Acta path. microbiol. scand. Section B. 81, 787--794, 1973

NITROBLUE-TETRAZOLIUM STAINING OF HUMAN NEUTROPHIL GRANULOCYTES

Effects of Stimulation with Pseudomonas Antigens in the Presence or Absence of Human Pseudomonas Precipitins

CHRISTIAN KOCII and NIELS HVIBY

’The University Clinic for Infectious Diseases, and Statens Seruminstitut, Department of Clinical Microbiology, Blegdamshospitalet. Copenhagen. The Department of Pediatrics TG, with the Department of Pedia,trics of Dronning Louises Bernehospital, Rigshospitalet,

University of Copenhagen, Denmark

Bacteria-free filtrates from Pseudomonas aeruginosa cultures were added to heparinized blood from 10 patients with multiple Pseudomonas precipitins and from 11 patients without such precipitins before carrying out a ni troblue-tetrazolium (NBT) test. Increased NBT staining could be induced in neutrophils from all of the patients with demonstrable Pseudo- rnonas precipitins but only in 3 of the patients without precipitins. This difference was found to be statistically significant. Using patients’ plasma but leucocytes from a normall control person it was found that this difference was dependent upon the origin of plasma but independent of the origin of leucocytes. These findinns indicate that increased NBT staining of neutrophils induced by bacterial antigen antibody interaction.

In 1968 Park et al. reported increased stain- ing in uitro of peripheral neutraphil granulo- cytes with the histochemical dye nitroblue- tetrazolium (NBT) in patients with bacterial infections, and these findings have been con- firmed by others (4, 6, 10). The NBT test as outlined by Park et al. (1968) has sub- sequently become widely used as a diagnostic aid in febrile disorders. It is well-established that increased NBT staining follows stimula- tion of phagocytosis and is dependent upon normal postphagocytic oxydative metabolic activity ( 2 ) , but the mechanisms in the in- fected patient responsible for the increased spontaneous NBT staining observed in vitro

Received 22.vi.73 Accepted 21.viii.73 Requests for reprints should be addressed to:

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

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products in vitro is in part dependent upon

are unexplained. Furthermore, i t is of some concern that unexpected “false” negative re- actions have been reported ( 3 ) which would throw serious doubt on the feasibility of this test in clinical routine. I t therefore seems important to elucidate the mechanisms by which increased NBT staining could be in- duced during bacterial infections.

Increased NBT staining may be induced in uitro in neutrophils by exposure of hepa- rinized blood to bacteria-free culture filtrates from different genera of bacteria (10). One of us has previously suggested that these culture filtrates contained antigens that could react with antibodies to form immune com- plexes that subsequently elicited the changes in the neutrophils responsible for increased NBT staining (9) . To further test this hypo- thesis, the following studies were undertaken :

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Two groups of patients with cystic fibrosis (CF) were selected on the basis of presence or absence of detectable precipitating anti- bodies against Pseudomonas aeruginosa. Bacteria-free culture filtrates from 2 different strains of Pseudomonas aeruginosa were then added to heparinized blood from the 2 groups of patients before carrying out the NBT test. In order to determine the importance of the origin of the leucocyte, the same experiments were repeated using patients' plasma but leucocytes from a normal control person. The results indicate that the increased NBT staining of neutrophils induced by bacterial products in vitro is in part dependent upon antigen-antibdy interaction and independent upon the origin of the leucocytes.

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

Crossed immunoelectrophoresis (c.i.e.) studies were carried out as described by Axelsen (1971) using 1 per cent agarose gel (Batch AGS 058 A, Litex, Glostrup, Denmark) in barbital buffer, pH 8.6, ionic strenght 0.05. The first dimension electrophoresis of 10 pl Pseudomonas aeruginosa standard antigen (St.Ag. (7)) was run for 1 hour a t 10 V per cm. The second dimension electro- phoresis was run for 20 hours applying 1-2 V per cm with patient serum included in the second dimension gel (12.5 pl per cm2). Thickness of gel: 1.5 mrn, dimension of plates: lox 10 cm. After the run, the plates were washed, dried, and stained with Coomassie brilliant blue R (Microme no. 1137, E. Gurr, Ltd., London, England) as described previously (7).

