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Luminol-dependent chemiluminescence analysis of opsonophagocytic dysfunctions
Journal of Immunological Methods, 23 (1978) 315--326 © Elsevier/North-Holland Biomedical Press
315
LUMINOL-DEPENDENT CHEMILUMINESCENCE ANALYSIS OF OPSONOPHAGOCYTIC DYSFUNCTIONS
MARK E. WILSON, MICHAEL A. TRUSH, KNOX VAN DYKE, JAMES M. KYLE, MARTHA D. MULLETT and WILLIAM A, NEAL Departments of Pharmacology and Pediatrics, West Virginia University Medical Center, Morgantown, WV 26506, U.S.A. (Received 27 December 1977, accepted 15 April 1978)
The use of luminol as a means of amplifying the native chemiluminescence responses of phagocytosing human polymorphonuclear leukocytes was studied. Parameters examined included the effects of luminol, leukocyte and particle (opsonized zymosan) concentration on luminol-dependent chemiluminescence response. The results indicate that all of the parameters studied influence the luminol-dependent chemiluminescence assay system and that, on the basis of experiments designed to assess both opsonic and phagocytosis defects, this system appears to reflect the same or similar metabolic events as does native chemiluminescence. The luminol-dependent chemiluminescence assay represents a significant improvement of the phagocyte chemiluminescence assay in that greater sensitivity is possible, and may therefore have clinical utility as a means of evaluating phagocytic function.
INTRODUCTION
P h a g o c y t o s i n g h u m a n p o l y m o r p h o n u c l e a r l e u k o c y t e s (PMN) generate a b u r s t of oxidative m e t a b o l i c activity w h i c h results in t h e increased p r o d u c t i o n o f several p o t e n t i a l l y bactericidal o x y g e n m e t a b o l i t e s including supero x i d e anion, h y d r o g e n p e r o x i d , h y d r o x y l radical and singlet o x y g e n (reviewed b y Cheson et al., 1977). T h e ability o f PMNs t o m o u n t a n o r m a l r e s p i r a t o r y burst is d e p e n d e n t u p o n b o t h h u m o r a l (opsonic) and cellular (oxidative m e t a b o l i c ) factors, and d e f e c t s in either o f these p a r a m e t e r s are f r e q u e n t l y associated with increased susceptibility to i n f e c t i o n (Baehner, 1 9 7 4 ) . T h e i m p o r t a n c e o f n o r m a l serum o p s o n i c and PMN m e t a b o l i c funct i o n in h o s t defense against i n f e c t i o n has ' p r o m p t e d the d e v e l o p m e n t o f in vitro clinical m e t h o d s designed t o assess p h a g o c y t i c f u n c t i o n . In view o f certain deficiencies i n h e r e n t in m a n y o f these c u r r e n t m e t h o d s ( F u e n f e r et al., 1 9 7 6 ) , n e w and m o r e reliable a p p r o a c h e s to the p r o b l e m o f evaluating p h a g o c y t i c c a p a c i t y m u s t be t a k e n . F o l l o w i n g t h e o b s e r v a t i o n t h a t p h a g o c y t o s i n g PMNs generate electronically e x c i t e d states (such as singlet o x y g e n or e x c i t e d c a r b o n y l groups), as manifested b y a m e a s u r a b l e c h e m i l u m i n e s c e n c e (CL) response (Allen et al., 1 9 7 2 ) , PMNs f r o m patients afflicted with c h r o n i c g r a n u l o m a t o u s disease
316 (CGD) were found to exhibit a markedly diminished CL response (Stjernholm et al., 1973). CGD PMNs are characterized by an inability to generate significant amounts of superoxide and hydrogen peroxide (Curnutte et al., 1974). Thus, defective oxidative metabolic activity of PMNs correlated with diminished CL responses. More recently, defective particle opsonization has also been shown to result in depressed CL responses elicited from normal PMNs (Stevens and Young, 1977). Premature human infants are reportedly deficient with respect to serum opsonic capacity, particularly with regard to gram-negative organisms which are opsonized via the alternative complement pathway (Stossel et al., 1973). We have previously employed the native CL assay in assessing the functional status of the alternative pathway in nonimmune neonatal serum and found a significant defect in opsonic capacity as compared to adult serum (Wilson et al., 1977). The ability of the CL assay to reflect opsonic and phagocytic dysfunctions suggested its potential application in the evaluation of phagocytic function. In certain clinical situations, however, particularly in reference to the premature newborn infant, blood volumes required to perform CL assays may be considered excessive. Thus, we attempted to modify the CL assay in order to decrease the required PMN concentration. Luminol (5-amino-2,3,-dihydro-l,4-phthalazinedione) is a cyclic hydrazide which is readily oxidized to an electronically excited intermediate which undergoes subsequent relaxation to ground state with concomitant p h o t o n emission (i.e., chemiluminescence). This c o m p o u n d has been employed in the detection of enzyme-catalyzed free radical production (Totter et al., 1960). The high quantum efficiency of luminol in reacting with oxidizing species such as are generated by phagocytosing PMNs suggested that this c o m p o u n d may be useful in amplifying the native CL response of phagocytic cells, an hypothesis which has recently been experimentally demonstrated (Allen and Loose, 1976). The luminol-dependent system also exhibits sensitivity to such agents as superoxide dismutase (which removes superoxide anion via an enzymatic dismutation reaction) and sodium benzoate (a hydroxyl radical scavenger), which are known to suppress native chemiluminescence (Allen and Loose, 1976; Van Dyke et al., 1977). These findings suggest that luminol-dependent chemiluminescence may reflect the same or similar oxidative metabolic events as does the native CL assay. The marked augmentation of native PMN CL responses achieved by the addition of luminol into the cell suspension medium led us to examine the possibility that luminol-dependent CL may impart sufficient sensitivity to the CL assay to render it useful under conditions in which only very small blood samples are available. This report describes conditions under which luminoldependent chemiluminescence analysis (LDCA) may be utilized in assessing opsonophagocytic function. The effect of varying luminol, PMN and particle (zymosan) concentration on luminol-amplified CL response was studied. Further, we have compared the abilities of native and luminol-dependent CL assays in reflecting both serum opsonic and PMN metabolic dysfunctions.
