Acta path. microbiol. scand. Section B. 82, 136142 , 1974

EFFECT OF SODIUM AZIDE UPON NORMAL AND PATHOLOGICAL

GRANULOCYTE FUNCTION

The University Clinic for Infectious Diseases, and Statens Seruminstitut, Department of Clinical Microbiology, Blegdamshospitalet, Copenhagen, Denmark

Using an improved method for the evaluation of the phagocytic function of human neutro- phi1 granulocytes in vitro, the effect of sodium azide (NaN,) upon the function of normal leucocytes and leucocytes from patients suffering from chronic granulomatous disease (C.G.D.) and from related carriers of C.G.D. has been investigated. 10-2 M NaN,, causes marked inhibition of intraleucocytic killing of Staphylococcus aureus by nonnal leucocytes without interfering with the ingestion-phase. The determination of intracellular recover- able bacteria is highly sensitive in terms of demonstrating defective intraleucocytic killing and it is preferable to determination of total recoverable bacteria from the system. l h i s has facilitated the detection of minor differences in intraleucocytic killing capacity between azide-treated normal leucocytes, C.G.D. leucocytes, and azide-treated C.G.D. leucocytes. Blocking of intraleucocytic killing with NaN, should prove a valuable tool in the investiga- tion of the function of granulocyte bactericidal systems in clinical conditions.

The importance of precise evaluation of the capacity of human neutrophil granulocytes to kill ingested micro-organisms has been em- phazised in later years by reports of con- genital and acquired defects in intraleuco- cytic killing ( 2 , 5, 7, 16, 2 2 ) . The combined activities of ingestion and killing can be stu- died in nit70 by incubation of the cells with a microbial test-organism followed by deter- mination at prescribed intervals of total re- maining viable organisms (intra- plus extra- cellular) by counting of colony-forming units ( C F U ) (4, 17). Distinction between the ingestion- and the killing-effect requires, however, exact determination of intracellular

Received 28.ix.73 Accepted 30.x.73 Requests for reprints should be addressed to:

C. Koch, M.D., Blegdamshospitalet, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark.

136

C F U which in turn depends upon effective elimination of non-ingested CFU from the system. This can be accomplished with the aid of fastacting antibiotics which do not penetrate the leucocytes (8, 2 1 ) . By simul- taneous determinations of the recoverable total CFU and recoverable intracellular C F U alone, the efficiency of both the ingestion and the intraleucocytic killing can thus be deter- mined with reasonable precision ( 15) .

The number of viable intracellular CFU depends upon the rate of ingestion and the rate of intraleucocytir killing. The degree of intraleucocytic killing could therefore be evaluated more exactly if the total number of ingested CFU was determined simultane- ously. This would be possible if intraleuco- cytic killing could be effectively prevented without interference with the rate of inges- tion. For this reason the present studies were

undertaken in which the effect of a metabolic inhibitor, sodium azide, with a previously de- monstrated blocking effect on intraleucocytic killing (12) was determined in an improved system recording both total and intracellular recoverable CFU ( 15) .

M A T E R I A L S A N D M E T H O D S

The technique employed is a modification of the method of Alexander et al. (1968) described in detail elsewhere (15) . Briefly, 2.5 x 106 polyrnor- phonuclear leucocytes were mixed with Staphylo- coccus a r c u s , strain 502A, in approximately 1 :1 ratio of cells to bacteria in gelatinized, heparinized Hank's balanced salt solution in the presence of 10 per cent normal human pooled serum. Incuba- tions were carried out in volumes of 2.0 ml a t 35" C with end over end rotation at 20 rev/min. For determination of total CFU 0.5 ml reaction mixture was withdrawn a t 2 and 4 hours, transferred to 4.5 ml distilled water for disruption of the cells followed by serial ten-fold dilutions and pour-plating. Colo- nies were counted after 48 hours' incubation at 35" C. For determinations of intracellular CFU pe- nicillin, 100 iu per ml, and streptomycin, 100 pg per ml, were added to separate tubes after 15 minutes' incubation. 0.5 and 1.0 ml were removed a t 2 and 4 hours, respectively, transferred to separate tubes followed by washing of the cells to get rid of the antibiotics, lysis in distilled water, serial dilu- tion, and pour-plating. Initial CFU was determined separately by serial dilution and pour-plating of the initial bacterial suspension. Sodium azide (Merck, Darmstadt, Germany), NaN,, in phos- phate-buffered saline was added in a volume of 20 pl to the reaction mixture prior to incubation, to give a final concentration as indicated in the re- sults. In experiments using phenylbutazone, this agent (Geigy, Basel, Switzerland) was dissolved in phosphate-buffered, gelatinized, heparinized Hank's balanced salt solution and further adjusted to physiological p H with 2.8 per cent sodium bi- carbonate. T h e final concentration of phenylbu- tazone was 2 mg per ml. The reaction mixtures of leucocytes and bacteria, were then made up in this medium and incubations carried out as de- scribed above.

