Acta path. microbiol. scand. Section B. 82, 127-135, 1974

NEUTROPHIL GRANULOCYTE FUNCTION IN VITRO

Evaluation of a fluid-phase leucocyte-bacteria reaction system

CHRISTIAN KOCH

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

A modified method for evaluation of human neutrophil granulocyte function in vitro, based upon the combined determination of total and intracellular surviving bacteria in a reaction system of leucocytes and Staphylococcus aureus, is described. Analysis of the system reveals a close relationship between the processes of ingestion and intraleucocytic killing on a functional level, and points towards dependence of intraleucocytic killing rate upon the rate of ingestion. Defects in intraleucocytic killing are disclosed readily by an increase in the number of surviving intracellular bacteria which will not be caused by increased ingestion under normal conditions. Greatly impaired ingestion will cause an increase in total surviving bacteria with a near normal number of intracellular surviving bacteria. O n the basis of these studies it can be concluded that this type of method is particularly suitable for the detection of defects in intraleucocytic killing whereas defects in ingestion are less readily disclosed.

Evaluation of the phagocytic function of neutrophilic granulocytes has received in- creased attention within later years and a number of congenital and acquired defects associated with decreased resistance to bacte- rial and fungal infections are being reported. These disease syndromes can be related to serum factors (2, 3, 4, 18, 2 5 ) or to the cells, and among the latter to isolated or combined defects in attachment and ingestion (6, 19, 24) or intraleucocytic killing of bacteria and fungi (1, 7, 16, 2 3 ) .

I n vitro incubation of leucocytes with bac- teria in the presence of autologous serum fol- lowed by colony counting of surviving bacte- ria will detect abnormalities related to serum factors, ingestion, and intraleucocytic killing

~~ ~

Received 26.ix.73 Accepted 30.x.73 Requests for reprints should be addressed to:

Chr. Koch, M.D., Blegdamshospitalet, Blegdam- vej 3, DR-2200 Copenhagen N, Denmark.

but is particularly suitable for detection of defective opsonization (15). The effect of variations in serum factors can be excluded by using a standard serum but distinction be- tween ingestion and intraleucocytic killing re- mains a major technical problem. In such a system the final indicator is the number of viable bacteria, or colony-forming units (CFU), and it is therefore clear that any recorded reduction in initially present CFU is dependent upon the combined rates of ingestion and killing. Effective elimination of non-ingested bacteria with antibiotics (8, 2 1 ) and blocking of further intraleucocytic kill- ing, subsequent to reaction of the cells with the bacteria (21 ) , have however been major improvements towards a more precise evalua- tion of the individual processes of ingestion and intraleucocytic killing.

This study describes a modification of the in vitro leucocyte function test of Alexander

127

et al. ( 1 9 6 8 ) and some studies done to de- termine the feasibility of this method in distinguishing between defects in ingestion and defects in intraleucocytic killing. Analysis of the results in terms of relationship between these two processes under normal conditions leads to a clear picture of the use and limita- tions of the method.

M A T E R I A 1 , S A N D M E T H O D S

Leucocytes. Venous blood was drawn into plastic syringes containing approximately 50 iu heparin (Leo, Copenhagen, containing 5 mg chlorbutol per ml) per ml of blood and mixed with 6 per cent dextran (MacrodexO', Pharmacia, Copenh., Mw: approximately 70,000, in 0.154 M saline), ratio blood to dextran: 1 :0.44 and left for sedimentation of erythrocytes, usually for 1 to 1% hour, at room temperature. The leucocyte-rich supernatant was withdrawn and the cells washed thrice in Hank's balanced salt solution containing 100 mg gelatin (Merck, Darmstadt, Germany) and 19.5 iu heparin without preservative per 100 ml. This basic culture medium was either adjusted to p H 7.45 by addi- tion of 2.8 per cent sodium bicarbonate or to approximately p H 7.45 by addition of 10 per cent standa.rd human serum (see below). Washing was carried out a t low-speed centrifugation, 250 x g, for five minutes. The final cellular pellet was re- suspended in this medium and adjusted to a con- centration of 5 x 10" polymorphonuclear leuco- cytes per ml by haemocytometer counting.