317 MATERIALS AND METHODS
Human subjects Human blood was obtained from healthy adult volunteers and from the chronic granulomatous disease patient by venipuncture. Premature neonatal blood (2--3 ml) was obtained via an indwelling umbilical catheter from an infant being maintained in the Newborn Intensive Care Unit of the West Virginia University Medical Center. In all cases informed consent was granted.
Leukocyte isolation Following collection of blood into heparinized vacutainer tubes, peripheral blood granulocytes were harvested by dextran sedimentation according to B o y u m (1968). The dextran--blood mixture was incubated for 30 min at 37°C, after which the upper leukocyte-rich plasma fraction was collected into polypropylene tubes and centrifuged for 5 min at 50 X g. The supernatant fraction was retained for subsequent particle opsonization procedures (vide infra). The cell pellet was disrupted by gentle vortexing and residual erythrocytes removed by a m m o n i u m chloride (0.83%) lysis. The ~anulocytes were washed once in complete Dulbecco's phosphate buffer (PBS, Grand Island), pH 7.2, containing 0.2% bovine serum albumin. The resulting pellets were resuspended in 1--2 ml of PBS and leukocyte yields determined by hemacytometer. PMN concentrations were adjusted according to experimental requirements, as indicated.
Particle opsonization Zymosan A (Sigma) was suspended in a few milliliters of physiological saline and placed in a boiling water bath for 30 min. Dextran-diluted plasma recovered from the leukocyte-rich fraction was centrifuged at 600 X g for 5 rain in order to remove cellular debris. Five milliliters of the plasma supernatant were then added to the boiled zymosan (20 mg) preparation and the suspension was incubated for 30 min at 37°C. The zymosan was centrifuged as above, washed once in PBS, and diluted to the final appropriate concentration in PBS. Preliminary studies indicated that when using undiluted or 1 : 5 diluted plasma for particle opsonization, no differences were observed in either the rate of activation of phagocytosis-associated CL or in peak CL responses over the range of particle concentrations examined (1--4 mg zymosan). Opsonization of zymosan using 10% plasma, on the other hand, provided a satisfactory separation of cell activation rates and was therefore employed in those experiments in which the effect of particle concentration on luminolamplified CL response was studied.
Luminol preparation 0.5 milligram of luminol (Aldrich Chemical) was dissolved in 0.05 ml of
318 d i m eth y l sulfoxide and diluted to a stock c o n c e n t r a t i o n of 10 mg% in PBS. The stock solution was then diluted, as indicated, in PBS.
Chemiluminescence assay Native and luminol-dependent CL assays were perform ed using a standard liquid scintillation c o u n t e r (Packard Model 2002) operated at ambient t e m p e r a t u r e in the out-of-coincidence mode, with a gain setting o f 60% and a wide open window. By eliminating the coincidence circuitry, the two bialkali p h o to mu l t i pl i e r tubes function independently, thus permitting the instrument to be used as a p h o t o n counter. Plastic scintillation vials and PBS were dark-adapted prior to use. PMNs and buffer were first added to the vials, which were then m o n i t o r e d for several minutes in the scintillation c o u n t e r until a stable background chemiluminescence (typically around 1 0 , 0 0 0 c p m ) was obtained. Luminol (0.1 ml of the indicated dilution) a n d / o r zymosan (suspended in 1 ml of PBS) were added to the vials in order to initiate phagocytosis-associated chemiluminescence. In all experiments the final reaction volume was 5 ml. Detection o f opsonophagocytic defects by a 'cross-matching' technique We have applied to the CL assay a technique e m p l o y e d by Form an and Stiehm (1969) in the analysis of opsonic and phagocytic defects which is based u p o n the 'cross-matching' of control and patient PMNs and serum opsonins in all possible combinations. Briefly, control and patient PMNs axe isolated simultaneously while a test particle, in this case zymosan, is being opsonized with control or patient serum. F o u r scintillation vials are prepared in which the first two contain patient PMNs and the second two vials control PMNs. CL responses are initiated via the addition o f zymosan opsonized with control serum to one vial of each pair and zymosan opsonized with patient serum to the remaining two vials. Phagocytic defects may be evaluated by comparing the CL responses of control and patient PMNs exposed to zymosan opsonized by control serum, whereas opsonic defects may be assessed by comparing the CL responses of control PMNs exposed to zymosan opsonized by control or patient serum. RESULTS
Effect o f luminol concentration on peak CL response The effect of varying luminol c o n c e n t r a t i o n upon peak PMN chemiluminescence response is shown in Fig. 1. Each scintillation vial contained 1 0 6 PMNs and 4 mg o f opsonized zymosan. The peak native CL response obtained with opsonized zymosan alone is provided for comparative purposes. Addition of luminol to resting PMNs simultaneously with zymosan markedly augmented the phagocytosis-associated CL response in a concent r a t i o n - d e p e n d e n t fashion over the range o f concentrations studied. The highest luminol c o n c e n t r a t i o n studied (1 : 125 dilution from stock) cor-
319
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r e s p o n d e d t o a f i n a l c o n c e n t r a t i o n in 1 0 -8 M. Fig. 1 d e m o n s t r a t e s t h a t t h i s the zymosan-induced response such coincidence mode (900,000 cpm) was c u m b e r s o m e f o r u s e in t i m e s t u d i e s .
