Journal o f Immunological Methods, 14 (1977) 303--311 © Elsevier/North-Holland Biomedical Press
303
KINETICS OF STAPHYLOCOCCAL OPSONIZATION, ATTACHMENT, INGESTION AND KILLING BY HUMAN POLYMORPHONUCLEAR LEUKOCYTES: A QUANTITATIVE ASSAY USING [3tt]THYMIDINE LABELED BACTERIA *
JAN VERHOEF 1, PHILLIP K. PETERSON 2 and PAUL G. QUIE 3 Departments o f Medicine and Pediatrics, University o f Minnesota School o f Medicine, Minneapolis, Minnesota 55455, U.S.A.
(Received 16 August 1976, accepted 5 October 1976)
A method has been developed for studying quantitatively the separate processes of bacterial opsonization, phagoeytosis, and killing by human polymorphonuelear leukocytes using [3H]thymidine labeled Staphylococcus aurcus. Phagoeytosis is determined by assaying for leukoeytes-associated radioactivity after differential eentrifugation and washing the leukocytes. Opsonization is studied by incubating bacteria with an opsonic source for varying durations and then adding leukocytes. By treatment of samples with the muralytie enzyme, lysostaphin, the attachment and ingestion phases of phagocytosis can be separated. Sampling for colony forming units after disruption of the leukoeytes permits the measurement of bacterial killing. Using this method, differences in the kinetics of staphylococcal opsonization by normal and C2 deficient sera were defined, opsonic influences on the attachment and ingestion phases of phagocytosis were delineated, and the influences of different opsonins and leukocyte populations on killing were determined.
INTRODUCTION The outcome of the interaction between polymorphonuclear (PMN) leuk o c y t e s a n d b a c t e r i a is d e t e r m i n e d b y t h e i n t e g r i t y o f t h r e e p r o c e s s e s : 1) b a c t e r i a l o p s o n i z a t i o n b y h e a t - s t a b l e a n d h e a t - l a b i l e s e r u m f a c t o r s , 2) p h a g o c y t o s i s , a n d 3) k i l l i n g . T h e p r o c e s s o f p h a g o c y t o s i s c a n b e f u r t h e r s e p a r a t e d into two phases, bacterial attachment to phagocytic cells and internalization ( i n g e s t i o n ) ( R a b i n o v i t c h , 1 9 6 7 ; G r i f f i n e t al., 1 9 7 5 ) . C o n s i d e r a b l e k n o w l edge has been gained of the bactericidal function of PMN leukocytes; much of this insight has come from studies of leukocytes from patients with bac* This work was supported in part by funds from the United States Public Health Services grants AI 06931 and AI 08821. ' Supported by the Netherlands Organization for the Advancement of Pure Research (ZWO). Present address: Laboratory for Microbiology, Cathariinesingel 59, Utrecht, Netherlands. 2 Recipient of Bristol Research Fellowship in Infections Diseases. American Legion Memorial Heart Research Professor.
304 tericidal defects (Klebanoff, 1975; Quie, 1975). Understanding of the process of opsonization and phagocytosis has also progressed (Stossel, 1975). Little is known, however, of the kinetics and exact interrelationships of these processes. Using [3H]thymidine labeled bacteria, a m e t h o d has been developed which allows simultaneous, i n d e p e n d e n t evaluation of the process o f opsonization, a t t a c h m e n t , ingestion and killing of staphylococci. MATERIALS AND METHODS
Bacterial strains and radioactive labeling Two Staphylococcal aureus strains, Cowan I and 502A were used. Onet e n th ml of an overnight culture of bacteria was inoculated into 10 ml Mueller--Hinton b r o t h (Difco, Detroit, MI) containing 0.02 mCi t hym i di ne methy l P H (specific activity 6.7 Ci/mmol, New England Nuclear, Boston, MA). After 18 h growth at 37°C, the bacteria were washed 3 times in phosphatebuffered saline, pH 7.4 (PBS). A final bacterial concent rat i on of 5 X 10 s colo n y forming units (CFU)/ml PBS was obtained using a s p e c t r o p h o t o m e t r i c m e t h o d confirmed by pour plate c ol ony counts.
Leukocytes F o r t y ml o f blood was drawn from healthy volunteers and from 5 patients with chronic granulomatous disease (CGD) in a syringe containing 200 U heparin. The e r y t h r o c y t e s were sedimented for 1 h in 6% dextran '70' (Cutter Labs., Berkeley, CA) in normal saline (10 ml blood: 3 ml saline). The leuk o c y t e rich plasma was withdrawn and centrifuged at 160 g for 5 min. The resulting pellet was washed twice in heparinized saline (10 U heparin/10 ml saline). Using a standard m e t hod, total and differential l eukocyt e counts were performed, and the final l e u k o c y t e pellet was resuspended to a concentration of 107 PMN l e ukoc yt e s / m l Hank's Balanced Salt Solution with 1% gelatin (HBSS).
