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Use of a unique chemiluminescence spectrometer in a study of factors influencing granulocyte light emission
Journal o f lmmunological Methods, 19 (1978) 279--287
279
© Elsevier/North-Holland Biomedical Press
USE O F A U N I Q U E C H E M I L U M I N E S C E N C E S P E C T R O M E T E R IN A S T U D Y O F F A C T O R S I N F L U E N C I N G G R A N U L O C Y T E L I G H T EMISSION *
BURTON R. ANDERSEN a and AVROM M. BRENDZEL b a Departments o f Medicine and Microbiology, VA West Side Hospital, and the University o f Illinois at the Medical Center, Chicago, IL, U.S.A. and b Bioengineering Program, University o f Illinois at Chicago Circle, Chicago, IL, U.S.A.
(Received 3 May 1977, accepted 26 July 1977)
Factors contributing to variability in chemiluminescence (CL) measurements of phagocytizing granulocytes were identified and controlled. Observed CL was decreased by light quenching caused by red blood cells, hemoglobin, phagocytizable particles, and granulocytes. Increases in CL were achieved by opsonization with heat labile and heat stable (antibody) serum factors, reaction temperatures in the range of 37--40°C, and mixing. The effects of controlled temperature variation and mixing were studied with a chemiluminescence spectrometer uniquely designed for such studies. Features of this spectrometer which make it more suitable than the previously employed scintillation spectrometers for the observation of granulocyte and other chemiluminescent systems include: (1) the ability to measure CL immediately upon reaction initiation; (2) simplicity of photomultiplier tube exchange; and (3) built-in optical filter holders for spectral analysis.
INTRODUCTION Allen et al. ( 1 9 7 2 ) were t h e first to d e m o n s t r a t e t h a t light is e m i t t e d by g r a n u l o c y t e s f o l l o w i n g p h a g o c y t o s i s , and t h a t the c h e m i l u m i n e s c e n c e (CL) p h e n o m e n o n is closely linked t o h e x o s e m o n o p h o s p h a t e s h u n t activity. T h e y p o s t u l a t e d t h a t electronically e x c i t e d singlet o x y g e n (~O2)is p r o d u c e d which acts as a m i c r o b i c i d a l agent. Allen ( 1 9 7 5 ) has d e m o n s t r a t e d t h a t the m y e l o p e r o x i d a s e - h a l i d e - H 2 0 2 s y s t e m described by K l e b a n o f f ( 1 9 6 8 ) prod u c e d CL in the presence o f c h l o r i d e or b r o m i d e ions. R o s e n and K l e b a n o f f ( 1 9 7 6 ) have p r o v i d e d evidence t h a t ~O2 is p r o d u c e d by the m y e l o p e r o x i d a s e halide-H202 system. It remains t o be d e m o n s t r a t e d , h o w e v e r , t h a t 102 is responsible f o r the observed light, and t h a t the light e m i t t i n g r e a c t i o n causes bacterial d e a t h . S o m e investigators have a t t e m p t e d t o use the CL p h e n o m e n o n as a p r o b e o f g r a n u l o c y t e f u n c t i o n . S t j e r n h o l m et al. ( 1 9 7 3 ) have s h o w n t h a t granuloc y t e s f r o m a p a t i e n t with c h r o n i c g r a n u l o m a t o u s disease were u n a b l e to
* This work was supported, in part, by West Side VA Hospital research funds, MRIS ~039~
280 generate light following phagocytosis. Bjorksten et al. (1976) demonstrated that granulocytes from patients treated with amphotericin B do not have normal CL activity. The full potential of CL as a research and clinical tool for studying granulocytes, however, has y e t to be realized because of uncontrolled variables and inadequate instrumentation. Because of the exceedingly low intensity of granulocyte CL, liquid scintillation spectrometers have been used in its measurement. However, the granulocyte environment within the scintillation spectrometer is definitely non-physiological, and may not be optimal for CL production. A spectrometer specifically designed to provide more physiological conditions for the measurement of granulocyte CL is described in this paper, and results are presented which could not be obtained with a standard scintillation spectrometer. Some other factors effecting CL determination will also be discussed. MATERIALS AND METHODS
Isolation o f white blood cells Heparinized human or canine blood (10 units heparin/ml of blood) was sedimented by adding 2 ml of Plasmagel (Roger Bellon Laboratories, Neuilly, France) to every 10 ml of blood. After standing for 30--60 min at 37°C, the supernatant fluid containing the leukocytes was removed. The cells were sedimented by centrifugation at 1,000 rpm (200g) for 10 min and the cells were washed with 30 ml of Hank's balanced salt solution (BSS). The red blood cells (RBC's) which contaminated the leukocyte preparation were removed by lysis with isotonic NH4C1 solution (Weening et al., 1974). This was accomplished by resuspending cells in 3--10 ml of ice-cold isotonic NH4C1 solution (0.155 M NH4C1, 10 mM KHCO3, 0.1 mM EDTA). After 10 min in this solution, the cells were centrifuged, washed once with 30 ml of BSS, and resuspended in BSS. Leukocyte and differential counts were performed and the granulocyte concentration adjusted to 106 cells per ml.