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320
dilution provided optimal amplification of the zymosan-induced CL responses and was therefore employed in all subsequent studies.
Effect o f PMN concentration on peak CL response Figure 2 reflects the relationship between peak CL response and PMN concentration, with luminol and zymosan (3 mg) concentrations held constant. Measurable CL responses to zymosan were observed at all PMN concentrations studied, with proportionately lower peak responses achieved with decreasing cell numbers. It seems likely that even lower PMN concentrations may be employed by increasing the luminol concentration, however this maneuver was not attempted. In comparing the peak responses of the various cell concentrations obtained with luminol amplification to that obtained with zymosan alone (refer to Fig. 1), it may be noted that an order of magnitude fewer PMNs produce a comparable peak CL in the luminolamplified system. Effect o f particle concentration on peak CL response Figure 3 depicts the temporal luminol-amplified CL curves obtained under conditions of varying particle concentration (1--4 mg of opsonized zymosan 16
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MINUTES AFTER ZYMOSAN ADDITION P M N CL analysis of neonatal o p s o n i c and p h a g o c y t i c defects using the cross-matching technique. N e o n a t a l b l o o d was obtained from a female infant o f approxim a t e l y 28 w e e k s gestation. Each vial contained 5 × 1 0 6 n e o n a t a l o r adult control P M N s and 4 m g z y m o s a n o p s o n i z e d with neonatal or control serum, as indicated. F i g . 5.
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in a final reaction volume of 5 ml). It may be seen that the rate o f cell activation is a function o f particle concentration (and consequently, o f the c e l l : p a r t i c l e ratio) over the range studied. The time at which peak CL occurs similarly varies with particle concentration, with lower concentrations activating PMN metabolism more slowly and producing a shift to the right of the temporal CL curve. It may be n o t e d that by opsonizing zymosan with 10% plasma instead of undiluted plasma both rate o f PMN activation and peak CL values are lowered. In particular, peak times are markedly shifted to the right and occur at approximately 60 min rather than at 5 - - 1 0 min as in previous experiments.
323
Assessing opsonophagocytic dysfunctions by LDCA Peripheral blood PMNs isolated from patients afflicted with chronic granulomatous disease of childhood generate defective CL. As these abnormal PMNs fail to generate substantial amounts of oxidizing species, it might then be anticipated that these cells would likewise fail to generate luminaldependent CL in the presence o f a particle such as opsonized zymosan. Figure 4 depicts the luminal-dependent CL responses of normal and CGD PMNs following addition of opsonized zymosan. As expected, CGD PMNs exhibit a markedly depressed CL response in the luminal-dependent CL assay as they do in the native CL assay. In fact, addition of up to 1 ml of the 10 mg% stock luminal solution did not augment the CL response of CGD PMNs.
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MINUTES A F T E R ZYMOSAN ADDITION Fig. 6. L u m i n a l - d e p e n d e n t CL analysis o f neonatal o p s o n i c and p h a g o c y t i c d e f e c t s using the cross-matching t e c h n i q u e . Each vial c o n t a i n e d 106 n e o n a t a l or adult control PMNs, 0.1 ml o f the 1 : 2 5 0 l u m i n a l dilution, and 3 mg z y m o s a n o p s o n i z e d w i t h n e o n a t a l or adult serum, as indicated.
