellular bacteria was found when the phagocytosis test was performed in the presence of sodium fluoride, a known inhibitor of ingestion (Karnovsky et al., 1970), and lysostaphin treatment of the granulocytes after phagocytosis of Staphylococcus aureus showed that only 13.4% of the total number of cell-associated bacteria were attached to the cell surface. From these control experiments it could be concluded that the decrease in the number of viable extracellular bacteria during incubation of granulocytes and bacteria under rotation, really represents the ingestion of micro-organisms. To describe the kinetics of phagocytosis in relation to the number of particles and phagocytic cells on the basis of enzyme kinetics, Stossel et al. (1972) used the uptake of oil droplets by granulocytes, Weisman et al. (1967) the phagocytosis of latex particles by Acanthamoeba, Michell et al. (1969) the uptake of starch particles or polystyrene spherules by monolayers of granulocytes, and Magnusson et al. (1977) the ingestion of heat-killed Saccharomyces cerevisiae and granulocytes. This type of approach implies that the rate of phagocytosis is proportional to both the logarithm of the concentration of particles and the logarithm of the concentration of phagocytic cells. Since the present study has shown that that rate of phagocytosis calculated for the initial 30 min represents the real rate of phagocytosis during the entire period of the assay, the initial rate was used throughout our calculations. Incubation of a known number of granulocytes with various concentrations of bacteria showed that the initial rate is proportional to the concentration of bacteria until a maximum is reached. Saturation kinetics for the micro-organisms could not be studied, however, because such high concentrations of bacteria were required that the decrease in the number of viable extracellular bacteria due to phagocytosis could not be measured accrurately. Furthermore, the use of higher concentrations than 1010 bacteria/ml leads to clumping of the micro-organisms, which also hampers accurate counting. With respect to the granulocyte concentration, the rate of phagocytosis is directly proportional to the number of cells above a certain minimum level. Con-
463
centrations of more than 108 granulocytes/ml cannot be used, because agglutination of the cells occurs. From the results of the calculations it may be concluded that it is also valid to apply enzyme kinetics to the phagocytosis of live bacteria by granulocytes, which is the best model for the situation in vivo at the site of an infection. The ingestion of almost all species of live bacteria is dependent on opsonization (Rabinovitch, 1968; Stossel et al., 1973; Griffin, Griffin & Silverstein, 1976; Michl et al., 1976). Therefore, for the study of the kinetics of phagocytosis, experiments must be performed in such a way that opsonization is not ratelimiting. In this respect two aspects have to be considered, namely the duration of pre-opsonization and the serum concentration for opsonization. The results of the present study show that regardless of the serum concentration, at least 15 min of pre-opsonization is required, because otherwise opsonization will be ratelimiting for the ingestion process. The concentration of the serum used for pre-opsonization or present during the entire period of phagocytosis proved to determine the maximum degree of ingestion of bacteria. Maximum ingestion of Staphylococcus aureus is only reached with a serum concentration of at least 2.5% and for Escherichia coli with a serum concentration of 5% or higher. These observations were made in sera from normal donors and granulocytes from healthy individuals, which means that when the phagocytosis-promoting activity of an unknown serum is investigated, a conclusion can only be drawn if the assay performed with bacteria (pre)opsonized with the same minimal serum concntration as that required for normal donor serum to obtain the maximum rate and degree of ingestion. Otherwise, a (partial) deficiency of opsonins may be obscured. It is clear from all this that if some other strain or species of bacteria is used to determine the opsonizing activity of an unknown serum, the minimum concentration required to obtain the maximum rate and degree of ingestion with normal donor serum must be determined first to serve as reference. The effect of temperature on the kinetics of phagocytosis was two-fold: first, an effect was observed on the ingestion process at temperatures from 40 to 330 and maximum ingestion in the temperature range of 33-39°, and second an effect on the opsonization of the micro-organisms was found at higher temperatures. The effect at 420 and higher was mainly due to inactivation of the heat-labile opsonins. Restoration of this activity gave a normal rate of phagocytosis.
