Quantitative neutrophil iodination — a simplified assay

Journal of Immunological Methods, 23 (1978) 335--340 © Elsevier/North-Holland Biomedical Press

335

Q U A N T I T A T I V E N E U T R O P H I L I O D I N A T IO N -- A SIMPLIFIED ASSAY

T.J. LENEHAN and J.F. GREALLY Immunology Dept., Trinity College, Dublin 2, Ireland (Received 21 December 1977, accepted 16 April 1978)

A modified method for the quantitative determination of neutrophil iodination is described. The system utilises fewer neutrophils thereby necessitating the taking of less blood. A glass fibre disc washing procedure is introduced. This reduces the hazards of handling radioactive precipitates and is time saving. It also has the advantages that it permits more efficient removal of unbound iodide as seen in background counts. Greater reproducibility of results was also obtained.

INTRODUCTION Phagocytosis of microorganisms by neutrophils initiates a sequence o f events in the cell which lead to microbial killing and degradation. T he phagocytic process is characterized by increased oxygen c o n s u m p t i o n by the neutrophil, increased hexose m o n o p h o s p h a t e pat hw ay activity and increased acid, h y d r o g e n p e r o x i d e (H202) and superoxide anion p r o d u c t i o n (Baehner et al., 1975). This series of metabolic changes is accompanied by degranulation where the lysosomal enzymes are discharged into the phagocytic vacuole (Bainton, 1971). Inactivation and degradation of the ingested particle follows. Th e lysosomal e n z y m e m y e l o p e r o x i d a s e (MPO) t oget her with H202 and an oxidisable c o f a c t o r such as halide combine to form a p o t e n t antimicrobial system (Klebanoff, 1970). Pincus and K l e b a n o f f (1971) described a quantitative assay to determine iodination mediated by the MPO-H202 system. T h e y d e m o n s t r a t e d no iodination by neutrophils from patients with chronic granulomatous disease (CGD). These patients lack the capacity to generate H202 during phagocytosis (Holmes et al., 1967). Reduced iodination was also f o u n d in patients with he r edi t ar y m y e l o p e r o x i d a s e deficiency and familial lip o ch r o m e histiocytosis. Thus neutrophil mediated iodination is a useful t ool for m o n i t o r i n g oxidative metabolism during the phagocytic and killing processes. T he aim o f this work was to m o d i f y and simplify the assay system. The system described is less time consuming and reduces the hazards o f working with radioactive material to a m i ni m um . L o w numbers o f neutrophils m ay be used in the assay system w i t h o u t affecting its sensitivity. The modified

336 washing p r o c e d u r e permits m o r e efficient removal of u n b o u n d label and has been f o u n d to reduce the scatter in individual determinations. MATERIALS AND METHODS

Isolation o f leukocytes Blood was taken from healthy adult volunteers, from a 7-year-old male (EG) with chronic granulomatous disease and from his mother. The blood was taken into 50 t~l of preservative-free heparin (3,000 units/ml). Plasmagel (Lab. Roger Bellon, Neuilly sur Seine, France) was mixed with the blood at a ratio o f 1 part to 4 parts of blood. The m i xt ure was allowed to sediment at 37°C f o r 15 min. The leukocyte-rich supernatant was washed 3 times in Hanks balanced salt solution (HBSS) containing 2 0 m M Hepes buffer (pH 7.3). The neutrophil c o u n t was adjusted to 5 X 106 cells/ml.

Preparation o f particles Th e particle of choice was C. albicans since this organism gave the best ratio o f test to background counts (unpublished observations). T he organism was grown in n ut r i ent broth for 4 days at 37°C. T he cell suspension was washed 3 times with HBSS and heated at 100°C for 30 min. The suspension was then adjusted to 5 X 106 cells/ml and stored at 4°C prior to use.

Measurement o f iodination The p r o ced u r e described by Pincus and K l ebanoff (1971) was modified as follows: The standard reaction m i xt ur e contained 5 X l 0 s neutrophils, 20% p o o led or autologous hum a n serum, 5 X l 0 s C. albicans and 0.2 pCi of sodium ~2Siodide (containing 40 nmol o f carrier sodium iodide) in a total volume o f 260 pl. T h e reaction m i x t u r e was incubated at 37°C for 60 min with co n tin u o u s mixing in siliconised glass tubes. Separate tubes were included to determine the non-specific binding (background) of label by serum and particles and to measure the activity to resting cells. In one set o f experiments the washing procedure o f Pincus and K l ebanoff (1971) was used. The reaction was stopped by the addition o f 1 ml o f cold 10% trichloracetic acid (TCA). The precipitate was centrifuged at 2000 X g f o r 5 min and washed 3 times with cold 10% TCA. The tubes containing washed precipitate were c o u n t e d in a gamma c o u n t e r (Nuclear Chicago Electronics Corp.). In a second set of experiments 100 gl aliquots were removed from the incubation tubes and placed on glass fibre discs (2.5 cm diameter GF/C Whatmans). Th e discs were immersed in cold 10% TCA to give a washing volume of 10 ml TCA per disc. The discs were swirled occasionally in a 500 ml glass beaker. After 5 min the fluid was poured o f f and fresh TCA added. The p r ocedur e was repeated three times. Following washing the discs were placed in plastic tubes and counted.