324 As previously discussed, premature human infants are deficient with respect to serum alternative pathway opsonic capacity. Results o f a typical experiment comparing normal adult and neonatal temporal native CL responses are presented in Fig. 5. Neonatal serum is seen to be deficient with respect to opsonization of zymosan, an alternative pathway activator. Further, a neonatal PMN defect in oxidative metabolism may also be noted by comparing the CL responses of adult and neonatal PMNs phagocytosing zymosan coated with adult serum opsonins. We then wished to determine whether luminol-dependent CL analysis of neonatal opsonic and PMN metabolic defects provided similar results to those observed in Fig. 5. Fig. 6 shows the results of a LDCA temporal response curve comparing a premature neonate to an adult control. As in the case of the CGD study, the LDCA system reflects similar defects as are observed in the native CL assay. DISCUSSION The importance of the phagocytic cell, particularly the polymorphonuclear leukocyte, in host defense against infection has led to the developm e n t of a n u m b e r of clinical tests designed to assess phagocytic function. Among the more widely employed of these tests is the nitro blue tetrazolium (NBT) test described by Baehner and Nathan (1968). Although showing initial promise as a clinical test for assessing phagocytic function, the NBT test has recently undergone a critical reappraisal as to its reliability, principally due to reports of false-positive and false-negative results in several clinical situations (Fuenfer et al., 1976). Thus, new and more reliable methods of evaluating phagocytic function are needed. The chemiluminescence assay described by Allen et al. (1972) has been employed in the detection of PMN metabolic defects in patients with chronic granulomatous disease of childhood (Stjernholm et al., 1973) and in patients with myeloperoxidase deficiency (Rosen and Klebanoff, 1976). Cooper et al. (1972) reported that a patient with a complete deficiency of leukocyte glucose-6-phosphate dehydrogenase presented in a manner similar to that characteristic of CGD patients, but upon further examination it was found that the PMN oxidase enzymes exhibited normal activity whereas reduced pyridine cofactors were not produced. As a direct consequence of this deficiency, the patient's leukocytes failed to generate adequate amounts of hydrogen peroxide. G-6-PDH deficiency thus would represent a further PMN metabolic dysfunction which may be detectable using the LDCA method. Recently the chemiluminescence assay has been utilized in the study of defective opsonization due to complement and/or type-specific (antibody) opsonic defects (Hemming et al., 1976; Stevens and Young, 1977; Wilson et al., 1977). Thus, the CL assay has been employed in the detection of both PMN metabolic and serum opsonic defects, and as such may represent a
325 p r o t o t y p e of a potentially useful clinical test for evaluating phagocytic function. We have characterized a luminol-dependent chemiluminescence assay (LDCA) with respect to luminol, PMN and particle concentration requirements and have also compared the results of LDCA studies performed on patients with opsonic deficiencies (neonates) or PMN metabolic deficiencies (CGD) with the results obtained by native CL assay analysis of these defects. Our findings suggest that LDCA and native CL assays are capable of detecting opsonophagocytic dysfunctions in a qualitatively similar manner. However, the LDCA m e t h o d exhibits at least an order of magnitude greater sensitivity in that comparable peak CL responses may be obtained in the luminol system while using 10-fold fewer cells. The application of the luminol-dependent CL assay as a clinical test for assessing phagocytic capacity permits a reduction in the minimal PMN concentration required by the native CL assay. By reducing assay cell concentration requirements a lowering of blood sample volumes is thus permitted. This becomes particularly significant in the case of the premature human infant, in which blood samples exceeding 3--5 ml may be ill-advised. This paper describes the potential application of a luminol-dependent chemiluminescence assay in the rapid initial assessment of opsonophagocytic capacity in a clinical setting. LDCA analysis holds several advantages over existing clinical tests for assessing phagocytic function, including: (1) the relative ease with which the assay may be performed; (2) the improved sensitivity over the native CL assay, thus decreasing blood volume requirements; (3) ability of LDCA to be performed using a standard liquid scintillation counter or an inexpensive Chem-Glow p h o t o m e t e r (Van Dyke et al., 1977); and (4) the ability of the LDCA m e t h o d to simultaneously assess overall phagocytic function, as well as serum opsonic capacity and PMN metabolic activity independently, using the cross-matching technique (see Materials and Methods section). ACKNOWLEDGMENTS The authors wish to extend thanks to Dr. Ruth Phillips of the Department of Pediatrics for her assistance in obtaining blood samples from the CGD patient. This research was supported by NIH Training Grant GM 07039-03 and by W.V.U. Institutional Grant 2-210-1615 (97). REFERENCES Allen, R.C., R.L. Stjernholm and R.H. Steele, 1972, Biochem. Biophys. Res. Commun. 47,679. Allen, R.C. and L.D. Loose, 1976, Biochem. Biophys. Res. Commun. 69,245. Baehner, R.L., 1974, J. Pediatr. 84, 317. Baehner, R.L. and D.G. Nathan, 1968, N. Engl. J. Med. 278,971. Boyum, A., 1968, Scand. J. Lab. Invest. 21, Supplementum 97.
326 Cheson, B.D., J.T. Curnutte and B.M. Babior, 1977, in: Progress in Clinical Immunology, vol. 3, ed. B.S. Schwartz (Grune and Stratton, New York) p. 1. Cooper, M.R., L.R. DeChatelet, C.E. McCall, M.F. LaVia, C.L. Spurr and R.L. Baehner, 1972, J. Clin. Invest. 51,769. Curnutte, J.T., D.M. Whitten and B.M. Babior, 1974, N. Engl. J. Med. 290, 593. Forman, M.L. and E.R. Stiehm, 1969, N. Engl. J. Med. 281,926. Fuenfer, M.M., K. Scott and H.C. Polk, Jr., 1976, J. Surg. Res. 21,207. Hemming, V.G., R.T. Hall, P.G. Rhodes, A.O. Shigeoka and H.R. Hill, 1976, J. Clin. Invest. 58, 1379. Rosen, H. and S.J. Klebanoff, 1976, J. Clin. Invest. 58, 50. Stevens, P. and L.S. Young, 1977, Infect. Immun. 16, 796. Stjernholm, R.L., R.C. Allen, R.H. Steele, W.W. Waring and J.A. Harris, 1973, Infect. Immun. 7, 313. Stossel, T.P., C.A. Alper and F.S. Rosen, 1973, Pediatrics 52, 134. Totter, J.R., E.C. Dugros and C. Riveiro, 1960, J. Biol. Chem. 235, 1839. Van Dyke, K., M. Trush, M. Wilson, P. Stealey and P. Miles, 1977, Microchem. J. 22,463. Wilson, M.E., M.A. Trush, K. Van Dyke, M.D. Mullett and W.A. Neal, 1977, Pediatr. Res. 11,496.