Blood was obtained from normal adult persons, from one female and four male patients with chronic granulomatous disease, and from eight re- lated female carriers of this disease. The results of leucocyte function studies in these patients and relatives have been presented previously ( 14).

10;; M 10 -

- - 10-6 -

0

2 4 h o u r s

Fig. 1 . Effect of increasing molarity of sodium azide (NaN,), as indicated in the figure, upon the total recoverable colony-forming units (CFU) Staph. aureus from a reaction system of normal leucocytes and Staph. aureus. Mean of two experiments.

R E S U L T S

Figure 1 shows the effect of sodium azide in graded concentrations upon the total number of CFU recoverable with normal leucocytes. Marked inhibition of the killing of Staph. a u r e u by normal leucocytes can be detected with M azide, and killing appears to be completely prevented by M. Figure 2 shows, however, that the number of recover- able intracellular CFU increases progressively with increasing concentration of azide up till lo-' M. Above this molarity, azide was bac- tericidal towards the test-organism. Figure 3 shows the effect of sodium azide upon normal cells compared to cells from patients and car- riers of chronic granulomatous disease (C.G. D.) if total CFU were recorded. Reduction in total CFU is essentially nil1 with cells from C.G.D. patients and azide, in concen- trations above 2 x M, renders normal cells a t least as inactive as C.G.D. cells. Cells from heterozygous carriers of C.G.D. are functionally intermediate to normal cells and C.G.D. patient cells or azide-treated normal cells.

M azide upon recover- able intracellular CFU from these three

The effect of

137

og CF U

6

5

4

3

0

NaN

-. M - 7 - - - -. 10-3 - -- 1

I / i-

-. -\ .

-. -1 .. 1

-. . -. -\

.

-

- -

0

1

2 L h o u r

F i g . 2. Effect of increasing molarity of sodium azide, as indicated in the figure, upon the intracellular recoverable colony-forming units (CFU) Staph. aureus from a reaction system of normal Ieucocytes and Staph. mureus. Mean of two experiments.

groups of cells is shown in Figure 4. There is a marked increase in intracellular CFU when normal cells are treated with azide (cir- cles). A slight increase in intracellular CFU was seen from untreated to azide-treated C.G.D. heterozygote cells (triangles). This is not surprising since C.G.D. carriers are sup- posed to harbour a normal and a defective cell population (24 ) and azide would render all the cells defective. Even if cells from the homozygote C.G.D. patients were used, azide seems to increase to a minor extent the number of recoverable intracellular C F U (squares). I n Figure 5, the effect of M sodium azide is compared with that of phe- nylbutazone, 2 mg per mi,-another known inhibitor of intraleucocytic killing ( 2 1,23). If total CFU were recorded, complete inhibi- tion of killing by normal cells seems to be ac- chieved with both agents, but if intracellular CFU were recorded, the blocking effect of a i d e seems to be slightly superior to that of phenylbutazone. Since, however, the effect of azide was slightly decreased in the simul-

138

taneous presence of phenylbutazone this might partly be explained by an inhibitory effect of phenylbutazone upon the ingestion, whereby fewer bacteria would be taken up in the cells, and therefore more bacteria killed by the antibiotics in the surrounding medium. Phenylbutazone thus inhibits both ingestion and intraleucocytic killing of E. coli by guinea pig polymorphonuclear leucocytes

T o study whether azide was bound to tht. cells , normal leucocytes were incubated for 30 minutes at 35" C with rotation in the presence and absence of 2.1 x 10 " M so- dium azide. The cells were then washed thrice a t low-speed centrifugation followed by reaction with the test-organism in the usual manner. The results shown in Figure 6 indicate that azide exerts no permanent effect upon resting cells. This suggests that azide either diffuses freely across the cell mem-