464
P. C. J. Leijh et al.
The fact that other investigators (Mandell, 1975; Craig & Suter, 1966) were unable to detect an effect of temperature on the phagocytosis of micro-organisms might be due to the fact that they did not pre-incubate the cells and bacteria at various temperatures. The present results are in conflict with the findings of Peterson et al. (1977), who found abnormal opsonization at 40, whereas attachment of the bacteria to the cells was mainly affected at 410. Previous reports on the phagocytosis of microorganisms based on microbiological assays (Cohn et al., 1959; Li, Mudd & Kapral, 1963; Quie et al., 1967; Solberg et al., 1973) do not deal with many of the interactions studied in the present investigation and show only the curve of the decrease in the number of viable extracellular bacteria, or describe the maximum degree of phagocytosis at various time points, or concern an over-all bactericidal index that includes both ingestion and intracellular killing. In general, if we calculate the initial rate of phagocytosis with their data and taking into account the granulocyte and bacteria concentrations they used, the results are in agreement with ours, but the present study provided more detailed information about the process ofphagocytosis over a wider range of bacteria and granulocyte concentrations. The present method of assessing the phagocytosis of micro-organisms made it possible to calculate the maximum number of bacteria ingested by a single cell. Since 5 x 106 granulocytes can phagocytose a maximum of 1 6 x 108 colony-forming units of Staphylococcus aureus and a colony-forming unit represents on average 1[47 bacteria-counted microscopically and microbiologically (unpublished observation)-the calculated average uptake is 47-3 staphylococci per granulocyte. The corresponding uptake of Escherichia coli, for which one colony-forming unit represents 1-54 bacteria, is 43-0 bacteria per cell. These data are close to those obtained by Bjorksten (1977), who used radiolabelled bacteria and found an uptake of 35-40 bacteria for Escherichia coli and 40 for Streptococcus group B per granulocyte. The uptake of 230 Staphylococcus aureus per granulocyte as calculated by Verbrugh, Peters, Perterson & Verhoef (1978) seems to us too high; this discrepancy is probably attributable to differences in the methods used. The values we obtained can be used to calculate the number of granuloctyes necessary to eliminate the bacteria from the site of an infection. If an inflammatory exudate contains 109 bacteria/ml (in an optimal medium, bacteria reach a concentration of
109-1010/ml), 2 x 107 granulocytes are needed to ingest the bacteria in I ml exudate if one cell ingests 50 micro-organisms. If one granulocyte ingests only 25 bacteria, this number is 4x 107. Since I ml blood contains on average 5 x 106 granulocytes, elimination of the bacteria in I ml exudate would require the migration of the granulocytes in 4 (or 8) ml blood to the site of the inflammation.
ACKNOWLEDGMENTS This study was partially supported by the Foundation for Medical Research Fungo, which is subsidized by The Netherlands Organization for the Advancement of Pure Research (ZWO) and by the J. A. Cohen Institute of Radiopathology and Radiation Protection.
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Kinetics ofphagocytosis by human granulocytes KARNOVSKY M.L., SIMMONS S., GLASS E.A., SHAFER A.W. & D'ARCY HART P. (1970) Metabolism of macrophages. In: Mononuclear Phagocytes (Ed. by R. van Furth), p. 103. Blackwell Scientific Publications, Oxford. Li I.W., MUDD S. & KAPRAL F.A. (1963) Dissociation of phagocytosis and intracellular killing of Staphylococcus aureus by human blood leukocytes. J. Immunol. 90, 804. MALE 0. (1946) On the Relation Between Alexin and
Opsonin. Munksgaard, Copenhagen. MAGNUSSON K. E., DAHLGREN C., STENDAHL 0. & SUNDQVIST T. (1977) Characteristics of the phagocytic process assessed by coulter counter. Acta pathol. microbiol. scand. Sect. C, 85, 215. MANDELL M. L. (1975) Effect of temperature on phagocytosis by human polymorphonuclear neutrophils. Infect. Immun. 12, 221. MICHELL R.H., PANCAKE S.J., NOSEWORTHY J. & KARNOVSKY M.L. (1969) Measurement of rate of phagocytosis: The use of cellular monolayers. J. cell. Biol. 40,216. MICHL J., OHLBAUM D.I. & SILVERSTEIN S.C. (1976) 2Deoxyglucose selectively inhibits Fc and complement receptor-mediated phagocytosis in mouse peritoneal macrophages. description of the inhibiting effect. J. exp Med. 144, 1465. NORMANN S.J. (1973) The kinetics of phagocytosis. J. reticuloendothel. Soc. 14, 587. PETERSON P.K., VERHOEF J. & QUIE P.G. (1977 Influence of temperature on opsonization and phagocytosis of staphylococci. Infect. Immun. 15. 175. QUIE P.G., WHITE J.G., HOLMES B. & GOOD R.A. (1967) In
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vitro bactericidal capacity of human polymorphonuclear leucocytes: diminished activity in chronic granulomatous disease of childhood. J. clin. Invest. 46, 668. RABINOVITCH M. (1968) Phagocytosis: the engulfment stage. Semin. Haematol. 5,134. SOLBERG C.O. & HELLUM K.B. (1973) Influence of serum on the bactericidal activity of neutrophil granulocytes. Acta pathol. microbiol. scand. Sect. B, 81, 621. STIFFEL C., MOUTON D. & Biozzi G. (1970) Kinetics of the phagocytic function of reticuloendothelial macrophages in vivo. In: Mononuclear Phagocytes (Ed. by R. van Furth), p. 335. Blackwell Scientific Publications, Oxford. STOSSEL T.P., MASON R.J., HARTWIG J. & VAUGHAN M. (1972) Quantitative studies of phagocytosis by polymorphonuclear leukocytes: use of emulsions to measure the initial rate of phagocytosis. J. clin. Invest. 51, 615. STOSSEL T.P., ALPER C.A. & ROSEN F.S. (1973) Serum-dependent phagocytosis of paraffin oil emulsified with bacterial lipopolysaccharide. J. exp Med. 137,690. TAN J.S., WATANAKUNAKORN C. & PHAIR J.P. (1971) A modified assay of neutrophil function: use of lysostaphin to differentiate defective phagocytosis from impaired intracellular killing. J. lab. clin. Med. 78, 316. VERBRUGH H.A., PETERS R., PETERSON P.K. & VERHOEF J. (1978) Phagocytosis and killing of staphylococci by human polymorphonuclear and mononuclear leucocytes. J. clin. Pathol. 31, 539. WEISMAN R.A. & KORN E.D. (1967) Phagocytosis of latex beads by Acanthamoeba. I. Biochemical properties. Biochemistry 6, 485.