337 RESULTS The quantitative neutrophil iodination test depends on the conversion o f inorganic iodide t o a T C A - p r e c i p i t a b l e f o r m a f t e r e x p o s u r e o f isolated n e u t r o p h i l s to o p s o n i s e d particles. T a b l e 1 i n d i c a t e s t h e r e q u i r e m e n t f o r l e u k o c y t e s , particles a n d s e r u m . N o i o d i n a t i o n o c c u r r e d w h e n t h e n e u t r o p h i l s w e r e h e a t t r e a t e d at 100°C f o r 5 m i n p r i o r t o testing. S e r u m p r o v i d e s t h e o p s o n i n s r e q u i r e d f o r p h a g o c y t o s i s a n d r e d u c e d i o d i n a t i o n o c c u r r e d in t h e a b s e n c e o f s e r u m . O p t i m u m i o d i n a t i o n t o o k place o n l y w h e n n e u t r o p h i l s , particles a n d s e r u m w e r e i n c u b a t e d t o g e t h e r in t h e p r e s e n c e o f N a ~2sI. C o m p a r i s o n o f t h e p r e c i p i t a t e washing a n d glass f i b r e filter m e t h o d indic a t e d t h a t m o r e e f f i c i e n t r e m o v a l o f u n b o u n d label was achieved using t h e l a t t e r m e t h o d . Using n o r m a l n e u t r o p h i l s , p r e c i p i t a t e washing was carried o u t 12 times, and glass f i b r e disc w a s h i n g a t o t a l o f 21 times. E a c h individ u a l t e s t was s a m p l e d in q u a d r u p l i c a t e . R e a c t a n t s w i t h o u t n e u t r o p h i l s ( b a c k g r o u n d c o u n t s ) gave m e a n values (±S.D.) o f 3.4 ± 2.8 a n d 1.56 ± 0.91 n m o l / 1 0 7 P M N / h ( T a b l e 1), f o r p r e c i p i t a t e a n d glass fibre disc washing r e s p e c t i v e l y . P r e c i p i t a t e w a s h i n g was c o n s i d e r a b l y less e f f e c t i v e at r e m o v i n g u n b o u n d label. T h e a m o u n t o f variat i o n b e t w e e n p r o c e d u r e s as m e a s u r e d b y t h e s t a n d a r d d e v i a t i o n is also g r e a t e r w h e r e t h e w h o l e p r e c i p i t a t e was washed. In t e s t m i x t u r e s w h i c h i n c l u d e d n e u t r o p h i l s , 16.6 ± 7.8 a n d 13.0 ± 3.7 n m o l iodide b o u n d / 107 P M N / h w e r e o b t a i n e d a f t e r washing b y t h e r e s p e c t i v e p r o c e d u r e s . Glass fibre disc w a s h i n g gives a b e t t e r t e s t t o b a c k g r o u n d r a t i o a n d m u c h less variat i o n in d i f f e r e n t tests. T h e r e s p e c t i v e n m o l e a m o u n t s o f iodide b o u n d t o p r o t e i n are given in T a b l e 1. N o significant loss o f p r e c i p i t a t e o c c u r r e d f r o m t h e glass fibre discs. This was e s t i m a t e d b y w a s h i n g discs f r o m o n e assay 3, 4, 5 or 6 times. N o differe n c e in c o u n t i n g levels was f o u n d in discs s u b m i t t e d t o a n y o f t h e f o u r w a s h i n g

TABLE 1 IODINATION BY INTACT NEUTROPHILS Test mixtures contained 5 × 10 s PMN, 5 × 10 s C. albicans and 20% normal pooled serum. Precipitate washing values are the mean of 12 experiments + S.D. Filter disc washing values are the mean of 21 experiments + S.D. Supplements

Precipitate washing

Filter disc washing

Serum + particles PMN + particles PMN + serum PMN + serum + particles

3.4 4.1 3.9 16.6

1.56 2.00 1.69 13.0

+_2.8 _+3.3 _+3.5 _+7.8

_+0.91 -+ 0.74 -+ 1.03 _+3.7

338 100-

.J

.|

t-Z

o 50o U_

0

3 oo • ouQ

Azide 2 mM

••

Cyanide

CGD

Mother

10 mM

(E G)

(EG)