315
LUMINOL-DEPENDENT CHEMILUMINESCENCE ANALYSIS OF OPSONOPHAGOCYTIC DYSFUNCTIONS
MARK E. WILSON, MICHAEL A. TRUSH, KNOX VAN DYKE, JAMES M. KYLE, MARTHA D. MULLETT and WILLIAM A, NEAL Departments of Pharmacology and Pediatrics, West Virginia University Medical Center, Morgantown, WV 26506, U.S.A. (Received 27 December 1977, accepted 15 April 1978)
The use of luminol as a means of amplifying the native chemiluminescence responses of phagocytosing human polymorphonuclear leukocytes was studied. Parameters examined included the effects of luminol, leukocyte and particle (opsonized zymosan) concentration on luminol-dependent chemiluminescence response. The results indicate that all of the parameters studied influence the luminol-dependent chemiluminescence assay system and that, on the basis of experiments designed to assess both opsonic and phagocytosis defects, this system appears to reflect the same or similar metabolic events as does native chemiluminescence. The luminol-dependent chemiluminescence assay represents a significant improvement of the phagocyte chemiluminescence assay in that greater sensitivity is possible, and may therefore have clinical utility as a means of evaluating phagocytic function.
INTRODUCTION
P h a g o c y t o s i n g h u m a n p o l y m o r p h o n u c l e a r l e u k o c y t e s (PMN) generate a b u r s t of oxidative m e t a b o l i c activity w h i c h results in t h e increased p r o d u c t i o n o f several p o t e n t i a l l y bactericidal o x y g e n m e t a b o l i t e s including supero x i d e anion, h y d r o g e n p e r o x i d , h y d r o x y l radical and singlet o x y g e n (reviewed b y Cheson et al., 1977). T h e ability o f PMNs t o m o u n t a n o r m a l r e s p i r a t o r y burst is d e p e n d e n t u p o n b o t h h u m o r a l (opsonic) and cellular (oxidative m e t a b o l i c ) factors, and d e f e c t s in either o f these p a r a m e t e r s are f r e q u e n t l y associated with increased susceptibility to i n f e c t i o n (Baehner, 1 9 7 4 ) . T h e i m p o r t a n c e o f n o r m a l serum o p s o n i c and PMN m e t a b o l i c funct i o n in h o s t defense against i n f e c t i o n has ' p r o m p t e d the d e v e l o p m e n t o f in vitro clinical m e t h o d s designed t o assess p h a g o c y t i c f u n c t i o n . In view o f certain deficiencies i n h e r e n t in m a n y o f these c u r r e n t m e t h o d s ( F u e n f e r et al., 1 9 7 6 ) , n e w and m o r e reliable a p p r o a c h e s to the p r o b l e m o f evaluating p h a g o c y t i c c a p a c i t y m u s t be t a k e n . F o l l o w i n g t h e o b s e r v a t i o n t h a t p h a g o c y t o s i n g PMNs generate electronically e x c i t e d states (such as singlet o x y g e n or e x c i t e d c a r b o n y l groups), as manifested b y a m e a s u r a b l e c h e m i l u m i n e s c e n c e (CL) response (Allen et al., 1 9 7 2 ) , PMNs f r o m patients afflicted with c h r o n i c g r a n u l o m a t o u s disease
316 (CGD) were found to exhibit a markedly diminished CL response (Stjernholm et al., 1973). CGD PMNs are characterized by an inability to generate significant amounts of superoxide and hydrogen peroxide (Curnutte et al., 1974). Thus, defective oxidative metabolic activity of PMNs correlated with diminished CL responses. More recently, defective particle opsonization has also been shown to result in depressed CL responses elicited from normal PMNs (Stevens and Young, 1977). Premature human infants are reportedly deficient with respect to serum opsonic capacity, particularly with regard to gram-negative organisms which are opsonized via the alternative complement pathway (Stossel et al., 1973). We have previously employed the native CL assay in assessing the functional status of the alternative pathway in nonimmune neonatal serum and found a significant defect in opsonic capacity as compared to adult serum (Wilson et al., 1977). The ability of the CL assay to reflect opsonic and phagocytic dysfunctions suggested its potential application in the evaluation of phagocytic function. In certain clinical situations, however, particularly in reference to the premature newborn infant, blood volumes required to perform CL assays may be considered excessive. Thus, we attempted to modify the CL assay in order to decrease the required PMN concentration. Luminol (5-amino-2,3,-dihydro-l,4-phthalazinedione) is a cyclic hydrazide which is readily oxidized to an electronically excited intermediate which undergoes subsequent relaxation to ground state with concomitant p h o t o n emission (i.e., chemiluminescence). This c o m p o u n d has been employed in the detection of enzyme-catalyzed free radical production (Totter et al., 1960). The high quantum efficiency of luminol in reacting with oxidizing species such as are generated by phagocytosing PMNs suggested that this c o m p o u n d may be useful in amplifying the native CL response of phagocytic cells, an hypothesis which has recently been experimentally demonstrated (Allen and Loose, 1976). The luminol-dependent system also exhibits sensitivity to such agents as superoxide dismutase (which removes superoxide anion via an enzymatic dismutation reaction) and sodium benzoate (a hydroxyl radical scavenger), which are known to suppress native chemiluminescence (Allen and Loose, 1976; Van Dyke et al., 1977). These findings suggest that luminol-dependent chemiluminescence may reflect the same or similar oxidative metabolic events as does the native CL assay. The marked augmentation of native PMN CL responses achieved by the addition of luminol into the cell suspension medium led us to examine the possibility that luminol-dependent CL may impart sufficient sensitivity to the CL assay to render it useful under conditions in which only very small blood samples are available. This report describes conditions under which luminoldependent chemiluminescence analysis (LDCA) may be utilized in assessing opsonophagocytic function. The effect of varying luminol, PMN and particle (zymosan) concentration on luminol-amplified CL response was studied. Further, we have compared the abilities of native and luminol-dependent CL assays in reflecting both serum opsonic and PMN metabolic dysfunctions.