Bacteria. Staphylococcus aureus, phage type 42 E + or strain 502.4 (kindly provided by Dr. K . Ro- srndahl, Statens Seruniinstitut) were grown over- night (approximately 18 hours) in meat infusion broth (Statens Seruniinstitut). The bacteria were washed thrice in 0.154 M saline by centrifugation a t 1,800 x g for 10 minutes, resuspended, and ad- justed visually to a concentration ranging from 3 to 6 x 108 colony-forming units ( C F U ) per ml. This suspension was diluted ten-fold two times in culture medium to give a final concentration of 3 to 6 x 10" CFU per nil.

Stnndnrd serum. Freshly isolated serum from no less than 15 healthy blood donors was pooled and stored in small aliqriots a t -20" C.

IncuOation mixturrs. 0.8 ml of the bacteria sus- pension was mixed with 1.0 ml leucocyte suspcn- sion and 0.2 ml standard serum. If the leucocytes were washed in medium with 10 per cent serum, the mixtures contained 0.8 ml bacterial suspension, 1.1 ml leucocyte suspension, and 0.1 ml serum. The final leucocyte concentration was 2.5 x 10" poly- morphonuclear leucocytes per nil with an approx- imately 1 :1 ratio to bacteria and a serum concen- tration of 10 per cent. Incubations were carried

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out in 110 by 16, or 100 by 14 mm polystyrene tubes with polyethylene screw-caps (Nunc, Roskil- de) at 35" C with end over end rotation a t 'LO rev/ min. Duplicate tubes were set up for determination of total GFU and intracellular CFU.

Determinution of total CFU. 0.5 ml of the in- cubation mixture was withdrawn a t 2 and 4 hours arid transferred to 4.5 nil distilled watcr followed in 5 minutes by vigorous pipetting to disrupt the cells. l'en-fold dilutions were made in 0.154 M saline and 1.0 ml of appropriate dilutions were mixed with 30 ml melted beef infusion agar (Sta- tens Seruniinstitut) a t 48" C and poured into Petri dishes with a diameter of 7 cm. Colonies were counted after 48 hours and calculations of initial number of CFU in the sample were based upon the dilutions containing the highest number of colonies that could be counted, usually between 100 and 1,000 colonies per plate.

Determination of intracellular CFU. '1'0 separate tubes sodium benzylpenicillin, 100 iu per nil (Leo, Copenh.) and streptomycin sulphate, 100 pg per ml (Novo, Copenh.) in a total volume of 40 pl were added after 15 minutes' incubation to kill re- maining non-ingested bacteria. At 2 and 4 hours 0.5 and 1.0 nil incubation mixture was withdrawn and transferred to small glass-tubes, centrifuged a t 250 x g for 5 minutes, and washed thrice i n culture medium to get rid of the antibiotics. The final cellular pellet was resuspended in 1.0 ml distilled water followed in 5 minutes by vigorous pipetting. Serial ten-fold dilutions were made hi 0.154 M saline and the rest of the procedure carried out as outlined above.

Determination of initial CFU. This was done by serial ten-fold dilutions and pour-plating of the initial bacterial suspension. In some experiments the effect of the antibiotics upon the bacteria in the absence of leucocytes was examined. Under these circumstances determination of surviving C F U was done after extensive washing and cen- trifugation a t 1,800 x g.

Strict aseptic technique was ensured throughout the entire experimental procedure.