Opsonins and bacterial opsonization Serum from 5 normal donors was pooled and kept frozen in 1 ml aliquots at --70°C. C2 deficient serum, obtained from a patient with inherited complete C2 deficiency, was kindly provided by Dr. Youngki Kim, University of Minnesota (Kim, Y., et al. Submitted for publication), and was kept frozen at --70°C in 0.2 ml aliquots. Shortly before use the aliquots were thawed and diluted to a final c onc e nt r at i on of 10% in HBSS. Heat-inactivated normal serum was prepared by heating aliquots at 56°C for 1 h and then diluting to a final c o n c e n t r a t i o n of 10%. In one series of experiments 0.15 ml of a bacterial c o n c e n t r a t i o n of 5 × 108 CFU/ml was added to each of four plastic tubes (12 × 75 mm, Falcon,
305 Oxnard, CA) containing 0.8 ml opsonin (normal, heat-inactivated and C2 deficient sera). After incubation for 1, 5, 15 and 60 rain at 37°C, 3 ml icecold PBS was added to each tube to stop opsonization followed by centrifuging at 1600 g for 15 min at 4°C. The supernatants were discarded and the bacterial pellets resuspended in 0.75 ml HBSS.
Phagocytosis mixtures Mixtures of 1.5 ml of leukocyte suspension, serum and bacteria were prepared in plastic tubes (12 X 75 mm, Falcon) in a volume ratio of 5 : 4 : 1. The final bacteria : PMN leukocyte ratio was approximately 10 : 1 in all experiments. In those experiments in which opsonized bacteria were added to the leukocytes, the same final volume and bacteria : leukocyte ratios were used. The phagocytosis mixtures were tumbled at 10 rpm at 37°C in a rotating rack (Fisher Roto-Rack, Fisher Scientific Co., Chicago, IL).
Determination o f PMN leukocyte bacterial uptake and killing Immediately after the phagocytosis mixtures were constituted, duplicate 5 pl samples were taken with an Eppendorf pipette to determine the total CFU added to the mixtures at time '0'. These samples were diluted in tubes containing 5 ml sterile water from which 5 gl samples were taken and plated in nutrient agar. After 48 h incubation at 37°C CFU were counted. To determine the leukocyte-associated bacterial population, duplicate 100 #l samples were taken from the phagocytosis mixtures with an Eppendorf pipette at 3, 10 and 20 min intervals and placed in 3 ml cold PBS in polypropylene vials (Bio-Vials, Beckman, Chicago, IL}. The vials were centrifuged for 5 min at 160 g at 4°C and the leukocyte pellets washed twice with ice cold PBS. The final leukocyte pellets were disrupted by vigorous mixing in 2.5 ml sterile distilled water. From these suspensions duplicate 5 pl samples were taken with an Eppendorf pipette to determine the viable leukocyteassociated bacterial population by plating in nutrient agar. CFU were counted after 48 h incubation at 37°C. The final 2.5 ml suspensions were centrifuged at 1600 g for 15 min, and the total leukocyte-associated population of bacteria (alive and dead) was then determined by solubilizing the pellets in 2.5 ml scintillation liquid (toluene containing fluoralloy (TLA, Beckman) and 20% Biosolve-3 (Beckman)) and counting in a liquid scintillation counter (Beckman LS-250). To determine the total bacteria-associated cpm (representing leukocyte-associated bacteria plus extracellular bacteria) duplicate 100 pl samples were taken at the end of the assay period, placed in 2.5 ml water and centrifuged at 1600 g for 15 min. The pellets were resuspended in 2.5 ml scintillation liquid and counted. An average of the duplicate values was used for all calculations. The percent of the total bacterial population that was phagocytized at a given sampling time (% uptake) was calculated using the formula:
306 % uptake =
cpm in leukocyte pellet cpm in total bacterial pellet X 100.
The phagocytized bacterial population that remained viable at a given sampling time was calculated according to the formula: % viable leukocyte-associated bacteria = % CFU in leukocyte pellet X 100, % uptake where the denominator was obtained from the above formula and the numerator was derived using the formula: CFU in leukocyte pellet at sampling time × 100. CFU at time '0' The killed population of bacteria was determined according to the formula: % killed leukocyte-associated bacteria = = % uptake --% viable leukocyte-associated bacteria.