( 2 3 ) .

c o n t r o l

2 L h o u r s

Fig. 3. Total recoverable colony-forming units (CFU) Staph. uureus from reaction systems of Staph. aureus and: 1 ) normal leucocytes ( 0 ; ten experiments in four persons), 2 ) leucocytes from patients with chronic granulomatous disease (C.G. D.) ( m ; eight experiments in five patients) 3 ) leucocytes from female carriers of C.G.D. (c.g.d.) (A; eight experiments in seven carriers), and 4 ) normal leucocytes in the presence of sodium azidr (NaN,), 2 x 10." M ( 0 ; four experiments in three persons) and 1 x 10-2 hl ( A ; two experi- ments in two persons).

109 C F U

6

5

4

3

0-- -- -0 I

0 1 I

2 4 h o u r s

Fig. 4. Intracellular recoverable colony-forming units ( C F U ) Staph. aureus from reaction systems of Staph. mureus and: 1 ) normal leucocytes ( 0 ; six experiments in four persons), 2 ) leucocytes from patients with chronic granulomatous disease (C.G. D.) ( m ; four experiments in three patients), and 3 ) leucocytes from female carriers of C.G.D. (c.g.d.) (A; three experiments in three carriers). Open symbols represent intracellular recoverable CFU from the corresponding leucocytes in the presence of sodium azide (NaN,), 1 x M (same number of experiments).

brane, but is not firmly bound to its site of action, when the cells are not engaged in phagocytosis, or that the membrane of rest- ing cells is not permeable to azide.

D I S C U S S I O N

Intracellular killing of a number of micro- organisms, including Staph. aureus, which is katalase positive, is probably largely mediated through the action of hydrogen peroxide, generated through the increased oxydative metabolism subsequent to ingestion, in conjunction with myeloperoxidase of the leucocytes and possibly an oxydizable co- factor (11, 18, 19). Sodium azide inhibits the antibacterial activity of an isolated sy- stem made up of these components and that of the intact cells (12) . The effect of azide

can be ascribed to inhibition of the enzymatic activity of the myeloperoxidase of the cells. Azide will also inhibit the iodination of bac- teria which can be brought about by H,O, in the presence of peroxidase and iodide ( 1 1 ).

In the present study, killing of Staph. aureus by normal leucocytes was completely inhibited by M azide if the total re- coverable CFU was used as the indicator (Figure 1 ) . By measuring recoverable intra- cellular CFU, the inhibition was shown to be even further increased from to lo-’ M azide since even more CFU could be re- covered intracellularly from cells treated with lo-? than cells treated with M azide (Figure 2 ) . Two possibilities could explain this finding. One is that the recording of re- coverable intracellular CFU is a more sen- sitive method of demonstrating decreased intraleucocytic killing, which at least in part can be explained by the elimination of con-

! t F !

-0 c o n t r o l --

0 I// , 2 L h o u r s

Fig. 5. Total recoverable colony-forming units (CFU) Staph. aureus (-) and intracellular recoverable CFU (- - - -) from reaction systems of Staph. aureus and: 1 ) normal leucocytes ( O ) , 2 ) normal leucocytes in the presence of phenyl- butazone (But ) , 2 mg per ml (A), 3 ) normal leu- cocytes in the presence of sodium azide (NaN,), 1 x 10-2 M ( ), and 4 ) normal leucocytes in the presence of But and NaN, in the same concen- trations ( V ).

139

-0 -.

L h o u r s 2

Fig. 6. Negative effect of pre-incubation (35” C / 30 min) of normal leucocytes with sodium azide, 2.1 x 10-3 M ( 0 ) compared to control leucocytes, pre-incubated without azide ( 0 ) . The cells were washed thrice after pre-incubation before addition to a reaction system of leucocytes and Staph. aureus. ~ indicates total recoverable colony- forming units (CFU) Staph. aureus and indicates intracellular recoverable CFU.

taminating non-ingested bacteria from the system. The other is that the ingestion is stimulated by azide by which an increased number of bacteria will be transferred from the outside to the interior of the cells, ren- dering them out of reach of the effect of the antibiotics. Sodium azide, in concentrations of 10-o M, can stimulate glucose C-1 oxida- tion by human leucocytes (13) which may be mediated via detoxification of accumulated H,O, through the glutathione system (20). The energy required for ingestion seems, however, to be supplied mainly through anae- robic glycolysis and not through an increased oxydation of glucose through the hexose mo- nophosphate pathway (9).