R E S U L T S

Stability of the test-system. Addition of gela- tin was found essential to secure a stable number of CFU. Omission of gelatin caused a tenfold decrease in CFU within one to two hours, which could be ascribed to agglutina- tion as demonstrated in direct microscopy and in control tubes without rotation. In the presence of 10 per cent serum and absence of leucocytes the number of C F U remained constant for two hours, increasing thereafter

nearly ten-fold from two to four hours indi- cating growth following a lag-phase. Viability of the leucocytes was repeatedly estimated by the trypan blue exclusion test which showed more than 98 per cent of the cells to be viable after four hours' incubation. The total number of leucocytes was found constant by haemocytometer counting although some ag- glutination of the cells occurred. During in- cubation a slight increase in p H t o approxi- mately 7.8 regularly occurred. The functional capability of the cells as measured by re- duction in total and intracellular CFU, how- ever, was unaffected by variations in the pH of the medium between 7.32 and 7.82.

Effect of antibiotics. Incubation of bacteria alone with penicillin and streptomycin in these concentrations caused over 99.9 per cent reduction in CFU within one hour. Nearly the same effect could be achieved by streptomycin alone, whereas penicillin alone caused only a little more than 10 per cent reduction in four hours. This is in accordance with the findings of Eagle (1948). The effect of penicillin and streptomycin in combination was dependent upon two factors: 1 ) the presence of serum; without serum, the effect was about ten-fold less. This may be caused by an enhanced effect of streptomycin upon bacteria in the serum-containing medium which promotes rapid bacterial growth, and 2 ) the p H of the medium; at acid p H the killing effect was decreased whereas killing was most efficient a t physiological pH. Under the experimental conditions of this method, effective elimination of non-ingested bacteria is thus achieved. O n the other hand, the in- ability of the antibiotics in these concentra- tions to influence the number of intracellular CFU has been amply demonstrated by the present technique (14) and related techniques (1, 8) in patients with a genetically de- termined defect in intraleucocytic killing: chronic granulomatous disease, and in exper- imental models (21). This is furthermore supported by the present experiments shown in Fig. 4 and 6, where intraleucocytic killing was blocked by 1 x lo-' M sodium a i d e (see below ) . 9 Aria path. microbial. wand. Section B. 82. 1

r I

2 L h o u r s

Fig. 1 . Reduction in total CFU S t a p h . aureus with changing number of CFU initially present and a constant number of neutrophil granulocytes: 2.5 x 106 per ml. Mean of three experiments.

I n o c u l u m CFU

-* 2.32 x 1 O7 \ 7--\ -

I I

I I

-_ -* 2 . 0 9 ~ 1 0 ~

I l l I

I 1 2 L h o u r s

Fig. 2. Intracellular recoverable CFU Staph. aureus with changing number of C F U initially present and a constant number of neutrophil granulocytes: 2.5 x 106 per ml. Mean of three experiments.

Effect of serum concentration. A concen- tration of 10 per cent serum was found to be optimal for promotion of ingestion in the present system as evidenced by optimal re- duction in total CFU. This is quite consistent with previous findings applying related techniques (1, 2 2 ) .

TABLE 1. Total and Intracellular Reroverablc Colony-forming Units (CFU) of Staphylococcus aureus at Four Hours’ Incubation in Relation to the Number of CFU Initially Present. Density of

Neutrophils: 2.5 x 106 per MI

Number of l’otal Intracellular CFU* per ml recoverable recoverable

initially present CFU CFU

2.26 x lo7 20.8 per cent 4 . 5 9 ~ 1 0 ~ 11.1 - 2 . 3 0 ~ loti 12.9 - 2.07 x lo5 14.3 -

2.32 x 107

2.09 x 105

3.48 x loG 2.1 1 x 106

0.25 per cent 0.12 -

0.068 -

0.055 -

* Colony-forming units Staphylococcus aureus.