Determination of the attached bacterial population
To lyse the extracellular population of bacteria, including those bacteria that were attached to but not ingested by the leukocytes, duplicate 100 pl samples were placed in PBS containing 1 pg/ml lysostaphin (Schwarz-Mann, Orangeburg, NY). These samples were then incubated at 37°C for 30 min followed by washing the leukocyte pellets twice in cold PBS. Simultaneous samples were processed in the standard manner to determine the total phagocytized bacterial population (attached plus ingested bacteria). Bacterial uptake was calculated as outlined above. The difference between the % uptake calculated from the samples placed in PBS and the samples placed in PBS containing lysostaphin was considered to represent the attached bacterial population. As less than 10% of the total cpm were leukocyte-associated at 20 min when bacteria were added to phagocytosis mixtures in the absence of an opsonic source (HBSS), it appeared that washing the leukocytes eliminated most of the extracellular bacteria and that lysostaphin lowered the leukocyte-associated cpm primarily by lysing attached bacteria. To confirm lysostaphin activity, control mixtures containing the same concentration of bacteria and serum but no leukocytes were also samples in PBS and PBS containing 1/lg/ml lysostaphin followed by incubation at 37°C for 30 min. After centrifuging at 1600 g, the bacterial pellets were suspended in scintillation liquid and counted.
307 RESULTS
Kinetics o f staphylococcal opsonization with normal, C2 deficient and heatinactivated sera The uptake o f S. aureus Cowan I by normal PMN leukocytes was studied after incubating the bacteria for 1, 5, 15 and 60 min in normal serum and in serum from a patient with C2 deficiency (fig. 1). When normal serum was used as an opsonic source, opsonization was completed within 5 min. No significant difference in the kinetics of phagocytosis could be detected using bacteria opsonized for 5, 15 and 60 rain (fig. la). In contrast, opsonization o f S. aureus Cowan I was markedly depressed with C2 deficient serum. After incubation of bacteria for 5 rain with C2 deficient serum only 39% of the bacteria were phagocytized com pa r ed with 80% phagocytosis after 5 rain incubation with normal serum. After incubation for 60 rain with C2 deficient serum, 69% of bacteria were phagocytized (fig. l b ) . When heat-inactivated serum served as an opsonic source, phagocytosis was significantly less than that observed with either normal or C2 deficient sera. As with normal serum, opsonization was complete after bacteria were incubated for 5 min with heat-inactivated serum (data not shown).
Kinetics o f staphylococcal attachment and ingestion Fig. 2 demonstrates the effect of lysostaphin on samples from phagocyto100
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Fig. 2. Effect of lysostaphin on measured leukocyte-associated radioactivity. 5’. aureus Cowan I was added to normal PMN leukocytes in the presence of normal and heat-inactivated sera. Samples were placed in PBS and in PBS containing lysostaphin, and after incubation, the leukocytes were washed and bacterial uptake was determined.
sis mixtures containing S. aureus Cowan I, normal PMN leukocytes, and normal or heat-inactivated sera. When samples from mixtures containing bacteria opsonized with normal serum were incubated in lysostaphin, there was approximately a 10% decrease in leukocyte-associated bacteria. Samples from control mixtures containing the same concentration of bacteria in normal serum but no leukocytes lost over 90% of centrifugeable (1600 g) radioactivity after incubation in lysostaphin. Therefore, it appeared that a large majority of the leukocyte-associated bacteria were ingested, and that a relatively small proportion were attached to the leukocytes. A relatively greater effect of lysostaphin on the total leucocyte-associated bacterial population was noted when bacteria were opsonized with heat-inactivated serum. After 20 min incubation with leukocytes, 32% of the leukocyte-associated bacterial population was removed by lysostaphin treatment of samples taken from the mixture containing bacteria and heat-inactivated serum compared with 10% removal when normal serum was used as the opsonic source.
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the effect of opsonins on PMN leukocyte staphylococcal killing, S. aureus 502A was added to leukocytes in the presence of normal and of heat-inactivated sera and bacterial uptake and viability were determined (fig. 3). A greater percentage of the total bacterial population was phagocytized when normal serum was used as an opsonin. Whereas 67% of the bacteria were phagocytized after 20 min incubation with leukocytes in the presence
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Fig. 3. E f f e c t o f o p s o n i c s o u r c e o n p h a g o c y t o s i s a n d killing o r s . a u r e u s 5 0 2 A. B a c t e r i a w e r e a d d e d t o n o r m a l P M N l e u k o c y t e s in t h e p r e s e n c e o f n o r m a l s e r u m a n d h e a t - i n a c t i v a t e d s e r u m . P h a g o c y t o s i s a n d v i a b l e l e u k o c y t e - a s s o c i a t e d b a c t e r i a w e r e a s s a y e d as described. Fig. 4. I n f l u e n c e o f l e u k o c y t e p o p u l a t i o n o n p h a g o e y t o s i s a n d killing o f S a u r e u s C o w a n I. B a c t e r i a w e r e a d d e d to n o r m a l d o n o r a n d C G D P M N l e u k o e y t e s in t h e p r e s e n c e o f n o r mal serum.