317 MATERIALS AND METHODS
Human subjects Human blood was obtained from healthy adult volunteers and from the chronic granulomatous disease patient by venipuncture. Premature neonatal blood (2--3 ml) was obtained via an indwelling umbilical catheter from an infant being maintained in the Newborn Intensive Care Unit of the West Virginia University Medical Center. In all cases informed consent was granted.
Leukocyte isolation Following collection of blood into heparinized vacutainer tubes, peripheral blood granulocytes were harvested by dextran sedimentation according to B o y u m (1968). The dextran--blood mixture was incubated for 30 min at 37°C, after which the upper leukocyte-rich plasma fraction was collected into polypropylene tubes and centrifuged for 5 min at 50 X g. The supernatant fraction was retained for subsequent particle opsonization procedures (vide infra). The cell pellet was disrupted by gentle vortexing and residual erythrocytes removed by a m m o n i u m chloride (0.83%) lysis. The ~anulocytes were washed once in complete Dulbecco's phosphate buffer (PBS, Grand Island), pH 7.2, containing 0.2% bovine serum albumin. The resulting pellets were resuspended in 1--2 ml of PBS and leukocyte yields determined by hemacytometer. PMN concentrations were adjusted according to experimental requirements, as indicated.
Particle opsonization Zymosan A (Sigma) was suspended in a few milliliters of physiological saline and placed in a boiling water bath for 30 min. Dextran-diluted plasma recovered from the leukocyte-rich fraction was centrifuged at 600 X g for 5 rain in order to remove cellular debris. Five milliliters of the plasma supernatant were then added to the boiled zymosan (20 mg) preparation and the suspension was incubated for 30 min at 37°C. The zymosan was centrifuged as above, washed once in PBS, and diluted to the final appropriate concentration in PBS. Preliminary studies indicated that when using undiluted or 1 : 5 diluted plasma for particle opsonization, no differences were observed in either the rate of activation of phagocytosis-associated CL or in peak CL responses over the range of particle concentrations examined (1--4 mg zymosan). Opsonization of zymosan using 10% plasma, on the other hand, provided a satisfactory separation of cell activation rates and was therefore employed in those experiments in which the effect of particle concentration on luminolamplified CL response was studied.
Luminol preparation 0.5 milligram of luminol (Aldrich Chemical) was dissolved in 0.05 ml of
318 d i m eth y l sulfoxide and diluted to a stock c o n c e n t r a t i o n of 10 mg% in PBS. The stock solution was then diluted, as indicated, in PBS.
Chemiluminescence assay Native and luminol-dependent CL assays were perform ed using a standard liquid scintillation c o u n t e r (Packard Model 2002) operated at ambient t e m p e r a t u r e in the out-of-coincidence mode, with a gain setting o f 60% and a wide open window. By eliminating the coincidence circuitry, the two bialkali p h o to mu l t i pl i e r tubes function independently, thus permitting the instrument to be used as a p h o t o n counter. Plastic scintillation vials and PBS were dark-adapted prior to use. PMNs and buffer were first added to the vials, which were then m o n i t o r e d for several minutes in the scintillation c o u n t e r until a stable background chemiluminescence (typically around 1 0 , 0 0 0 c p m ) was obtained. Luminol (0.1 ml of the indicated dilution) a n d / o r zymosan (suspended in 1 ml of PBS) were added to the vials in order to initiate phagocytosis-associated chemiluminescence. In all experiments the final reaction volume was 5 ml. Detection o f opsonophagocytic defects by a 'cross-matching' technique We have applied to the CL assay a technique e m p l o y e d by Form an and Stiehm (1969) in the analysis of opsonic and phagocytic defects which is based u p o n the 'cross-matching' of control and patient PMNs and serum opsonins in all possible combinations. Briefly, control and patient PMNs axe isolated simultaneously while a test particle, in this case zymosan, is being opsonized with control or patient serum. F o u r scintillation vials are prepared in which the first two contain patient PMNs and the second two vials control PMNs. CL responses are initiated via the addition o f zymosan opsonized with control serum to one vial of each pair and zymosan opsonized with patient serum to the remaining two vials. Phagocytic defects may be evaluated by comparing the CL responses of control and patient PMNs exposed to zymosan opsonized by control serum, whereas opsonic defects may be assessed by comparing the CL responses of control PMNs exposed to zymosan opsonized by control or patient serum. RESULTS
Effect o f luminol concentration on peak CL response The effect of varying luminol c o n c e n t r a t i o n upon peak PMN chemiluminescence response is shown in Fig. 1. Each scintillation vial contained 1 0 6 PMNs and 4 mg o f opsonized zymosan. The peak native CL response obtained with opsonized zymosan alone is provided for comparative purposes. Addition of luminol to resting PMNs simultaneously with zymosan markedly augmented the phagocytosis-associated CL response in a concent r a t i o n - d e p e n d e n t fashion over the range o f concentrations studied. The highest luminol c o n c e n t r a t i o n studied (1 : 125 dilution from stock) cor-
319
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320
dilution provided optimal amplification of the zymosan-induced CL responses and was therefore employed in all subsequent studies.