Ef fect of density of bacteria. Fig. 1 shows the effect of varying the number of initial CFU upon the number of total recoverable CFU. Fig. 2 showing the effect upon the number of intracellular, recoverable CFU. Over a 100-fold range in initial CFU, a nearly constant proportion can be recovered total and intracellular a t 4 hours. The per- centage of recoverable CFU at 4 hours in these experiments are given in Table 1. These data are consistent with a proportional increase both in ingestion and intraleucocytic killing with increasing density of bacteria in the suspension. The results shown in Fig. 1, however, would also be consistent with an increase in per cent ingested bacteria and a similar decrease in per cent killed, but in that case a much higher percentage of initial CFU would be recovered intracellular at high density than at low density of initial bacteria since increased ingestion and decreased kill- ing both would increase intracellular CFU.

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I o g CF U

6

5

L

3

./* 2 . 5 x ‘03

2 L h o u r s

Fig. 3. Recoverable total (-) and intracellular ( - - -) CFU Staph. aureus with changing number of neutrophil granulocytes a s indicated in the fig- ure. Mean of three experiments.

I t is quite clear from the data presented in Fig. 2 that this is not the case.

Effect of density of leucocytcs. Fig. 3 shows that, over a nearly 100-fold range of concen- tration of neutrophils, the number of intra- cellular CFU after 4 hours varies only very little, whereas the number of total CFU is greatly increased with decreasling density of neutrophils. The percentage of recoverable CFU at 4 hours in these experiments are given in Table 2. Since the data presented in Figs. 1 and 2 and Table 1 indicate that the number of bacteria to be killed is directly related to the number being ingested (i.e. a constant proportion of ingested bacteria are being killed over this range) it is to be ex- pected that a higher number of bacteria are being ingested when the density of neutro- phils is increased and an equally higher num- ber are being killed, resulting in a nearly con- stant number of recoverable intracellular CFU. The results presented in Fig. 3 are quite in agreement with this. Still, as in the experi- ments shown in Fig. 1 and 2 there is a slight tendency towards more effective killing if few bacteria are ingested. The increase from 2 to

4 hours in total CFU a t 2.5 x lo3 neutro- phils is caused by inadequate ingestion since growth of non-ingested bacteria can be ob- served (Fig. 3 ) .

5 -

TABLE 2. Tota l and Intracellular Recoverable C F U Staph. aureus at Four Hours Incubation in Relation to the Number of Neutrophil Granulo-

cytes Present in the Incubation Mixture

I *\. I

I i

Total Intracellular recoverable recoverable

CFU* CFU

Number of neutrophil

per ml

2 x 107 2.17 per cent 0.28 per cent 2 . 5 ~ 1 0 O 23.8 - 0.23 -

2 . 5 ~ lo5 475.8 - 0.074 -

Colony-forming units Staphylococcus aureus.

Effect of sodium azide. Fig. 4 shows the effect of addition of sodium azide (Merck, Darmstadt, Germany), 1 x lo-' M, to the incubation mixture a t the start of the reac- tion. Reduction in total C F U is completely abolished which is clearly due to defective

I og CF U

intraleucocytic killing as evidenced by the extremely high number of recoverable intra- cellular CFU. Contamination with viable non-ingested CFU was excluded in control experiments which showed that although kill- ing of the bacteria with penicillin and strep- tomycin was less efficient in the presence of sodium azide still more than 99 per cent were killed within two hours. Other control ex- periments using bacteria and sodium azide alone showed that azide at this concentration did not kill the bacteria within four hours, but multiplication was effectively prevented.

Effect of heat-inactivation of serum. Fig. 5 shows that heat-inactivation (56"C/30 min) of serum led to markedly impaired reduction in total CFU due to inadequate ingestion; in fact, ample growth of non-ingested bacteria after 2 hours could be demonstrated. In spite of diminished ingestion, however, the number of intracellular CFU is nearly the same in tubes with heat-inactivated and in tubes with fresh frozen serum. These data parallel the experiments in which varying density of leucocytes was used (Fig. 3 and Table 2)

l o g C F U

6

5

L

2 L h o u r s

Fig. 4 . Recoverable total (-) and intracellular ( - - -) CFU Staph. aureus with untreated neutro- phi1 granulocytes (0) and treated with 1 x 10-2 M sodium azide (A). Density of neutrophils: 2.5 x 106 per ml.