o f normal serum, only 25% were taken up when heat-inactivated serum was used. Killing by PMN leukocytes also appeared to be decreased when heatinactivated serum was used as an opsonic source. A p p r o x i m a t e l y twice as many leukocyte-associated bacteria were found to be viable in the m i xt ure with heat-inactivated serum at 3, 10 and 20 min when compared with the viable leukocyte-associated bacteria in the mixture containing normal serum. Similar data was obtained when S. a u r e u s Cowan I was studied. To study the influence of different l e u k o c y t e populations on staphylococcal killing, PMN leukocytes from 5 patients with CGD, a disease with a known intraleukocytic staphylocidal def e c t (Quie et al., 1967), were compared with leukocytes from healthy volunteers. Fig. 4 is representative of the results obtained in this experiment. Phagocytosis of S. a u r e u s Cowan I was n o t f o u n d to differ significantly when leukocytes from the patients were c o m p a r e d with control cells. There was, however, a greater percentage of surviving staphylococci when the bacteria were phagocytized by leukocytes from patients with GGD. Incubation of samples in lysostaphin resulted in comparable decreases in leukocyte-associated bacteria indicating that the size of the attached, non-ingested bacterial population was similar for both patient and normal d o n o r leukocytes (data not presented ).
310 DISCUSSION The kinetics of S. a u r e u s opsonization, a t t a c h m e n t , ingestion and killing were studied quantitatively using [ 3H]thymidine labeled bacteria and different opsonic sources and PMN l e u k o c y t e populations. By incubating bacteria with normal serum, C2 deficient serum and heat-inactivated serum for varying durations of time prior to adding normal PMN leukocytes, differences in the opsonie capacities of these sera could be defined. Within 5 min opsonization with normal and heat-inactivated sera was complete. Opsonization with C2 deficient serum proceeded at a slower rate; staphylococci opsonized for 60 min were significantly better phagocytized than were bacteria opsonized for 5 and 15 min. These findings are consistent with previous observations that the classical c o m p l e m e n t pathway is essential for optimal opsonization of some S. a u r e u s strains and that opsonization in the absence of this pathway b u t in the presence of the alternative pathway occurs at a slower rate (Forsgren and Quie, 1974; Verhoef et al., submitted for publication). To separate the two phases of phagocytosis (at t achm ent and ingestion), samples from phagocytosis mixtures were incubated in lysostaphin to remove attached, non-ingested leukocyte-associated bacteria. Lysostaphin is a staphylococcal muralytic e n z y m e that does not enter leukocytes (Tan et al., 1971). When bacteria were opsonized with normal serum, a p p r o x i m a t e l y 10% of the leukocyte-associated bacteria were removed by lysostaphin treatment. When bacteria opsonized with heat-inactivated serum were studied, a greater percentage of leukocyte-associated bacteria (approximately 30%) were removed by lysostaphin. These findings suggest that bacteria opsonized in the absence of c o m p l e m e n t (heat-inactivated serum) are not only less efficiently attached to PMN leukocytes, but are also internalized at a slower rate than are bacteria opsonized in the presence of c o m p l e m e n t (normal serum). Bacterial killing by PMN leukocytes was assessed, after t horough washing o f the leukocytes, by disrupting the cells with water and sampling for CFU. Using this technique, it was found that a greater percent of leukocyte-associated bacteria were alive when bacteria were opsonized with heat-inactivated serum than when bacteria were opsonized with normal serum. The explanation of this finding may lie in the previous observation that bacteria opsonized with heat-inactivated serum are internalized at a slower rate than are bacteria opsonized with normal serum, and they would presumably t h er eb y be killed at a slower rate. When the killing capacity of PMN leukocytes from patients with CGD was c o mp ar ed with normal d o n o r leukocytes, a greater percent of bacteria associated with the patients' leukocytes were viable. This is consistent with the known staphylocidal defect in leukocytes from patients with this disease (Quie et al., 1967). No significant difference in either a t t a c h m e n t or ingestion could be de t e c t e d when CGD leukocytes were compared with d o n o r leukoeytes. The enhanced phagocytosis of CGD leukocytes reported elsewhere (Biggar, 1975) may be the result of methodological differences. A
311
false impression of increased phagocytosis by CGD leukocytes might arise if only the numbers of viable leukocyte-associated bacteria are compared. We are currently using the methods described to investigate other bacterial strains and phagocyte cell populations from patients with a variety of impaired host defense problems. REFERENCES
Biggar, W.D., 1975, Lancet i, 991. Forsgren, A. and P.G. Quie, 1974, Infect. Immunol. 1O, 402. Griffin, F.M., J.A. Griffin, J.E. Leider and S.C. Silverstein, 1975, J. Exp. Med. 142, 1263. Klebanoff, S.J., 1975, Semin. Hematol. 12, 117. Quie, P.G., J.G. White, B. Holmes and R.A. Good, 1967, J. Clin. Invest. 46, 668. Quie, P.G., 1975, Semin. Hematol. 12, 143. Rabinovitch, M., 1967, Exp. Cell Res. 46, 19. Stossel, T.P., 1975, Semin. Hematol. 12, 83. Tan, J.S., C. Watanakunakorn and J.P. Phair, 1971, J. Lab. Clin. Med. 70, 316.