Effect o f PMN concentration on peak CL response Figure 2 reflects the relationship between peak CL response and PMN concentration, with luminol and zymosan (3 mg) concentrations held constant. Measurable CL responses to zymosan were observed at all PMN concentrations studied, with proportionately lower peak responses achieved with decreasing cell numbers. It seems likely that even lower PMN concentrations may be employed by increasing the luminol concentration, however this maneuver was not attempted. In comparing the peak responses of the various cell concentrations obtained with luminol amplification to that obtained with zymosan alone (refer to Fig. 1), it may be noted that an order of magnitude fewer PMNs produce a comparable peak CL in the luminolamplified system. Effect o f particle concentration on peak CL response Figure 3 depicts the temporal luminol-amplified CL curves obtained under conditions of varying particle concentration (1--4 mg of opsonized zymosan 16
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MINUTES AFTER ZYMOSAN ADDITION P M N CL analysis of neonatal o p s o n i c and p h a g o c y t i c defects using the cross-matching technique. N e o n a t a l b l o o d was obtained from a female infant o f approxim a t e l y 28 w e e k s gestation. Each vial contained 5 × 1 0 6 n e o n a t a l o r adult control P M N s and 4 m g z y m o s a n o p s o n i z e d with neonatal or control serum, as indicated. F i g . 5.
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in a final reaction volume of 5 ml). It may be seen that the rate o f cell activation is a function o f particle concentration (and consequently, o f the c e l l : p a r t i c l e ratio) over the range studied. The time at which peak CL occurs similarly varies with particle concentration, with lower concentrations activating PMN metabolism more slowly and producing a shift to the right of the temporal CL curve. It may be n o t e d that by opsonizing zymosan with 10% plasma instead of undiluted plasma both rate o f PMN activation and peak CL values are lowered. In particular, peak times are markedly shifted to the right and occur at approximately 60 min rather than at 5 - - 1 0 min as in previous experiments.
323
Assessing opsonophagocytic dysfunctions by LDCA Peripheral blood PMNs isolated from patients afflicted with chronic granulomatous disease of childhood generate defective CL. As these abnormal PMNs fail to generate substantial amounts of oxidizing species, it might then be anticipated that these cells would likewise fail to generate luminaldependent CL in the presence o f a particle such as opsonized zymosan. Figure 4 depicts the luminal-dependent CL responses of normal and CGD PMNs following addition of opsonized zymosan. As expected, CGD PMNs exhibit a markedly depressed CL response in the luminal-dependent CL assay as they do in the native CL assay. In fact, addition of up to 1 ml of the 10 mg% stock luminal solution did not augment the CL response of CGD PMNs.
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MINUTES A F T E R ZYMOSAN ADDITION Fig. 6. L u m i n a l - d e p e n d e n t CL analysis o f neonatal o p s o n i c and p h a g o c y t i c d e f e c t s using the cross-matching t e c h n i q u e . Each vial c o n t a i n e d 106 n e o n a t a l or adult control PMNs, 0.1 ml o f the 1 : 2 5 0 l u m i n a l dilution, and 3 mg z y m o s a n o p s o n i z e d w i t h n e o n a t a l or adult serum, as indicated.