The immunodiffusion (id.) studies were carried out as double diffusions according to the plate technique of Ochterlony (1967). The diffusion was carried out in 1 per cent agarose gel (same batch as that used for c.i.e.) in 0.154 M NaCl supported by glass plates: lox 10 cm, thickness of gel: 2 mm. The diffusion took place in humid atmosphere at room temperature for 6 days. A combined system was used with a central rectan- gular trough: 60 x 2 mm, containing 0.2 ml of the Pseudomonas fihtrate, and 20 g1 patients' serum placed in 4 mm wells at a distance of 5 mm from each other and the central trough. After the diffusion, the plates were washed, dried, and stained as outlined for the c.i.e.

The St.Ag. consisted of water-soluble con- stituents obtained by sonication of 4 different strains of Pseudomonas aeruginosa: 0-groups 3, 5, 6, and 11. The preparation procedure and prop- erties of the St.Ag. have been described pre-

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viously ( 7 ) . I t contains a t least 55 different anti- gens (7) and is therefore considered especially well-suited for the purpose of revealing Pseudo- monas precipitins in human serum. In pilot studies, St.Ag. was able to induce increased NBT staining in a normal control person in whom demonstrable precipitins against St.Ag. were absent, indicating that other mechanisms in addition to immune complexes might possibly be able to induce in- creased NBT staining (toxins?). This is subject to further studies, but in the present study St.Ag. was consequently considered less suitable for an elucida- tion of the role of immune complexes in the induction of increased NBT response.

The Pseudomonas aeruginosa filtrntes were ob- tained from 2 strains: one strain (designated P - I ) was isolated from a patient with chronic bronchitis and pneumonia due to this organism. This strain has not been 0-group typed. The other strain (designated P-2) is identical to one of ,the four strains used for making the St.Ag. and belongs to 0-group 6. I t was isolated from a patient with CF. Both strains were obtained from tracheal aspirates and cultured in meat infusion broth enriched with 10 per cent horse serum (serum broth, Statens Seruminstitut, Copenhagen, Denmark). After 24 hours incubation a t 35" C the cultures were centrifuged at 1800 x g and the supernatants passed through 0.45 pm Millipore@ filters and stored in small aliquots at -20" C.

The P-1 and P-2 filtrates were compared to St.Ag. by means of a rabbit antiserum (St.Ab.) raised against St.Ag. (7). St.Ab. was absorbed with P-1 and P-2 by mixing equal volumes of St.Ab. and P-1 or P-2 or 0.154 M NaCl as control. The absorption took place at 37" C for 1 hour. The results of the absorptions were then evaluated by c.i.e. of 10 pl St.Ag. against the absorbed St.Ab. (10 pl per cm2) according to the above outlined methods for c.i.e. By comparison with the result of the control c.i.e. showing the St.Ag.-St.Ab. re- ference pattern, it was found that both P-1 and P-2 had absorbed antibodies corresponding to 1 of the 55 precipitates visible on the control c.i.e. and P-1 in addition had absorbed antibodies correspond- ing to another of the precipitates. This finding indicated that P-1 and P-2 contained antigenic materials Corresponding to 2 respectively 1 of the antigens of St.Ag. This was confirmed by i.d. studies. No precipitates could be demonstrated by c.i.e., however, when P-1 or P-2 was run against St.Ab. or patients' sera. The reason for this has not been found.

Patients. Twenty-one patients with CF were in- cluded. The diagnostic criteria have been described previously ( 7 ) . All patients were examined and followed as out-patients every month including bacteriological examination of tracheal secretion (7). In 10 of the patients, designated CF + Ab., circulating precipitating antibodies against the

PERCENTA- I

s SB P-1 P-2

Fig. I . Results of directly stimulated (aireot) NHT tests in 10 CF patients containing precipitins against Pseudomonas aeruginosa St.Ag. in c.i.e. Ea,ch patient was tested in four different conditions: 1 ) Spon- taneous NBT test ( S ) , - 2) with the culture medium used for the filtrates (serum broth), 5 p1 per 0.1 ml blood ( S B ) , - 3) bacteria-free filtrate of Pseudomonar aeruginosa culture, strain 1, 5 fiI per 0.1 ml blood [P-1). and - 4 ) filtrate from strain 2 (P-2). The resullts from each patient are connected . , with lines, and each value represents one NBT test condition.