303
KINETICS OF STAPHYLOCOCCAL OPSONIZATION, ATTACHMENT, INGESTION AND KILLING BY HUMAN POLYMORPHONUCLEAR LEUKOCYTES: A QUANTITATIVE ASSAY USING [3tt]THYMIDINE LABELED BACTERIA *
JAN VERHOEF 1, PHILLIP K. PETERSON 2 and PAUL G. QUIE 3 Departments o f Medicine and Pediatrics, University o f Minnesota School o f Medicine, Minneapolis, Minnesota 55455, U.S.A.
(Received 16 August 1976, accepted 5 October 1976)
A method has been developed for studying quantitatively the separate processes of bacterial opsonization, phagoeytosis, and killing by human polymorphonuelear leukocytes using [3H]thymidine labeled Staphylococcus aurcus. Phagoeytosis is determined by assaying for leukoeytes-associated radioactivity after differential eentrifugation and washing the leukocytes. Opsonization is studied by incubating bacteria with an opsonic source for varying durations and then adding leukocytes. By treatment of samples with the muralytie enzyme, lysostaphin, the attachment and ingestion phases of phagocytosis can be separated. Sampling for colony forming units after disruption of the leukoeytes permits the measurement of bacterial killing. Using this method, differences in the kinetics of staphylococcal opsonization by normal and C2 deficient sera were defined, opsonic influences on the attachment and ingestion phases of phagocytosis were delineated, and the influences of different opsonins and leukocyte populations on killing were determined.
INTRODUCTION The outcome of the interaction between polymorphonuclear (PMN) leuk o c y t e s a n d b a c t e r i a is d e t e r m i n e d b y t h e i n t e g r i t y o f t h r e e p r o c e s s e s : 1) b a c t e r i a l o p s o n i z a t i o n b y h e a t - s t a b l e a n d h e a t - l a b i l e s e r u m f a c t o r s , 2) p h a g o c y t o s i s , a n d 3) k i l l i n g . T h e p r o c e s s o f p h a g o c y t o s i s c a n b e f u r t h e r s e p a r a t e d into two phases, bacterial attachment to phagocytic cells and internalization ( i n g e s t i o n ) ( R a b i n o v i t c h , 1 9 6 7 ; G r i f f i n e t al., 1 9 7 5 ) . C o n s i d e r a b l e k n o w l edge has been gained of the bactericidal function of PMN leukocytes; much of this insight has come from studies of leukocytes from patients with bac* This work was supported in part by funds from the United States Public Health Services grants AI 06931 and AI 08821. ' Supported by the Netherlands Organization for the Advancement of Pure Research (ZWO). Present address: Laboratory for Microbiology, Cathariinesingel 59, Utrecht, Netherlands. 2 Recipient of Bristol Research Fellowship in Infections Diseases. American Legion Memorial Heart Research Professor.
304 tericidal defects (Klebanoff, 1975; Quie, 1975). Understanding of the process of opsonization and phagocytosis has also progressed (Stossel, 1975). Little is known, however, of the kinetics and exact interrelationships of these processes. Using [3H]thymidine labeled bacteria, a m e t h o d has been developed which allows simultaneous, i n d e p e n d e n t evaluation of the process o f opsonization, a t t a c h m e n t , ingestion and killing of staphylococci. MATERIALS AND METHODS
Bacterial strains and radioactive labeling Two Staphylococcal aureus strains, Cowan I and 502A were used. Onet e n th ml of an overnight culture of bacteria was inoculated into 10 ml Mueller--Hinton b r o t h (Difco, Detroit, MI) containing 0.02 mCi t hym i di ne methy l P H (specific activity 6.7 Ci/mmol, New England Nuclear, Boston, MA). After 18 h growth at 37°C, the bacteria were washed 3 times in phosphatebuffered saline, pH 7.4 (PBS). A final bacterial concent rat i on of 5 X 10 s colo n y forming units (CFU)/ml PBS was obtained using a s p e c t r o p h o t o m e t r i c m e t h o d confirmed by pour plate c ol ony counts.
Leukocytes F o r t y ml o f blood was drawn from healthy volunteers and from 5 patients with chronic granulomatous disease (CGD) in a syringe containing 200 U heparin. The e r y t h r o c y t e s were sedimented for 1 h in 6% dextran '70' (Cutter Labs., Berkeley, CA) in normal saline (10 ml blood: 3 ml saline). The leuk o c y t e rich plasma was withdrawn and centrifuged at 160 g for 5 min. The resulting pellet was washed twice in heparinized saline (10 U heparin/10 ml saline). Using a standard m e t hod, total and differential l eukocyt e counts were performed, and the final l e u k o c y t e pellet was resuspended to a concentration of 107 PMN l e ukoc yt e s / m l Hank's Balanced Salt Solution with 1% gelatin (HBSS).