Phagocy tic particles Commercial baker's yeast was suspended in saline and boiled for 10 min. The suspension was filtered 4 times through gauze and the concentration of yeast particles adjusted according to the requirements of the particular experiment. Pseudomonas aeruginosa was inoculated into trypticase soy broth and allowed to grow overnight at 37°C. The optical density at a wave length of 550 nm was determined. The linear relationship between optical density and the numbers of organisms had been previously determined. The organisms were centrifuged at 3,000 rpm (1,700 g) for 20 min and the culture supernatant was removed and discarded. The organisms were adjusted to the appropriate concentration by the addition of BSS.
281
Chemiluminescence using a scintillation spectrometer In early experiments, CL determinations were performed in a scintillation spectrometer. The reaction chambers consisted of glass scintillation vials that had been dark adapted prior to use. The reaction mixture consisted of 5 ml of leukocytes (1 X 106 granulocytes per ml) plus 0.5 ml of the suspension containing the particles (yeast or P. aeruginosa). Generally, the particle suspension had previously been incubated with 0.1 ml of serum at 37°C for 30 min. In some experiments the serum incubation was deleted. The particle and leukocyte suspensions were added to the vial in near darkness. The vial was then inverted to assure mixing and placed in a liquid scintillation spectrometer (Packard TriCarb, Model 3380). Samples to be assayed in a single run were read sequentially following the addition of phagocytizable particles. Emitted light was measured as counts per min (cpm) with spectrometer refrigeration and coincidence circuits off, and both photomultiplier tubes (PMT) functioning. The gain was set to m a x i m u m and the lower discriminator threshold at 20. At the beginning of each run, the background levels of an e m p t y scintillation vial and a vial containing leukocytes only were determined. CL measurements were corrected by subtraction of the background light level of the e m p t y vial.
Chemiluminescence spectrometer Because of limitations in scintillation spectrometers when used for CL determinations, a new instrument was designed which uses the light sensing and electronic components of a Packard Tri Carb scintillation spectrometer {Model 3380), but incorporates the PMT (I{CA 4501/V3) into a unique light tight chamber. The PMT type can be readily changed in this instrument. The major new feature of the instrument is a carousel {fig. 1), machined from an aluminum block, which holds 12 reaction vials (Pico Vials ¢ 6 0 0 0 1 6 6 ,
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282 Packard I n s t r u m e n t Co.). Any of the 12 vials can be positioned in front of the PMT by rotating the carousel. The back of each vial holder in the aluminum block has been polished to reflect light back toward the PMT. All of the vials can be maintained at the same preset temperature (in particular, at 37°C) by a heating element e m b e d d e d in the carousel (Fenwal temperature control unit series 194, a m at ched probe, and a chromalox tubular element). Magnetic mixers (8--12 volts, 0.3 amp) are located under each of the 12 vial positions so t ha t granulocytes can be kept suspended and well oxygenated during the course of a long rung. Mumetal shielding was used to minimize magnetic effects on t he PMT. R u b b e r septa injection ports located over the reaction vials permit the vials to be dark adapted in the c ha m ber prior to adding reactants. CL readings may be made immediately u p o n injection of reactants. While the carousel is loaded with vials, a shutter covers the PMT face to p r o t e c t it from ro om light. Optical filters can be placed in retainers that position the filter between the reaction vial and the PMT. E m i t t ed light is measured as counts per preset time interval. Background counts obtained from unstimulated granulocytes were subtracted from the values obtained during the CL experiment. Particle and granulocyte preparation with this s p e c t r o m e t e r is the same as that for the scintillation s p e c t r o m e t e r e x c e p t that particles are injected into the reaction vial through the rubber ports. A carbon-14 scintillation standard was used to evaluate the p e r f o r m a n c e o f this instrument. RESULTS Light quenching The degree o f light quenching o f CL by contaminating RBC's is shown in fig. 2. Small changes in the RBC c o n c e n t r a t i o n had marked effects on the CL stimulated by yeast particles. Following this observation all the l e u k o c y t e preparations were treated to remove the RBC's by lysis. Free hemoglobin had a quenching effect similar to intact RBC's, and therefore, serum used as an opsonin in these studies was from non-hemolysed blood. Particle : cell ratio Experiments were p e r f o r m e d using varying particle : cell ratios in order to establish optimal conditions for this system. In the e x p e r i m e n t shown in fig. 3, yeast incubated with normal canine serum was added to normal canine granulocytes. A yeast : granulocyte ratio of 50 : 1 gave greater CL than 5 : 1 or 500 : 1. At the 500 : i level, the decrease in measured light was probably due to quenching caused by the turbid suspension of yeast particles.
283 k4o Yeast Granulccyte RotlO e : 500 I
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Fig. 2. E f f e c t o f changes in RBC c o n c e n t r a t i o n o n the m e a s u r e m e n t o f light e m i t t e d by canine granulocytes f o l l o w i n g the addition o f serum o p s o n i z e d yeast. Light was measured in a scintillation spectrometer at a m b i e n t temperature. Particle : cell ratio = 50 : 1. Fig. 3. Differences in CL w i t h varying yeast : granulocyte ratios. N o r m a l canine granuloc y t e s were used in a scintillation spectrometer at a m b i e n t temperature. Yeast particles were o p s o n i z e d w i t h normal serum.