The bars represent mean values of the tests in each

St.Ag. had previously been detected by c.i.e. All patients in this group had harboured mucoid strains of Pseludomonas aeruginosa for a t least 2 years (mean: 2.5 years). The second group, designated CF - Ab., comprised 11 patients in none of whom precipitating antibodies against St.Ag. had pre- viously been detected. Pseudomonas aeruginosa had never been isolated from any of the CF - Ab. patients. Average control period in the clinic was 4 years for C F + Ah., and 3.9 years for C F - Ab. The mean age was 10.5 years for CF + .4b. (range: 6.5 to 2 1 ) and 9.2 for CF - Ab. (range: 3.5 to 16.5). CF + Ab. included 7 male and 3 female patients and CF - Ab. 6 male and 5 female patients. Heparinized blood, plasma, and serum used in the present studies were obtained simul- taneously from all 21 patients who all were afebrile and out-patients at the time of examina- tion.

The N B T tests were carried out according to Park et al. (1968) with the modifications described earlier (9) . For direct stimulation (direct te j t s )

5 p l filtrate was added to 0.1 ml heparinized (approximately 50 i.u. heparin per ml blood) freshly drawn venous blood from the patients im- mediately before the NBT tests. For stimulation of heterologous neutrophils (indirect tes t s ) , 0.3 ml freshly isolated heparinized plasma from the pa- tients was pre-incubated with 20 pl filtrate for 60 minutes a t 35" C. Blood cells from 0.3 ml heparin- ized freshly drawn venous blood from a normal person were washed 3 times a t low-speed centrifu- gation (250 x g ) in Hank's balanced salt solu- tion containing 1 mg gelatin and 19.5 i.u. heparin per ml, p H 7.45. The final cellular pellet was re- suspended in the 0.3 ml patient plasma pre- incubated as outlined, and the NBT tests carried out immediately. The same normal person was used as donor of heterologous blood cells in all indirewt tests. His serum contained no detectable precipitat- ing antibodies against the St.Ag. in c.i.e. or id., and he had never experienced infection with Pseudomonas aeruginosa. Direct tests were done with 1 ) P-1, and 2 ) P-2 filtrate, 3 ) without any

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PER C E NTA - GE OF NB.1 STAIN IN G NEUTRO- PHlLS

20

10

S SB P -1 P -2 F i g . 2. Result of direct NBT tests in 1 1 CF patients without demonstrable precipitins against the St.Ag. in c.i.e. Test-conditions and symbols as in Figure 1.

addition (spontaneous NBT test corresponding to the ordinary NBT tests), and 4.) with 5 pl serum broth, the last 2 test-conditions serving as con- trol values. Indirect tests were done with P-1, and P-2, and with serum broth serving as control value.

In each test, 500 consequtive neutrophils from 2 smears were counted and evaluated in blind by one person. Statistical calculations were carried out using the Mann-Whitney rank sum test and Spear- man’s correlation coefficient R (Documenta Geigy) .

R E S U L T S

All of the CF + Ab. contained multiple precipitating antibodies against the Pseudo- monas aeruginosa St. Ag. in c. i. e. The mean number of precipitins was 20 with a range of 4 to 50. In none of the CF - Ab. sera could any precipitins against the St. Ag. be detected. All sera were further tested by i. d. against the P-1 and P-2 filtrates. Lines of precipitation could be demonstrated in 9 of the 10 CF + Ab. sera against P-2 and in 5 against P-1. None of the sera contained more than 1 demonstrable precipitin by this

method. Precipitins against either P-1 or P-2 could not be demonstrated by i.d. in any of the 1 1 C F - Ab. sera.

The results of the direct NBT tests in the 2 groups of patients are given in Fig. 1 and 2. Addition of both P-1 and P-2 to heparinized blood from CF + Ab. patients caused an increase in the percentage of NBT staining neutrophils compared to the control tests in all 10 patients (Fig. 1). Statistical calcula- tions of the results in the CF + Ab. group showed that these increases were significant ( p < 0.01). The results obtained with P-1 were significantly correlated to those ob- tained with P-2 (R = 0.7576, p < 0.01).

In the CF - Ab. group increased staining could only be induced in 3 patients by P-2, and only in 1 of these by P-1 (Fig. 2). In the CF - Ab. group as a whole there was no significant effect of stimulation with either P-1 or P-2 compared to control tests

The results of the indirect tests are given ( p > 0.10).

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PER C EN TA - GE OF N.BT S TA I N I N G NEUTRO- PHIL S

20

SB P -1 P -2

Fig. 3. Results of indirect stimulation (indirect tests) of heterologous neutrophils from one normal person with plasma from 10 CF patients containing precipitins against Pseudomonas aeruginosa St.Ag. in c.i.e. Before addition of patients’ plasma to heterologous neutrophils plasma-portions of 0.3 ml were pre-incubated with either culture medium used for the filtrates (serum broth), 20 pl (SB), bacteria-free filtrate of Pseudomonar aeruginosa culture, strain 1 (P- I ) , 20 pl, or filtrate from strain 2 ( P - 2 ) , 20 gl. The results from each patient are connected with lines and the bars represent mean values of all 10 patients for each test-condition.