Opsonins and bacterial opsonization Serum from 5 normal donors was pooled and kept frozen in 1 ml aliquots at --70°C. C2 deficient serum, obtained from a patient with inherited complete C2 deficiency, was kindly provided by Dr. Youngki Kim, University of Minnesota (Kim, Y., et al. Submitted for publication), and was kept frozen at --70°C in 0.2 ml aliquots. Shortly before use the aliquots were thawed and diluted to a final c onc e nt r at i on of 10% in HBSS. Heat-inactivated normal serum was prepared by heating aliquots at 56°C for 1 h and then diluting to a final c o n c e n t r a t i o n of 10%. In one series of experiments 0.15 ml of a bacterial c o n c e n t r a t i o n of 5 × 108 CFU/ml was added to each of four plastic tubes (12 × 75 mm, Falcon,
305 Oxnard, CA) containing 0.8 ml opsonin (normal, heat-inactivated and C2 deficient sera). After incubation for 1, 5, 15 and 60 rain at 37°C, 3 ml icecold PBS was added to each tube to stop opsonization followed by centrifuging at 1600 g for 15 min at 4°C. The supernatants were discarded and the bacterial pellets resuspended in 0.75 ml HBSS.
Phagocytosis mixtures Mixtures of 1.5 ml of leukocyte suspension, serum and bacteria were prepared in plastic tubes (12 X 75 mm, Falcon) in a volume ratio of 5 : 4 : 1. The final bacteria : PMN leukocyte ratio was approximately 10 : 1 in all experiments. In those experiments in which opsonized bacteria were added to the leukocytes, the same final volume and bacteria : leukocyte ratios were used. The phagocytosis mixtures were tumbled at 10 rpm at 37°C in a rotating rack (Fisher Roto-Rack, Fisher Scientific Co., Chicago, IL).
Determination o f PMN leukocyte bacterial uptake and killing Immediately after the phagocytosis mixtures were constituted, duplicate 5 pl samples were taken with an Eppendorf pipette to determine the total CFU added to the mixtures at time '0'. These samples were diluted in tubes containing 5 ml sterile water from which 5 gl samples were taken and plated in nutrient agar. After 48 h incubation at 37°C CFU were counted. To determine the leukocyte-associated bacterial population, duplicate 100 #l samples were taken from the phagocytosis mixtures with an Eppendorf pipette at 3, 10 and 20 min intervals and placed in 3 ml cold PBS in polypropylene vials (Bio-Vials, Beckman, Chicago, IL}. The vials were centrifuged for 5 min at 160 g at 4°C and the leukocyte pellets washed twice with ice cold PBS. The final leukocyte pellets were disrupted by vigorous mixing in 2.5 ml sterile distilled water. From these suspensions duplicate 5 pl samples were taken with an Eppendorf pipette to determine the viable leukocyteassociated bacterial population by plating in nutrient agar. CFU were counted after 48 h incubation at 37°C. The final 2.5 ml suspensions were centrifuged at 1600 g for 15 min, and the total leukocyte-associated population of bacteria (alive and dead) was then determined by solubilizing the pellets in 2.5 ml scintillation liquid (toluene containing fluoralloy (TLA, Beckman) and 20% Biosolve-3 (Beckman)) and counting in a liquid scintillation counter (Beckman LS-250). To determine the total bacteria-associated cpm (representing leukocyte-associated bacteria plus extracellular bacteria) duplicate 100 pl samples were taken at the end of the assay period, placed in 2.5 ml water and centrifuged at 1600 g for 15 min. The pellets were resuspended in 2.5 ml scintillation liquid and counted. An average of the duplicate values was used for all calculations. The percent of the total bacterial population that was phagocytized at a given sampling time (% uptake) was calculated using the formula:
306 % uptake =
cpm in leukocyte pellet cpm in total bacterial pellet X 100.
The phagocytized bacterial population that remained viable at a given sampling time was calculated according to the formula: % viable leukocyte-associated bacteria = % CFU in leukocyte pellet X 100, % uptake where the denominator was obtained from the above formula and the numerator was derived using the formula: CFU in leukocyte pellet at sampling time × 100. CFU at time '0' The killed population of bacteria was determined according to the formula: % killed leukocyte-associated bacteria = = % uptake --% viable leukocyte-associated bacteria.