Effect o f serum Fig. 4 d e m o n s t r a t e s t h a t CL is a u g m e n t e d in the presence o f serum. In this e x p e r i m e n t the particles c o n s i s t e d o f a suspension o f washed viable P. aeruginosa, t y p e 6, in a particle : cell ratio o f 50 : 1. Serum from a d o g immu-
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Fig. 4. The e f f e c t of i m m u n e and n o n - i m m u n e (control), h e a t e d , and u n h e a t e d canine serum on the CL of granulocytes in the presence o f p s e u d o m o n a s . Performed in a scintillation spectrometer at a m b i e n t temperature. Particle : cell ratio = 50 : 1.
284
lipopolysaccharide (Parke-Davis) gave highs nized with type 6-pseudomonas serum. Heated serum (56°C 30 min) showe levels of CL than non-immune decreased CL. Some CL above background occurred even in the absence c serum. In most experiments the increased levels of CL associated with irr mune serum occurred within the first 15-20 min. Duplicate experiment showed similar results. Temperature In studies conducted with the chemiluminescence spectrometer (CLS), th effect of temperature on CL was explored. Because of the time required tl reach temperature equilibrium the number of runs that could be performer with a single granulocyte preparation was limited. For this reason many sepa rate runs were performed comparing CL at several different temperatures Mixing was continuous during each run. Only human granulocytes were usec in these observations. Results are shown in fig. 5. It can be seen that the peak response of CL was highest at 40°C (fig. 5C)
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Fig. 6. Effect of mixing on the CL of human granulocytes. Serum opsonized yeast (particle : cell ratio = 50 : 1) was used to stimulate CL. Light was measured in the CLS. CL at 41°C was n o t t e s t e d , b u t at 42°C t h e r e was a significant r e d u c t i o n . T h e p e a k r e s p o n s e o c c u r r e d w i t h i n 4 m i n o f t h e t i m e t h a t particles were a d d e d , e x c e p t t h a t a t 2 5 ° C p e a k r e s p o n s e o c c u r r e d at a p p r o x i m a t e l y 9 m i n (fig. 5B). In fig. 5D it can b e seen t h a t a l t h o u g h t h e p e a k CL r e s p o n s e was g r e a t e r at 40°C t h a n at 37°C, the curves cross a t a b o u t 10 m i n a f t e r t h e a d d i t i o n o f particles. In a 60 m i n r u n t h e t o t a l CL at 37°C was g r e a t e r t h a n at 40°C.
Mix ing T h e i m p o r t a n c e o f m i x i n g as p e r f o r m e d in t h e CLS is seen in Fig. 6. T h e CL o f h u m a n g r a n u l o c y t e s at 3 9 ° C , f o l l o w i n g t h e a d d i t i o n o f s e r u m o p s o n ized y e a s t was o b s e r v e d w i t h c o n t i n u o u s m i x i n g . A s e c o n d s a m p l e o f granul o c y t e s was m i x e d f o r a f e w s e c o n d s f o l l o w i n g t h e a d d i t i o n o f o p s o n i z e d y e a s t in o r d e r t o insure e v e n d i s t r i b u t i o n o f particles and cells. F o r t h e n e x t 20 m i n CL was m e a s u r e d w i t h t h e m i x e r off. T h e p e a k r e s p o n s e o f t h e unm i x e d s a m p l e was m u c h l o w e r t h a n t h e m i x e d s a m p l e . When t h e m i x e r was r e s t a r t e d a s e c o n d p e a k o f CL o c c u r r e d w h i c h was higher t h a n t h e first peak. In o t h e r e x p e r i m e n t s , relatively little d i f f e r e n c e was seen in CL d u e to variat i o n in m i x i n g speed. DISCUSSION T h e i m p o r t a n c e o f light q u e n c h i n g b y c o m m o n c o n t a m i n a n t s o f t h e s y s t e m such as RBCs a n d h e m o g l o b i n h a v e b e e n illustrated in this s t u d y .