- -

-

-

-

in Figures 3 and 4. Plasma from 8 of the 10 CF + Ab. patients, pre-incubated with either P-1 or P-2, caused increased NBT staining in heterologous neutrophils compared to control tests with serum broth (Fig. 3) . These results too were found to be statistically significant for the CF + Ab. group (p<O.Ol) and the results obtained with P-1 were statistically correlated to those obtained with P-2 (R = 0.7213, p < 0.05). The 2 CF + Ab. plasma which did not induce increased staining following pre-incubation with P- 1 and P-2 did not differ from that in the rest of the group with respect to precipitins. They contained 5 and 7 precipitins in c.i.e. and

both had precipitins against P-1, one also against P-2, in i.d. In the CF-Ab. group, plasma from only 3 of the 11 patients pre- incubated with P-2, and one of those also with P-I, induced increased staining in heterologous neutrophils (Fig. 4 ) . These were from the patients who also reacted with in- creased staining in direct tests. (Fig. 2 ) . In the CF-Ab. group as a whole, however, there was no statistically significant effect of pre-incubation of plasma with either P-1 or P-2 compared to control tests ( p > 0.10). There was no statistically significant relation- ship between the number of precipitins in the CF + Ab. group and the degree of increased

79 1

PERCENTA- GE OF NB.7:- STAIN I N G NEUTRO - PHILS

t

-

-

SB P -1 P -2 Fig. 4. Results of indirect #tests using plasma from 1 1 CF patients without demonstrable precipitins against the St.Ag. Test-conditions and symbols as in Fig. 3.

NBT staining in either direct or indirect tests, It is noteworthy, however, that indirect tests showed the highest degree of increase if plasma from 2 patients containing the highest number of precipitins in c. i. e. was used, i. e. 39 and 50 lines of precipitation.

D I S C U S S I O N

The design of the present study was aimed at a demonstration by means of a sensitive method of circulating precipitins against Pseudomonas aeruginosa in patients sera ( 7 ) . By this method, the patients were classed in 2 groups: one with such precipitins and one without. The correlation of presence of antibodies to infection with Pseudomonas aeruginosa has been discussed previously ( 7 ) .

These 2 groups were then examined with

regard to the effect of 2 bacteria-free filtrates of Pseudomonas aeruginosa cultures upon the number of NBT staining neutrophils in vitro. These filtrates were selected from a panel of filtrates derived from different genera of bacteria. A number of normal persons and patients suffering from various disorders have been tested in direct tests with this panel and it has been found that some filtrates will induce increased NBT staining in all persons tested, whereas other filtrates will induce increased staining in some but not all persons (9) . A similar individual response pattern has also been noted by others (10).

P-1 and P-2 contained 2 and 1, respectively, of the 55 antigens present in St. Ag. but might contain antigens not present in St. Ag. However, the i. d. studies showed that 90

792

per cent of the CF + Ab. patients harboured detectable precipitins against P-2 and 50 per cent against P-1, whereas none of the CF - Ab. patients harboured detectable pre- cipitins against P-1 or P-2.

In the present study it was found that P-1 and P-2 could regularly induce increased staining in neutrophils from the patients with demonstrable circulating antibodies against Pseudomonas aeruginosa in c. i. e. and seldom in neutrophils from patients without such antibodies. The results of the indirect tests furthermore indicate that this difference was dependent on the origin of the plasma but in- dependent of the origin of the leucocytes, since nearly the same results were obtained when patients’ plasma were pre-incubated with the filtrates and thereafter exposed to heterologous neutrophils. These findings in- dicate that the primary event when increased NBT staining is induced in uitro by bacterial products is interaction of bacterial antigens with antibodies.