Determination of the attached bacterial population
To lyse the extracellular population of bacteria, including those bacteria that were attached to but not ingested by the leukocytes, duplicate 100 pl samples were placed in PBS containing 1 pg/ml lysostaphin (Schwarz-Mann, Orangeburg, NY). These samples were then incubated at 37°C for 30 min followed by washing the leukocyte pellets twice in cold PBS. Simultaneous samples were processed in the standard manner to determine the total phagocytized bacterial population (attached plus ingested bacteria). Bacterial uptake was calculated as outlined above. The difference between the % uptake calculated from the samples placed in PBS and the samples placed in PBS containing lysostaphin was considered to represent the attached bacterial population. As less than 10% of the total cpm were leukocyte-associated at 20 min when bacteria were added to phagocytosis mixtures in the absence of an opsonic source (HBSS), it appeared that washing the leukocytes eliminated most of the extracellular bacteria and that lysostaphin lowered the leukocyte-associated cpm primarily by lysing attached bacteria. To confirm lysostaphin activity, control mixtures containing the same concentration of bacteria and serum but no leukocytes were also samples in PBS and PBS containing 1/lg/ml lysostaphin followed by incubation at 37°C for 30 min. After centrifuging at 1600 g, the bacterial pellets were suspended in scintillation liquid and counted.
307 RESULTS
Kinetics o f staphylococcal opsonization with normal, C2 deficient and heatinactivated sera The uptake o f S. aureus Cowan I by normal PMN leukocytes was studied after incubating the bacteria for 1, 5, 15 and 60 min in normal serum and in serum from a patient with C2 deficiency (fig. 1). When normal serum was used as an opsonic source, opsonization was completed within 5 min. No significant difference in the kinetics of phagocytosis could be detected using bacteria opsonized for 5, 15 and 60 rain (fig. la). In contrast, opsonization o f S. aureus Cowan I was markedly depressed with C2 deficient serum. After incubation of bacteria for 5 rain with C2 deficient serum only 39% of the bacteria were phagocytized com pa r ed with 80% phagocytosis after 5 rain incubation with normal serum. After incubation for 60 rain with C2 deficient serum, 69% of bacteria were phagocytized (fig. l b ) . When heat-inactivated serum served as an opsonic source, phagocytosis was significantly less than that observed with either normal or C2 deficient sera. As with normal serum, opsonization was complete after bacteria were incubated for 5 min with heat-inactivated serum (data not shown).
Kinetics o f staphylococcal attachment and ingestion Fig. 2 demonstrates the effect of lysostaphin on samples from phagocyto100
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Fig. 2. Effect of lysostaphin on measured leukocyte-associated radioactivity. 5’. aureus Cowan I was added to normal PMN leukocytes in the presence of normal and heat-inactivated sera. Samples were placed in PBS and in PBS containing lysostaphin, and after incubation, the leukocytes were washed and bacterial uptake was determined.
sis mixtures containing S. aureus Cowan I, normal PMN leukocytes, and normal or heat-inactivated sera. When samples from mixtures containing bacteria opsonized with normal serum were incubated in lysostaphin, there was approximately a 10% decrease in leukocyte-associated bacteria. Samples from control mixtures containing the same concentration of bacteria in normal serum but no leukocytes lost over 90% of centrifugeable (1600 g) radioactivity after incubation in lysostaphin. Therefore, it appeared that a large majority of the leukocyte-associated bacteria were ingested, and that a relatively small proportion were attached to the leukocytes. A relatively greater effect of lysostaphin on the total leucocyte-associated bacterial population was noted when bacteria were opsonized with heat-inactivated serum. After 20 min incubation with leukocytes, 32% of the leukocyte-associated bacterial population was removed by lysostaphin treatment of samples taken from the mixture containing bacteria and heat-inactivated serum compared with 10% removal when normal serum was used as the opsonic source.
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the effect of opsonins on PMN leukocyte staphylococcal killing, S. aureus 502A was added to leukocytes in the presence of normal and of heat-inactivated sera and bacterial uptake and viability were determined (fig. 3). A greater percentage of the total bacterial population was phagocytized when normal serum was used as an opsonin. Whereas 67% of the bacteria were phagocytized after 20 min incubation with leukocytes in the presence
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Fig. 3. E f f e c t o f o p s o n i c s o u r c e o n p h a g o c y t o s i s a n d killing o r s . a u r e u s 5 0 2 A. B a c t e r i a w e r e a d d e d t o n o r m a l P M N l e u k o c y t e s in t h e p r e s e n c e o f n o r m a l s e r u m a n d h e a t - i n a c t i v a t e d s e r u m . P h a g o c y t o s i s a n d v i a b l e l e u k o c y t e - a s s o c i a t e d b a c t e r i a w e r e a s s a y e d as described. Fig. 4. I n f l u e n c e o f l e u k o c y t e p o p u l a t i o n o n p h a g o e y t o s i s a n d killing o f S a u r e u s C o w a n I. B a c t e r i a w e r e a d d e d to n o r m a l d o n o r a n d C G D P M N l e u k o e y t e s in t h e p r e s e n c e o f n o r mal serum.