286 Another factor which requires careful control is the particle : cell ratio. As demonstrated, there is an optimal ratio which is probably determined by the balance between the greater likelihood of phagocytosis as the particle : cell ratio increases and the increase in light quenching and scattering which occurs as the number of particles increases. Factors which influence the rate of phagocytosis, such as serum opsonins, must be considered in designing CL experiments since phagocytosis appears to be the major stimulus of granulocyte CL. The a m o u n t of CL following the addition of pseudomonas incubated with various sera correlated well with the expected effect of the sera on phagocytosis. Serum with anti-pseudomonas antibodies was associated with the greatest light production. Heating the serum reduced CL. Pseudomonas w i t h o u t serum stimulated very little CL. These observations are in agreement with those of Allen (1977), Grebner et al. (1977), and Hemming et al. (1976), who showed that CL is directly related to changes in the phagocytic ability of granulocytes. The effects of temperature variation and sample mixing can be readily demonstrated with the CLS. It is apparent from our studies and those of Nelson et al. (1976) that CL is lower at room temperature than at 37°C. The marked temperature effects which we have observed could explain at least in part the day-to-day variability experienced when performing CL determinations in scintillation spectrometers, which lack temperature controls. In the present study the level of CL increases with the temperature to the range of 37--40°C before beginning to decline. A consistent finding is that at 40°C the peak of CL is greater than at 37°C; however, after the first 10 min of the run, the CL at 37°C is greater. If the run continues for 60 min the total CL at 37°C is greater than at 40°C. The significance of this observation in terms of the functional ability of granulocytes in afebrile versus febrile patients is unclear. Peterson et al. (1976) have shown that phagocytosis of bacteria was depressed at 41°C when compared with results at 37°C. Although we did not measure CL at 41°C, the decrease that we observed at 42°C could have been due to a reduction in the phagocytic ability of the granulocytes. Allen (1973) has shown that intermittent mixing during the course of a CL experiment causes transient increases in CL, probably due to enhanced oxygenation of the granulocytes. Our observations support this conclusion. Since mixing increases oxygen levels in an aqueous solution we would expect that continuous mixing would favor the higher levels of oxygen in solution required by granulocytes for the post-phagocytic increase in cell metabolism. Mixing also increases the potential for contact between granulocytes and particles which should increase phagocytosis. In an unmixed vial granulocytes and particles tend to settle to the bottom. This results in both quenching due to high granulocyte and particle concentration and in suboptimal light collection by the PMT. Results from previous studies (Allen et al., 1972; Stjernholm et al., 1973) have revealed that CL may follow a biphasic pattern. We have similar unpublished observations for unmixed suspensions at ambient temperature.
287 Biphasic curves, h o w e v e r , have n e v e r been observed in the CLS w h e n continu o u s m i x i n g and t e m p e r a t u r e c o n t r o l has b e e n m a i n t a i n e d . This suggests t h a t u n e v e n o x y g e n a t i o n or t e m p e r a t u r e changes m a y have caused these biphasic CL patterns. T h e r e are additional advantages o f using a CLS. Because o f the system o f injection p o r t s , it is possible t o have t h e glass r e a c t i o n vial in place and dark a d a p t e d well b e f o r e t h e r e a c t i o n takes place. In o u r e x p e r i e n c e , glass fluorescence, which is likely t o o c c u r w h e n scintillation s p e c t r o m e t e r s are used, p r e v e n t s a c c u m u l a t i o n o f m e a n i n g f u l d a t a for a p p r o x i m a t e l y the first 30 sec o f the CL run. A n o t h e r d e l a y in the c o l l e c t i o n o f d a t a results f r o m the time lost in lowering t h e p a r t i c l e ~ c e l l m i x t u r e into the PMT c h a m b e r o f the scintillation s p e c t r o m e t e r . These delays d o n o t o c c u r w h e n the CLS is used. The light emission o f s o d i u m h y p o c h l o r i t e - h y d r o g e n p e r o x i d e , which o c c u r s within a few seconds o f mixing, has b e e n d e t e c t e d using the CLS (unpublished observations), indicating the utility o f the injection ports. F u r t h e r advantages o f t h e CLS are d u e t o the ease o f access t o the PMT and the carousel. T h e PMT can easily be replaced so t h a t PMTs having spectral sensitivities d i f f e r e n t f r o m t h o s e used in scintillation s p e c t r o m e t e r s m a y be emp l o y e d , and optical filters can be i n t e r p o s e d b e t w e e n the sample vial and PMT to p r o v i d e a rough spectral analysis o f e m i t t e d light. C u r r e n t l y , tests are u n d e r w a y t o d e t e r m i n e t h e clinical usefulness o f the CLS in evaluating the f u n c t i o n a l ability o f g r a n u l o c y t e s . T h e CLS also can be used to s t u d y o t h e r c h e m i l u m i n e s c e n t cells such as m o n o c y t e s and platelets (Mills et al., 1977). ACKNOWLEDGEMENTS Mr. Russ Schavey and Dr. L e r o y E v e r e t t o f t h e Packard I n s t r u m e n t Co., and Messrs. G e o r g e Mertz and A1 Cerveny, University o f Illinois at the Medical Center, p r o v i d e d help in designing and building the c h e m i l u m i n e s c e n c e s p e c t r o m e t e r . T h e Packard I n s t r u m e n t C o m p a n y generously supplied m a n y o f the c o m p o n e n t s f o r t h e light d e t e c t i o n system. We gratefully a c k n o w l e d g e the t e c h n i c a l assistance o f Mrs. Lilia Kizlaitis and Mr. H a r o l d Amirault. REFERENCES Allen, R., 1973, Ph.D. Thesis, Tulane University. Allen, R., 1975, Biochem. Biophys. Res. Commun. 63,675. Allen, R., 1977, Inf. Immun. 15,828. Allen, R., R. Stjernholm and R. Steele, 1972, Biochem. Biophys. Res. Commun. 47, 679. Bjorksten, B., C. Ray and P. Quie, 1976, Inf. Immun. 14,315. Grebner, J., E. Mills, B. Gray and P. Quie, 1977, J. Lab. Clin. Med. 89, 153. Hemming, V., R. Hall, P. Rhodes, A. Shigeoka and H. Hill, 1976, J. Clin. Invest. 58, 1379. Klebanoff, S., 1968, J. Bacteriol. 95, 2131. Mills, E., J. Gerrard, C. Clawson, P. Quie and J. White, 1977, Fed. Proc. 36,350. Nelson, R.D., E. Mills, R. Simmons and P. Quie, 1976, Inf. Immun. 14, 129. Peterson, P., J. Verhoef, L. Sabath and P. Quie, 1976, Inf. Immun. 14,496. Rosen, H. and S. Klebanoff, 1976, Fed. Proe. 35, 1391. Stjernholm, R., R. Allen, R. Steele, W. Waring and J. Harris, 1973, Inf. Immun. 7,313. Weening, R., D. Roos and J. Loos, 1974, J. Lab. Clin. Med. 83,570.