9,

2 L h o u r s

Fig. 5 . liecoverable total (--) and intracellular ( - - - ) CFU Staph. aureus in presence of heat- inactivated (56"C/30 min) serum (0) and fresh frozen serum (a). Density of neutrophils: 2.5 x 10'; per ml. Mean of three experiments.

131

and further support the finding that relative- ly fewer are being killed within the observa- tion time if ingestion of bacteria is reduced. Fig. 6 shows that the difference between azide-treated and untreated cells is somewhat larger in the presence of fresh frozen serum than in the presence of heat-inactivated se- rum. Since this difference will be an expres- sion of the activity of intraleucocytic killing, these findings could indicate an enhancing effect of heat-labile factors upon intraleuco- cytic killing. These limited data, however, do not allow any conclusions as to this important question. This experiment also shows that ingestion is still possible in the presence of heated serum although to a reduced extent as seen from the number of intracellular CFU that could be recovered from the azide- treated cells (open triangles).

Reproducibility of the method. An es-

l o g CFU

6

5

L

3

2

c

1

i- i

-.3 I /"--- --

i f I 1 l l

I1 I1

2 L h o u r s

Fig. 6. Recoverable total (----) and intracellular ( - - - ) CFU Staph . aurerrs in presence of heat- inactivated (56"C/30 min) serum (0) and fresh frozen serum (a). A indicates sodium a i d e ( 1 x 10.2 M ) treated cells, and A indicates azide-treated cells in presence of heat-inactivated serum. Density of neutrophils: 2.5 x loG per ml.

132

/

I I

2 L h o u r s

Fig. 7. Mean and range of determinations 01 total (-) and intracellular ( - - - ) CFU Staph . nureus from nine identical incubation mixtures from one leucocyte population. Dmsity of neutro- phils: 2.5 x 10" per ml.

timate of the reproducibility of the method was done by running nine identically pro- cessed tubes from a single population of leu- cocytes for determination of intracellular CFU and nine for determination of total CFU. The results are shown in Fig. 7 which gives the mean and the range for each set of determinations. Good reproducibility of a single cell suspension is evident.

D I S C U S S I O N

The present results indicate a near linear re- lationship between the rate of ingestion and the rate of intraleucocytic killing of this test- organism by human neutrophil granulocytes. Results that would support these findings have been reported by others using related techniques. Alexander et al. (1968) reported variations in total CFU at 5 hours from 0.18 to 1.7 per cent over a range of 1.5 x lo5 to 1.5 x lo' initial C F U per ml. SolbPrg (1972 b ) found a variation from 2.0 to 2.8 per cent a t 2 hours over a range of 1 x lo5 to 1 x 10' initial C F U per ml. The present

variation ranged from 14.3 to 20.8 per cent over a range of 2.23 x lo5 to 2.94 x lo7 initial CFU per ml a t 4 hours (Table 1 ) . In contrast, variations in the density of leucocytes ranging from 5 x 10' to 5 x lo6 neutrophils per ml was found by Alexander et al. (1968) to give a variation in total CFU from 106 to 1.9 per cent at 4 hours which should be compared to the present values of 475.8 to 2.17 per cent total CFU over a range of 2.5 x 10:' to 2.0 x lo7 neutrophils per ml (Table 2 ) . The important point demonstrated in the present studies is, however, that over this range of density of neutrophils, the num- ber of recoverable intracellular CFU did only vary from 0.074 to 0.28 per cent in spite of the large variation in total CFU (Fig. 3 and Table 2 ) . Evaluation of data depicted graph- ically by Solberg ( 1972 b ) , however, indicates a similar pattern of large variations in total CFU and minor variations in intracellular CFU with changing density of neutrophils. The apparent discrepancy between the pre- sent study and those cited above concerning total surviving CFU is readily explained on the basis of differences in density of neutro- phils, system for mechanical mixing and, pos- sibly, the leucocyte separation procedure ( 1 ,