o f normal serum, only 25% were taken up when heat-inactivated serum was used. Killing by PMN leukocytes also appeared to be decreased when heatinactivated serum was used as an opsonic source. A p p r o x i m a t e l y twice as many leukocyte-associated bacteria were found to be viable in the m i xt ure with heat-inactivated serum at 3, 10 and 20 min when compared with the viable leukocyte-associated bacteria in the mixture containing normal serum. Similar data was obtained when S. a u r e u s Cowan I was studied. To study the influence of different l e u k o c y t e populations on staphylococcal killing, PMN leukocytes from 5 patients with CGD, a disease with a known intraleukocytic staphylocidal def e c t (Quie et al., 1967), were compared with leukocytes from healthy volunteers. Fig. 4 is representative of the results obtained in this experiment. Phagocytosis of S. a u r e u s Cowan I was n o t f o u n d to differ significantly when leukocytes from the patients were c o m p a r e d with control cells. There was, however, a greater percentage of surviving staphylococci when the bacteria were phagocytized by leukocytes from patients with GGD. Incubation of samples in lysostaphin resulted in comparable decreases in leukocyte-associated bacteria indicating that the size of the attached, non-ingested bacterial population was similar for both patient and normal d o n o r leukocytes (data not presented ).
310 DISCUSSION The kinetics of S. a u r e u s opsonization, a t t a c h m e n t , ingestion and killing were studied quantitatively using [ 3H]thymidine labeled bacteria and different opsonic sources and PMN l e u k o c y t e populations. By incubating bacteria with normal serum, C2 deficient serum and heat-inactivated serum for varying durations of time prior to adding normal PMN leukocytes, differences in the opsonie capacities of these sera could be defined. Within 5 min opsonization with normal and heat-inactivated sera was complete. Opsonization with C2 deficient serum proceeded at a slower rate; staphylococci opsonized for 60 min were significantly better phagocytized than were bacteria opsonized for 5 and 15 min. These findings are consistent with previous observations that the classical c o m p l e m e n t pathway is essential for optimal opsonization of some S. a u r e u s strains and that opsonization in the absence of this pathway b u t in the presence of the alternative pathway occurs at a slower rate (Forsgren and Quie, 1974; Verhoef et al., submitted for publication). To separate the two phases of phagocytosis (at t achm ent and ingestion), samples from phagocytosis mixtures were incubated in lysostaphin to remove attached, non-ingested leukocyte-associated bacteria. Lysostaphin is a staphylococcal muralytic e n z y m e that does not enter leukocytes (Tan et al., 1971). When bacteria were opsonized with normal serum, a p p r o x i m a t e l y 10% of the leukocyte-associated bacteria were removed by lysostaphin treatment. When bacteria opsonized with heat-inactivated serum were studied, a greater percentage of leukocyte-associated bacteria (approximately 30%) were removed by lysostaphin. These findings suggest that bacteria opsonized in the absence of c o m p l e m e n t (heat-inactivated serum) are not only less efficiently attached to PMN leukocytes, but are also internalized at a slower rate than are bacteria opsonized in the presence of c o m p l e m e n t (normal serum). Bacterial killing by PMN leukocytes was assessed, after t horough washing o f the leukocytes, by disrupting the cells with water and sampling for CFU. Using this technique, it was found that a greater percent of leukocyte-associated bacteria were alive when bacteria were opsonized with heat-inactivated serum than when bacteria were opsonized with normal serum. The explanation of this finding may lie in the previous observation that bacteria opsonized with heat-inactivated serum are internalized at a slower rate than are bacteria opsonized with normal serum, and they would presumably t h er eb y be killed at a slower rate. When the killing capacity of PMN leukocytes from patients with CGD was c o mp ar ed with normal d o n o r leukocytes, a greater percent of bacteria associated with the patients' leukocytes were viable. This is consistent with the known staphylocidal defect in leukocytes from patients with this disease (Quie et al., 1967). No significant difference in either a t t a c h m e n t or ingestion could be de t e c t e d when CGD leukocytes were compared with d o n o r leukoeytes. The enhanced phagocytosis of CGD leukocytes reported elsewhere (Biggar, 1975) may be the result of methodological differences. A
311
false impression of increased phagocytosis by CGD leukocytes might arise if only the numbers of viable leukocyte-associated bacteria are compared. We are currently using the methods described to investigate other bacterial strains and phagocyte cell populations from patients with a variety of impaired host defense problems. REFERENCES
Biggar, W.D., 1975, Lancet i, 991. Forsgren, A. and P.G. Quie, 1974, Infect. Immunol. 1O, 402. Griffin, F.M., J.A. Griffin, J.E. Leider and S.C. Silverstein, 1975, J. Exp. Med. 142, 1263. Klebanoff, S.J., 1975, Semin. Hematol. 12, 117. Quie, P.G., J.G. White, B. Holmes and R.A. Good, 1967, J. Clin. Invest. 46, 668. Quie, P.G., 1975, Semin. Hematol. 12, 143. Rabinovitch, M., 1967, Exp. Cell Res. 46, 19. Stossel, T.P., 1975, Semin. Hematol. 12, 83. Tan, J.S., C. Watanakunakorn and J.P. Phair, 1971, J. Lab. Clin. Med. 70, 316.