279
© Elsevier/North-Holland Biomedical Press
USE O F A U N I Q U E C H E M I L U M I N E S C E N C E S P E C T R O M E T E R IN A S T U D Y O F F A C T O R S I N F L U E N C I N G G R A N U L O C Y T E L I G H T EMISSION *
BURTON R. ANDERSEN a and AVROM M. BRENDZEL b a Departments o f Medicine and Microbiology, VA West Side Hospital, and the University o f Illinois at the Medical Center, Chicago, IL, U.S.A. and b Bioengineering Program, University o f Illinois at Chicago Circle, Chicago, IL, U.S.A.
(Received 3 May 1977, accepted 26 July 1977)
Factors contributing to variability in chemiluminescence (CL) measurements of phagocytizing granulocytes were identified and controlled. Observed CL was decreased by light quenching caused by red blood cells, hemoglobin, phagocytizable particles, and granulocytes. Increases in CL were achieved by opsonization with heat labile and heat stable (antibody) serum factors, reaction temperatures in the range of 37--40°C, and mixing. The effects of controlled temperature variation and mixing were studied with a chemiluminescence spectrometer uniquely designed for such studies. Features of this spectrometer which make it more suitable than the previously employed scintillation spectrometers for the observation of granulocyte and other chemiluminescent systems include: (1) the ability to measure CL immediately upon reaction initiation; (2) simplicity of photomultiplier tube exchange; and (3) built-in optical filter holders for spectral analysis.
INTRODUCTION Allen et al. ( 1 9 7 2 ) were t h e first to d e m o n s t r a t e t h a t light is e m i t t e d by g r a n u l o c y t e s f o l l o w i n g p h a g o c y t o s i s , and t h a t the c h e m i l u m i n e s c e n c e (CL) p h e n o m e n o n is closely linked t o h e x o s e m o n o p h o s p h a t e s h u n t activity. T h e y p o s t u l a t e d t h a t electronically e x c i t e d singlet o x y g e n (~O2)is p r o d u c e d which acts as a m i c r o b i c i d a l agent. Allen ( 1 9 7 5 ) has d e m o n s t r a t e d t h a t the m y e l o p e r o x i d a s e - h a l i d e - H 2 0 2 s y s t e m described by K l e b a n o f f ( 1 9 6 8 ) prod u c e d CL in the presence o f c h l o r i d e or b r o m i d e ions. R o s e n and K l e b a n o f f ( 1 9 7 6 ) have p r o v i d e d evidence t h a t ~O2 is p r o d u c e d by the m y e l o p e r o x i d a s e halide-H202 system. It remains t o be d e m o n s t r a t e d , h o w e v e r , t h a t 102 is responsible f o r the observed light, and t h a t the light e m i t t i n g r e a c t i o n causes bacterial d e a t h . S o m e investigators have a t t e m p t e d t o use the CL p h e n o m e n o n as a p r o b e o f g r a n u l o c y t e f u n c t i o n . S t j e r n h o l m et al. ( 1 9 7 3 ) have s h o w n t h a t granuloc y t e s f r o m a p a t i e n t with c h r o n i c g r a n u l o m a t o u s disease were u n a b l e to
* This work was supported, in part, by West Side VA Hospital research funds, MRIS ~039~
280 generate light following phagocytosis. Bjorksten et al. (1976) demonstrated that granulocytes from patients treated with amphotericin B do not have normal CL activity. The full potential of CL as a research and clinical tool for studying granulocytes, however, has y e t to be realized because of uncontrolled variables and inadequate instrumentation. Because of the exceedingly low intensity of granulocyte CL, liquid scintillation spectrometers have been used in its measurement. However, the granulocyte environment within the scintillation spectrometer is definitely non-physiological, and may not be optimal for CL production. A spectrometer specifically designed to provide more physiological conditions for the measurement of granulocyte CL is described in this paper, and results are presented which could not be obtained with a standard scintillation spectrometer. Some other factors effecting CL determination will also be discussed. MATERIALS AND METHODS
Isolation o f white blood cells Heparinized human or canine blood (10 units heparin/ml of blood) was sedimented by adding 2 ml of Plasmagel (Roger Bellon Laboratories, Neuilly, France) to every 10 ml of blood. After standing for 30--60 min at 37°C, the supernatant fluid containing the leukocytes was removed. The cells were sedimented by centrifugation at 1,000 rpm (200g) for 10 min and the cells were washed with 30 ml of Hank's balanced salt solution (BSS). The red blood cells (RBC's) which contaminated the leukocyte preparation were removed by lysis with isotonic NH4C1 solution (Weening et al., 1974). This was accomplished by resuspending cells in 3--10 ml of ice-cold isotonic NH4C1 solution (0.155 M NH4C1, 10 mM KHCO3, 0.1 mM EDTA). After 10 min in this solution, the cells were centrifuged, washed once with 30 ml of BSS, and resuspended in BSS. Leukocyte and differential counts were performed and the granulocyte concentration adjusted to 106 cells per ml.