Biochemical studies of phagocytizing neu- trophils offer a possible explanation of the relationship between rate of ingestion and rate of intraleucocytic killing indicated in the present studies. Both anaerobic glycolysis and respiration increase during phagocytosis but, while glycolysis seems to supply the energy for ingestion, effective killing is dependent upon normal oxygen consumption, hydrogen peroxide formation, and flow of glucose through the hexose monophosphate shunt ( 9 ) . Several models linking these metabolic changes have been suggested (for extensive reviews see ref. 10 and 12). There is, how- ever, general agreement that the burst of oxydative metabolism necessary for effective killing follows very rapidly after ingestion.

The present findings indicate a linkage of the metabolic changes associated with inges- tion to those associated with intraleucocytic

2 2 ) .

killing a t a functional level. The degree of stimulation of metabolic activity may be de- termined by the number of bacteria ingested, and may again directly control the amount of energy released for intraleucocytic killing. Biochemical studies have in fact indicated a relationship between degree of particle in- gestion and increment in oxydative meta- bolism ( l l ) . Furthermore, there is evidence that initial intraleucocytic killing may be limited by the amount of hydrogen peroxide supplied via respiration (13 ) . The amount of stimulation of oxydative metabolism, how- ever may, vary for the different micro-organ- isms to be ingested ( 1 7 , 20).

The important point for the practical ap- plication of this and related techniques illu- strated by the present studies is that increased ingestion does not lead to an increased num- ber of recoverable intracellular CFU under normal conditions. Defects in intraleucocytic killing are readily disclosed by an increased number of intracellular CFU as demonstrated by the present and related techniques in pa- tients and carriers of the neutrophil defect: chronic granulomatous disease ( 1 , 14). This is also in the present study demonstrated in the experiments with sodium azide treated cells (Figs. 4 and 6 ) , and in other studies over the effect of experimental blockade of intraleucocytic killing (21 ) .

Minor changes in the ingestion rate will hardly be detected by the present method. Since total surviving CFU, at the employed density of neutrophils (2.5 x los per ml), rarely exceed 20 per cent at 4 hours, it follows that a t least 80 per cent are being ingested and even more when the density of neutro- phils is increased (Fig. 3 ) . Thus, depending upon the density of neutrophils, ingestion proceeds optimally or suboptimally due to mechanical promotion of contact between cells and bacteria. Furthermore, defective in- gestion due to defects in random or directed migration of neutrophils (6, 19, 24) would hardly be detected. The experiments with heat-inactivated serum (Figs. 5 and 6 ) show, however, that impairment of ingestion will in the present system be revealed by decreased

133

reduction in total CFU with minor, or no, alterations in intracellular CFU provided intraleucocytic killing proceeds normally. This is consistent with the earlier mentioned usefullness of the recording of total CFU for the detection of defective opsonization (15).

This work was suppoited by grants from T h e Michaelsen Foundation, T h e Danish Medical Re- search Council and T h e Foundation for the A d - vancement of Medical Science, Copenhagen, Den- mark. Mrs. Ulla H ~ i b y is thanked for excellent technical assistance. C. Koch is holder of a re- search grant from T h e Michaelsen Foundation.

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6. Edelson, P . J., Stites, D . P . , Gold , S. & Fu- denberg, H . H . : Disorders of neutrophil func- tion. Defects in the early stages of the phago- cytic process. Clin. Exp. Immunol. 13: 21 -28, 1973.

7. Holrnes, B., Quie , P. G., Windhorst , D . B . & Good, R . A . : Fatal granulomatous disease of childhood: an inborn abnormality of phago- cytic function. Lancet I: 1225-1228, 1966.

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