Previous studies in this laboratory have in- dicated that increased NBT staining may be brought about by interaction of bacterial antigens with antibodies with the formation of immune complexes, and that high-mole- cular weight complexes are more effective than low-molecular weight in this respect ( 9 ) . Since the technique employed in the present study for detection of precipitating anti- bodies is highly sensitive ( 7 ) it is interesting that increased staining could be induced in homologous and heterologous neutrophils with blood or plasma from a few patients without Pseudomonas precipitins demonstra- ble in c.i.e. (St. Ag.) or in i. d. (P-1 and P-2) (Fig. 2 and 4 ) . The reason for this is not clear; one possibility is that low-con- centrated, or non-precipitating immune com- plexes may also induce increased staining. Relevant to this is the finding that it has been possible by means of c. i. e. to de- monstrate antibodies in concentrated pooled normal human gammaglobulin precipitating with one of the antigens present in the St. Ag. (8). In unconcentrated normal sera these antibodies are not demonstrable (8) whereas

agglutinating antibodies against Pseudomonas aeruginosa have been demonstrated in such sera ( 5 ) .

The present study indicates that interaction of bacterial antigens with corresponding anti- bodies is able to induce increased NBT stain- ing of human neutrophils in vitro and seems to support our previous findings ( 9 ) . I t remains to be studied whether this mechanism is responsible for the increased spontaneous NBT staining seen during bacterial infections. If, however, such a mechanism was operative in uiuo to induce increased NBT staining, the finding of “false” negative reactions in infected patients might be offered an explan- ation based upon the absence of antibodies against the invading organism, or conversely, absence of bacterial antigens from the blood stream.

Several questions should be answered to elucidate the possible interaction of immune complexes with neutrophils as a mechanism inducing increased NBT staining. These would include the size, concentration, and possibly specificity in terms of class of im- munoglobulin involved plus the role of heat- labile factors such as complement (9) . I t is to be hoped that such information might help to explain the wide range 0.f values of NBT tests seen in infected patients and further clarify the usefulness of this test in clinical practice.

This work was supported by grants from T h e Michaelsen Foundation, T h e Danish Medical Re- search Council, T h e Foundation for the Advance- ment of Medical Science, T h e Thorvald Madsen Legat, and Landsforeningen ti1 bekampelse af Cystisk Fibrose. Mrs. Ulla Hoiby and Mrs. Annie Bethien are thanked for skilful technical assistance.

R E F E R E N C E S

1. Axelsen, N . H.: Human precipiiins against a microorgamnisrn (Candida albicans) demon- strated by means of quantitative immuno- electrophoresis. Clin. Exp. Irnmunol. 9: 749- 752, 1971.

2. Baehner, R. L. & Nathan, D. G.: Quantita- tive nitroblue tetrazoliurn test in chronic granulomatous disease. New Engl. J. hled. 278: 971-976, 1968.

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3. Esposito, R. & DeLalla, F.: N.B.T. test in bacterial meningitis. Lancet I : 747-748, 1972.

4. Feigin, R. D., Shackelford, P. G., Choi, S . C., Flake, K. K., Franklin, Ir., F. A . & Eisen- berg, C . S.: Nitroblue tetrazolium dye test as an aid in the differential diagnosis of febrile disorders, J. Pediat. 78: 230-237, 1971.

5. Gaines, S. & Landy, M . : Prevalence of anti- bodies to Pseudcmonas in normal human sera. J. Bacteriol. 69: 628-633, 1955.

6. Humbert, J. R., Marks, M. I . , Hathaway, W. E. & Thoren, C . H.: The histochemical nitro- blue tetrazolium reduction test in the differen- tial diagnosis of acute infections. Pediatrics 48: 259-267, 1971.

7. Hsiby, N . & Axelsen, N . H.: Identification and quantitation of precipitins against Pseudo- monas aeruginosa in patients with cystic fibrosis by means of crossed immunoelectro-

phoresis with intermediate gel. Acta path. microbiol. scand. Sect. B 82: 298-308, 1973.

8. Hsiby, N.: Unpublished observations. 9. Koch, C.: Studies on the nitroblue-tetrazolium

staining induced in human neutrophils by bacterial products. Acta path. microbiol. scand. Sect. B 82: 266-268, 1973.

10. Matula, G. & Paterson, P. Y.: Spontaneous in vitro reduction of nitroblue tetrazolium by neutrophils of adult patients with bacterial infection. New Engl. J. Med. 285: 311-317, 1971.

11. Ouchterlony, U. : Immunodiffusion and im- munoelectrophoresis. In : Weir, M. (Ed.) : Handbook of experimental immunology. 1. ed. Blackwell Scientific Publications, Oxford 1967,

12. Park, B. H., Fikrig, S . M . & Smithwick, E. M.: Infection and nitroblue-tetrazolium reduction by neutrophils. Lancet II: 532-534, 1968.

p. 655-706.

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