Intraleucocytic killing of Staph. aureus, which are katalase positive, seems to be completely abolished in leucocytes from pa- tients with C.G.D. (8). The finding that azide treatment of C.G.D. cells caused a

further increase in the number of intracellular recoverable CFU might therefore suggests a stimulation of ingestion (Figure 4 ) . Stimula- tion of metabolic activity by inhibition of myeloperoxidase and accumulation of H,O, however, would not be expected to occur in these cells since C.G.D. leucocytes are incap- able of manifesting the increase in oxydative metabolism, including increased H,O, pro- duction that normally follows the ingestion of particles (9, 10). It is more likely that the present technique of recording intracellular CFU is sensitive to such a degree that i t de- tect a slight residual killing of Staph. aureus in C.G.D. leucocytes inhibited by azide. This slight killing of the bacteria in C.G.D. cells might be mediated by the action of the myelo- peroxidase of the cells in combination with H,O, produced by the bacteria in excess of that broken down by the katalase produced by the bacteria per se. Indeed, such a me- chanism of “metabolic suicide” of the bac- teria has been suggested to account for the striking ability of C.G.D. leucocytes to kill certain katalase negative micro-organisms

The defect of C.G.D. leucocytes was in the present experiments found to be slightly more pronounced than that of azide-treated nor- mal cells (Figure 4). A bactericidal effect of H,O,, not mediated through the action of myeloperoxidase, has been suggested ( 13). This would fit in with the present finding, that C.G.D. leucocytes that do not generate H,O, at all, are more defective than myelo- peroxidase-inhibited normal cells. Support for this idea comes from the important find- ing that the killing defect in cells from pa- tients with a genetic absence of myeloperoxi- dase is not as pronounced as that induced in normal cells by azide-treatment (12). The present study does not indicate a similar proli- feration of azide-insensitive bactericidal sy- stems to occur in C.G.D. leuocytes, since the defect of these cells was at least as profound as that of azide-treated normal cells (Figure 4 ) . Nor would this be expected if a compen- satory azide-insensitive bactericidal system was H,O,-dependent.

(6) .

140

I t has recently been suggested that super- oxide, O,-, may be formed in human leuco- cytes during ingestion of particles, and might possess bactericidal activity ( 3 ) . I t is indi- cated by the present studies showing complete inhibition of intraleucocytic killing by inhibi- tion of myeloperoxidase with sodium azide that if energy-release from 0,- is important for killing this would be via reduction to H,O, and further reaction of H,O, with m yeloperoxidase.

The results of the studies shown in Figure 6 indicate that azide exerts no permanent effect on resting cells. One possibility to be mentioned is that the resting cell membrane is not permeable to azide. In that case it would seem likely that azide is taken up along with the ingested particles from the sur- rounding medium and azide would then be located in critical position in the phagocytic vacuole if myeloperoxidase acts in the vacuole after liberation of granular contents into the vacuole.

The point of importance for the practical application of sodium azide in an in vitro phagocytic system is the finding that this agent effectively blocks intraleucocytic kill- ing of Staph. uureus without interfering with the ingestion-phase. A slight stimulation of the ingestion could be indicated by some of the data, but the experiments with azide- treated leucocytes from C.G.D. patients do not support this suggestion. The blocking ef- fect of azide was found to be at least as pro- nounced as that induced by phenylbutazone which has been used in another modification of the technique aimed at a more precise eva- luation of granulocyte function (21 ). The present studies further illustrate the scnsiti- vity by which defects in intraleucocytic kill- ing can be disclosed by measuring intracellu- lar recoverable CFU, illustrating also the superiority of this principle over the isolated recording of total recoverable CFU. Finally, since the azide-sensitive antibacterial system seems to be by far the most important of many possible systems for the initial inactiva- tion of a number of micro-organisms, the comparison between azide-treated and un-

treated cells should be valuable in the evalua- tion of this system under clinical conditions.

This work was supported by grants from T h e Michaelsen Foundation, T h e Danish Medical Rese- arch Council, and T h e Foundation for the A d - vancement of Medical Science, Copenhagen, Den- mark. Mrs. Ulla Heiby is thanked for excellent technical assistance. C . Koch is holder of a research grant from T h e Michaelsen Foundation.

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