324 As previously discussed, premature human infants are deficient with respect to serum alternative pathway opsonic capacity. Results o f a typical experiment comparing normal adult and neonatal temporal native CL responses are presented in Fig. 5. Neonatal serum is seen to be deficient with respect to opsonization of zymosan, an alternative pathway activator. Further, a neonatal PMN defect in oxidative metabolism may also be noted by comparing the CL responses of adult and neonatal PMNs phagocytosing zymosan coated with adult serum opsonins. We then wished to determine whether luminol-dependent CL analysis of neonatal opsonic and PMN metabolic defects provided similar results to those observed in Fig. 5. Fig. 6 shows the results of a LDCA temporal response curve comparing a premature neonate to an adult control. As in the case of the CGD study, the LDCA system reflects similar defects as are observed in the native CL assay. DISCUSSION The importance of the phagocytic cell, particularly the polymorphonuclear leukocyte, in host defense against infection has led to the developm e n t of a n u m b e r of clinical tests designed to assess phagocytic function. Among the more widely employed of these tests is the nitro blue tetrazolium (NBT) test described by Baehner and Nathan (1968). Although showing initial promise as a clinical test for assessing phagocytic function, the NBT test has recently undergone a critical reappraisal as to its reliability, principally due to reports of false-positive and false-negative results in several clinical situations (Fuenfer et al., 1976). Thus, new and more reliable methods of evaluating phagocytic function are needed. The chemiluminescence assay described by Allen et al. (1972) has been employed in the detection of PMN metabolic defects in patients with chronic granulomatous disease of childhood (Stjernholm et al., 1973) and in patients with myeloperoxidase deficiency (Rosen and Klebanoff, 1976). Cooper et al. (1972) reported that a patient with a complete deficiency of leukocyte glucose-6-phosphate dehydrogenase presented in a manner similar to that characteristic of CGD patients, but upon further examination it was found that the PMN oxidase enzymes exhibited normal activity whereas reduced pyridine cofactors were not produced. As a direct consequence of this deficiency, the patient's leukocytes failed to generate adequate amounts of hydrogen peroxide. G-6-PDH deficiency thus would represent a further PMN metabolic dysfunction which may be detectable using the LDCA method. Recently the chemiluminescence assay has been utilized in the study of defective opsonization due to complement and/or type-specific (antibody) opsonic defects (Hemming et al., 1976; Stevens and Young, 1977; Wilson et al., 1977). Thus, the CL assay has been employed in the detection of both PMN metabolic and serum opsonic defects, and as such may represent a
325 p r o t o t y p e of a potentially useful clinical test for evaluating phagocytic function. We have characterized a luminol-dependent chemiluminescence assay (LDCA) with respect to luminol, PMN and particle concentration requirements and have also compared the results of LDCA studies performed on patients with opsonic deficiencies (neonates) or PMN metabolic deficiencies (CGD) with the results obtained by native CL assay analysis of these defects. Our findings suggest that LDCA and native CL assays are capable of detecting opsonophagocytic dysfunctions in a qualitatively similar manner. However, the LDCA m e t h o d exhibits at least an order of magnitude greater sensitivity in that comparable peak CL responses may be obtained in the luminol system while using 10-fold fewer cells. The application of the luminol-dependent CL assay as a clinical test for assessing phagocytic capacity permits a reduction in the minimal PMN concentration required by the native CL assay. By reducing assay cell concentration requirements a lowering of blood sample volumes is thus permitted. This becomes particularly significant in the case of the premature human infant, in which blood samples exceeding 3--5 ml may be ill-advised. This paper describes the potential application of a luminol-dependent chemiluminescence assay in the rapid initial assessment of opsonophagocytic capacity in a clinical setting. LDCA analysis holds several advantages over existing clinical tests for assessing phagocytic function, including: (1) the relative ease with which the assay may be performed; (2) the improved sensitivity over the native CL assay, thus decreasing blood volume requirements; (3) ability of LDCA to be performed using a standard liquid scintillation counter or an inexpensive Chem-Glow p h o t o m e t e r (Van Dyke et al., 1977); and (4) the ability of the LDCA m e t h o d to simultaneously assess overall phagocytic function, as well as serum opsonic capacity and PMN metabolic activity independently, using the cross-matching technique (see Materials and Methods section). ACKNOWLEDGMENTS The authors wish to extend thanks to Dr. Ruth Phillips of the Department of Pediatrics for her assistance in obtaining blood samples from the CGD patient. This research was supported by NIH Training Grant GM 07039-03 and by W.V.U. Institutional Grant 2-210-1615 (97). REFERENCES Allen, R.C., R.L. Stjernholm and R.H. Steele, 1972, Biochem. Biophys. Res. Commun. 47,679. Allen, R.C. and L.D. Loose, 1976, Biochem. Biophys. Res. Commun. 69,245. Baehner, R.L., 1974, J. Pediatr. 84, 317. Baehner, R.L. and D.G. Nathan, 1968, N. Engl. J. Med. 278,971. Boyum, A., 1968, Scand. J. Lab. Invest. 21, Supplementum 97.
326 Cheson, B.D., J.T. Curnutte and B.M. Babior, 1977, in: Progress in Clinical Immunology, vol. 3, ed. B.S. Schwartz (Grune and Stratton, New York) p. 1. Cooper, M.R., L.R. DeChatelet, C.E. McCall, M.F. LaVia, C.L. Spurr and R.L. Baehner, 1972, J. Clin. Invest. 51,769. Curnutte, J.T., D.M. Whitten and B.M. Babior, 1974, N. Engl. J. Med. 290, 593. Forman, M.L. and E.R. Stiehm, 1969, N. Engl. J. Med. 281,926. Fuenfer, M.M., K. Scott and H.C. Polk, Jr., 1976, J. Surg. Res. 21,207. Hemming, V.G., R.T. Hall, P.G. Rhodes, A.O. Shigeoka and H.R. Hill, 1976, J. Clin. Invest. 58, 1379. Rosen, H. and S.J. Klebanoff, 1976, J. Clin. Invest. 58, 50. Stevens, P. and L.S. Young, 1977, Infect. Immun. 16, 796. Stjernholm, R.L., R.C. Allen, R.H. Steele, W.W. Waring and J.A. Harris, 1973, Infect. Immun. 7, 313. Stossel, T.P., C.A. Alper and F.S. Rosen, 1973, Pediatrics 52, 134. Totter, J.R., E.C. Dugros and C. Riveiro, 1960, J. Biol. Chem. 235, 1839. Van Dyke, K., M. Trush, M. Wilson, P. Stealey and P. Miles, 1977, Microchem. J. 22,463. Wilson, M.E., M.A. Trush, K. Van Dyke, M.D. Mullett and W.A. Neal, 1977, Pediatr. Res. 11,496.