Phagocy tic particles Commercial baker's yeast was suspended in saline and boiled for 10 min. The suspension was filtered 4 times through gauze and the concentration of yeast particles adjusted according to the requirements of the particular experiment. Pseudomonas aeruginosa was inoculated into trypticase soy broth and allowed to grow overnight at 37°C. The optical density at a wave length of 550 nm was determined. The linear relationship between optical density and the numbers of organisms had been previously determined. The organisms were centrifuged at 3,000 rpm (1,700 g) for 20 min and the culture supernatant was removed and discarded. The organisms were adjusted to the appropriate concentration by the addition of BSS.
281
Chemiluminescence using a scintillation spectrometer In early experiments, CL determinations were performed in a scintillation spectrometer. The reaction chambers consisted of glass scintillation vials that had been dark adapted prior to use. The reaction mixture consisted of 5 ml of leukocytes (1 X 106 granulocytes per ml) plus 0.5 ml of the suspension containing the particles (yeast or P. aeruginosa). Generally, the particle suspension had previously been incubated with 0.1 ml of serum at 37°C for 30 min. In some experiments the serum incubation was deleted. The particle and leukocyte suspensions were added to the vial in near darkness. The vial was then inverted to assure mixing and placed in a liquid scintillation spectrometer (Packard TriCarb, Model 3380). Samples to be assayed in a single run were read sequentially following the addition of phagocytizable particles. Emitted light was measured as counts per min (cpm) with spectrometer refrigeration and coincidence circuits off, and both photomultiplier tubes (PMT) functioning. The gain was set to m a x i m u m and the lower discriminator threshold at 20. At the beginning of each run, the background levels of an e m p t y scintillation vial and a vial containing leukocytes only were determined. CL measurements were corrected by subtraction of the background light level of the e m p t y vial.
Chemiluminescence spectrometer Because of limitations in scintillation spectrometers when used for CL determinations, a new instrument was designed which uses the light sensing and electronic components of a Packard Tri Carb scintillation spectrometer {Model 3380), but incorporates the PMT (I{CA 4501/V3) into a unique light tight chamber. The PMT type can be readily changed in this instrument. The major new feature of the instrument is a carousel {fig. 1), machined from an aluminum block, which holds 12 reaction vials (Pico Vials ¢ 6 0 0 0 1 6 6 ,
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282 Packard I n s t r u m e n t Co.). Any of the 12 vials can be positioned in front of the PMT by rotating the carousel. The back of each vial holder in the aluminum block has been polished to reflect light back toward the PMT. All of the vials can be maintained at the same preset temperature (in particular, at 37°C) by a heating element e m b e d d e d in the carousel (Fenwal temperature control unit series 194, a m at ched probe, and a chromalox tubular element). Magnetic mixers (8--12 volts, 0.3 amp) are located under each of the 12 vial positions so t ha t granulocytes can be kept suspended and well oxygenated during the course of a long rung. Mumetal shielding was used to minimize magnetic effects on t he PMT. R u b b e r septa injection ports located over the reaction vials permit the vials to be dark adapted in the c ha m ber prior to adding reactants. CL readings may be made immediately u p o n injection of reactants. While the carousel is loaded with vials, a shutter covers the PMT face to p r o t e c t it from ro om light. Optical filters can be placed in retainers that position the filter between the reaction vial and the PMT. E m i t t ed light is measured as counts per preset time interval. Background counts obtained from unstimulated granulocytes were subtracted from the values obtained during the CL experiment. Particle and granulocyte preparation with this s p e c t r o m e t e r is the same as that for the scintillation s p e c t r o m e t e r e x c e p t that particles are injected into the reaction vial through the rubber ports. A carbon-14 scintillation standard was used to evaluate the p e r f o r m a n c e o f this instrument. RESULTS Light quenching The degree o f light quenching o f CL by contaminating RBC's is shown in fig. 2. Small changes in the RBC c o n c e n t r a t i o n had marked effects on the CL stimulated by yeast particles. Following this observation all the l e u k o c y t e preparations were treated to remove the RBC's by lysis. Free hemoglobin had a quenching effect similar to intact RBC's, and therefore, serum used as an opsonin in these studies was from non-hemolysed blood. Particle : cell ratio Experiments were p e r f o r m e d using varying particle : cell ratios in order to establish optimal conditions for this system. In the e x p e r i m e n t shown in fig. 3, yeast incubated with normal canine serum was added to normal canine granulocytes. A yeast : granulocyte ratio of 50 : 1 gave greater CL than 5 : 1 or 500 : 1. At the 500 : i level, the decrease in measured light was probably due to quenching caused by the turbid suspension of yeast particles.