Fig. 1. The effect of metabolic inhibitors azide and cyanide on iodination by neutrophils. Minimal iodination by CGD neutrophils and intermediate iodination by neutrophils of the patient's mother is indicated. Each point represents a single determination. The mean bar is indicated for each experiment.

procedures. It appears unlikely therefore t hat any significant loss of precipitate from the discs takes place in this system. Iodination was totally inhibited by p r e t r e a t m e n t o f neutrophils with 10 mM potassium cyanide or 2 mM sodium azide or by simultaneous addition o f the inhibitors to the incubation mixtures (Fig. 1). Iodination b y leukocytes isolated from a male child (EG) with CGD was less than 2% of control values where heat treated C. albicans was the test particle (Fig. 1). Similar results were obtained when the test particle was S t a p h y l o c o c c u s aureus (Oxford). L e u k o c y t e s from the child's m o t h e r on two occasions gave iodination levels o f 63% and 68% of cont rol values. DISCUSSION The q u an titati on of l e u k o c y t e iodination during phagocytosis provides a useful investigative t ool f or the investigation o f neutrophil function defects, particularly defects involving the MPO-H202 systems (K l ebanoff and Clarke, 1977). Oth er killing systems such as the cationic protein system described by Spitznagel and Chi (1963) and the m o n o c y t e antifungal system described by Lehrer (1975) are n o t taken a c c ount o f in the quantitative iodination system. However, neutrophil d y s f u n c t i o n associated with a specific defect in cationic proteins has n o t ye t been detected in man.

339 It now appears that hydrogen peroxide and its derivatives play a central role in inactivation of ingested microorganisms. The MPO-H202-halide system may augment the killing process. Indeed iodide is not an absolute requirement for the system. Chloride is present in the neutrophil at higher concentration than iodide and Klebanoff (1968) demonstrated that chloride could be utilised by the MPO-system. Nevertheless, quantitative iodination is a useful monitor of the MPO-H202 system. Large amounts of H202 are produced close to the neutrophil cell membrane during phagocytosis (Root and Stossel, 1974). It diffuses into the phagocytic vacuole where MPO will normally be present following degranulation. Iodide is concentrated preferentially by neutrophils (Siegel and Sachs, 1964) and diffuses into the phagocytic vacuole. That this process takes place is indicated by the finding of R o o t and Stossel (1974) that the majority of TCA-precipitable iodide is present in the phagocytic vacuole. The method described in this paper has the advantage of requiring lower numbers of neutrophils than other methods described. Thus less blood need be taken from the patient. This may be important and desirable where y o u n g children and babies are involved. Iodination is dependent on the presence of neutrophils. It is inhibited by the metabolic inhibitors sodium azide and potassium cyanide. Iodination by the neutrophils of a male child with CGD was less than 2% of control values and his mother's iodination system was 63--68% of normal. Thus by using 5 × 10 s PMN's in the test system one does not affect the iodination function. The washing of whole radioactive precipitates by centrifugation presents hazards. Where large numbers of tests are involved, m a n y manipulations are required for washing. Centrifugation is hazardous from the possibility of breakage and aerosol formation. It was also found that aggregation of the precipitate during centrifugation did n o t permit efficient washing as seen in the high background counts. By washing on glass fibre discs most of these hazards are removed. The p r o c e d u r e was less time consuming. Greater reproducibility of results and lower backgrounds were obtained, indicating more efficient removal of u n b o u n d label. The procedure as described, therefore, has the advantages of requiring fewer cells, is less time consuming and safer than previously reported methods. Its allows greater reproducibility and is more amenable to use in multiple testing. ACKNOWLEDGEMENTS We wish to acknowledge support for this work from the Medical Research Council of Ireland and the Laboratory Medicine Development Fund, Trinity College.

340 REFERENCES Baehner, R.L., S.K. Murrmann, J. Davis and R.B. Johnston Jr., 1975, J. Clin. Invest. 56, 571. Bainton, D.F., 1971, in: Phagocytic Mechanisms in Health and Disease, eds. R.C. Williams and H.H. Fudenberg (Intercontinental Medical Book Corp., New York) p. 123. Holmes, B., A.R. Page and R.A. Good, 1967, J. Clin. Invest. 46, 1422. Klebanoff, S.J., 1968, J. Bacteriol. 95, 2131. Klebanoff, S.J., 1970, Science 169, 1095. Klebanoff, S.J. and R.A. Clark, 1977, J. Lab. Clin. Med. 89, 675. Lehrer, R.I., 1975, J. Clin. Invest. 55, 338. Pincus, S.H. and S.J. Klebanoff, 1971, N. Engl. J. Med. 284,744. Root, R.K. and T.P. Stossel, 1974, J. Clin. Invest. 53, 1207. Siegel, E. and B.A. Sachs, 1964, J. Clin. Endocr. Metab. 24,313. Spitznagel, J.K. and N.Y. Chi, 1963, Am. J. Pathol. 45,697.