283 k4o Yeast Granulccyte RotlO e : 500 I
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Effect o f serum Fig. 4 d e m o n s t r a t e s t h a t CL is a u g m e n t e d in the presence o f serum. In this e x p e r i m e n t the particles c o n s i s t e d o f a suspension o f washed viable P. aeruginosa, t y p e 6, in a particle : cell ratio o f 50 : 1. Serum from a d o g immu-
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284
lipopolysaccharide (Parke-Davis) gave highs nized with type 6-pseudomonas serum. Heated serum (56°C 30 min) showe levels of CL than non-immune decreased CL. Some CL above background occurred even in the absence c serum. In most experiments the increased levels of CL associated with irr mune serum occurred within the first 15-20 min. Duplicate experiment showed similar results. Temperature In studies conducted with the chemiluminescence spectrometer (CLS), th effect of temperature on CL was explored. Because of the time required tl reach temperature equilibrium the number of runs that could be performer with a single granulocyte preparation was limited. For this reason many sepa rate runs were performed comparing CL at several different temperatures Mixing was continuous during each run. Only human granulocytes were usec in these observations. Results are shown in fig. 5. It can be seen that the peak response of CL was highest at 40°C (fig. 5C)
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285 120-
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Fig. 6. Effect of mixing on the CL of human granulocytes. Serum opsonized yeast (particle : cell ratio = 50 : 1) was used to stimulate CL. Light was measured in the CLS. CL at 41°C was n o t t e s t e d , b u t at 42°C t h e r e was a significant r e d u c t i o n . T h e p e a k r e s p o n s e o c c u r r e d w i t h i n 4 m i n o f t h e t i m e t h a t particles were a d d e d , e x c e p t t h a t a t 2 5 ° C p e a k r e s p o n s e o c c u r r e d at a p p r o x i m a t e l y 9 m i n (fig. 5B). In fig. 5D it can b e seen t h a t a l t h o u g h t h e p e a k CL r e s p o n s e was g r e a t e r at 40°C t h a n at 37°C, the curves cross a t a b o u t 10 m i n a f t e r t h e a d d i t i o n o f particles. In a 60 m i n r u n t h e t o t a l CL at 37°C was g r e a t e r t h a n at 40°C.
Mix ing T h e i m p o r t a n c e o f m i x i n g as p e r f o r m e d in t h e CLS is seen in Fig. 6. T h e CL o f h u m a n g r a n u l o c y t e s at 3 9 ° C , f o l l o w i n g t h e a d d i t i o n o f s e r u m o p s o n ized y e a s t was o b s e r v e d w i t h c o n t i n u o u s m i x i n g . A s e c o n d s a m p l e o f granul o c y t e s was m i x e d f o r a f e w s e c o n d s f o l l o w i n g t h e a d d i t i o n o f o p s o n i z e d y e a s t in o r d e r t o insure e v e n d i s t r i b u t i o n o f particles and cells. F o r t h e n e x t 20 m i n CL was m e a s u r e d w i t h t h e m i x e r off. T h e p e a k r e s p o n s e o f t h e unm i x e d s a m p l e was m u c h l o w e r t h a n t h e m i x e d s a m p l e . When t h e m i x e r was r e s t a r t e d a s e c o n d p e a k o f CL o c c u r r e d w h i c h was higher t h a n t h e first peak. In o t h e r e x p e r i m e n t s , relatively little d i f f e r e n c e was seen in CL d u e to variat i o n in m i x i n g speed. DISCUSSION T h e i m p o r t a n c e o f light q u e n c h i n g b y c o m m o n c o n t a m i n a n t s o f t h e s y s t e m such as RBCs a n d h e m o g l o b i n h a v e b e e n illustrated in this s t u d y .
286 Another factor which requires careful control is the particle : cell ratio. As demonstrated, there is an optimal ratio which is probably determined by the balance between the greater likelihood of phagocytosis as the particle : cell ratio increases and the increase in light quenching and scattering which occurs as the number of particles increases. Factors which influence the rate of phagocytosis, such as serum opsonins, must be considered in designing CL experiments since phagocytosis appears to be the major stimulus of granulocyte CL. The a m o u n t of CL following the addition of pseudomonas incubated with various sera correlated well with the expected effect of the sera on phagocytosis. Serum with anti-pseudomonas antibodies was associated with the greatest light production. Heating the serum reduced CL. Pseudomonas w i t h o u t serum stimulated very little CL. These observations are in agreement with those of Allen (1977), Grebner et al. (1977), and Hemming et al. (1976), who showed that CL is directly related to changes in the phagocytic ability of granulocytes. The effects of temperature variation and sample mixing can be readily demonstrated with the CLS. It is apparent from our studies and those of Nelson et al. (1976) that CL is lower at room temperature than at 37°C. The marked temperature effects which we have observed could explain at least in part the day-to-day variability experienced when performing CL determinations in scintillation spectrometers, which lack temperature controls. In the present study the level of CL increases with the temperature to the range of 37--40°C before beginning to decline. A consistent finding is that at 40°C the peak of CL is greater than at 37°C; however, after the first 10 min of the run, the CL at 37°C is greater. If the run continues for 60 min the total CL at 37°C is greater than at 40°C. The significance of this observation in terms of the functional ability of granulocytes in afebrile versus febrile patients is unclear. Peterson et al. (1976) have shown that phagocytosis of bacteria was depressed at 41°C when compared with results at 37°C. Although we did not measure CL at 41°C, the decrease that we observed at 42°C could have been due to a reduction in the phagocytic ability of the granulocytes. Allen (1973) has shown that intermittent mixing during the course of a CL experiment causes transient increases in CL, probably due to enhanced oxygenation of the granulocytes. Our observations support this conclusion. Since mixing increases oxygen levels in an aqueous solution we would expect that continuous mixing would favor the higher levels of oxygen in solution required by granulocytes for the post-phagocytic increase in cell metabolism. Mixing also increases the potential for contact between granulocytes and particles which should increase phagocytosis. In an unmixed vial granulocytes and particles tend to settle to the bottom. This results in both quenching due to high granulocyte and particle concentration and in suboptimal light collection by the PMT. Results from previous studies (Allen et al., 1972; Stjernholm et al., 1973) have revealed that CL may follow a biphasic pattern. We have similar unpublished observations for unmixed suspensions at ambient temperature.
287 Biphasic curves, h o w e v e r , have n e v e r been observed in the CLS w h e n continu o u s m i x i n g and t e m p e r a t u r e c o n t r o l has b e e n m a i n t a i n e d . This suggests t h a t u n e v e n o x y g e n a t i o n or t e m p e r a t u r e changes m a y have caused these biphasic CL patterns. T h e r e are additional advantages o f using a CLS. Because o f the system o f injection p o r t s , it is possible t o have t h e glass r e a c t i o n vial in place and dark a d a p t e d well b e f o r e t h e r e a c t i o n takes place. In o u r e x p e r i e n c e , glass fluorescence, which is likely t o o c c u r w h e n scintillation s p e c t r o m e t e r s are used, p r e v e n t s a c c u m u l a t i o n o f m e a n i n g f u l d a t a for a p p r o x i m a t e l y the first 30 sec o f the CL run. A n o t h e r d e l a y in the c o l l e c t i o n o f d a t a results f r o m the time lost in lowering t h e p a r t i c l e ~ c e l l m i x t u r e into the PMT c h a m b e r o f the scintillation s p e c t r o m e t e r . These delays d o n o t o c c u r w h e n the CLS is used. The light emission o f s o d i u m h y p o c h l o r i t e - h y d r o g e n p e r o x i d e , which o c c u r s within a few seconds o f mixing, has b e e n d e t e c t e d using the CLS (unpublished observations), indicating the utility o f the injection ports. F u r t h e r advantages o f t h e CLS are d u e t o the ease o f access t o the PMT and the carousel. T h e PMT can easily be replaced so t h a t PMTs having spectral sensitivities d i f f e r e n t f r o m t h o s e used in scintillation s p e c t r o m e t e r s m a y be emp l o y e d , and optical filters can be i n t e r p o s e d b e t w e e n the sample vial and PMT to p r o v i d e a rough spectral analysis o f e m i t t e d light. C u r r e n t l y , tests are u n d e r w a y t o d e t e r m i n e t h e clinical usefulness o f the CLS in evaluating the f u n c t i o n a l ability o f g r a n u l o c y t e s . T h e CLS also can be used to s t u d y o t h e r c h e m i l u m i n e s c e n t cells such as m o n o c y t e s and platelets (Mills et al., 1977). ACKNOWLEDGEMENTS Mr. Russ Schavey and Dr. L e r o y E v e r e t t o f t h e Packard I n s t r u m e n t Co., and Messrs. G e o r g e Mertz and A1 Cerveny, University o f Illinois at the Medical Center, p r o v i d e d help in designing and building the c h e m i l u m i n e s c e n c e s p e c t r o m e t e r . T h e Packard I n s t r u m e n t C o m p a n y generously supplied m a n y o f the c o m p o n e n t s f o r t h e light d e t e c t i o n system. We gratefully a c k n o w l e d g e the t e c h n i c a l assistance o f Mrs. Lilia Kizlaitis and Mr. H a r o l d Amirault. REFERENCES Allen, R., 1973, Ph.D. Thesis, Tulane University. Allen, R., 1975, Biochem. Biophys. Res. Commun. 63,675. Allen, R., 1977, Inf. Immun. 15,828. Allen, R., R. Stjernholm and R. Steele, 1972, Biochem. Biophys. Res. Commun. 47, 679. Bjorksten, B., C. Ray and P. Quie, 1976, Inf. Immun. 14,315. Grebner, J., E. Mills, B. Gray and P. Quie, 1977, J. Lab. Clin. Med. 89, 153. Hemming, V., R. Hall, P. Rhodes, A. Shigeoka and H. Hill, 1976, J. Clin. Invest. 58, 1379. Klebanoff, S., 1968, J. Bacteriol. 95, 2131. Mills, E., J. Gerrard, C. Clawson, P. Quie and J. White, 1977, Fed. Proc. 36,350. Nelson, R.D., E. Mills, R. Simmons and P. Quie, 1976, Inf. Immun. 14, 129. Peterson, P., J. Verhoef, L. Sabath and P. Quie, 1976, Inf. Immun. 14,496. Rosen, H. and S. Klebanoff, 1976, Fed. Proe. 35, 1391. Stjernholm, R., R. Allen, R. Steele, W. Waring and J. Harris, 1973, Inf. Immun. 7,313. Weening, R., D. Roos and J. Loos, 1974, J. Lab. Clin. Med. 83,570.