The interaction of Brucella Melitensis 16-M and caprine polymorphonuclear leukocytes

Comp. lmmun. Microbiol. in/ect. Dis. Vol. 13, No. 2, pp. 59~5, 1990 Printed in Great Britain. All rights reserved

014%957|/90 $3.00 + 0.00 Copyright ~3 1990 Pergamon Press plc

THE INTERACTION OF BRUCELLA MELITENSIS 16-M A N D C A P R I N E P O L Y M O R P H O N U C L E A R LEUKOCYTES M . C. GALLEGO ~ a n d M . A . LAPEI~A2 ~Department of Pathology, Veterinary Faculty, University of Murcia, Spain and 2State Polytechnical Institute No 3. Murcia, Spain (Received 7 November 1989) Abstract--In the present work, a study about the phagocytosis and intracellular killing of the polymorphonuclear leukocytes (PNMLs) of goat in animals clinically healthy, vaccinated and inoculated experimentally with Brucella melitensis has been done. During 6 weeks postvaccination and postinfection, the evolution in these animals has been studied. Although animals were inoculated or vaccinated, there was influence in the phagocytosis and intracellular killing phases, even though a very low indices in this last phase, and in every event were given. The nitroblue tetrazolium (NBT) reduction indices in PMNLs were also investigated with different fractions of B. melitensis. Very low indices were given, and no influence of a postvaccination or postinfection state was found. Finally, the serum bactericidal action in every animal was studied with Brucella melitensis and this effect was not found. Key words: Ingestion, intracellular killing, Brucella melitensis, polymorphonuclear leukocytes, goat, nitroblue tetrazolium. L'INTERACTION PHAGOCYTES

DE

BRUCELLA

MELITENSIS

POLYMORPHONUCLI~AIRES

16-M E T D E S DE

CHt~VRES

R~sum~-Dans ce travail, nous avons fait une 6tude de la phagocytose et de la bactericidie intracellulaire par les polymorphonucl6aires neutrophiles (PMNLs) de ch6vre avec des animaux cliniquement sains, vaccin6s et infect/~s exp6rimentalement par Brucella melitensis. Nous avons &udi6 l'6volution de ces animaux pendant les six semaines postvaccination et postinfection. Le fair que les animaux 6taient vaccin6s ou infect6s a influ6 sur la phagocytose et sur la bact6ricidie, bien que cette derni6re phase ait eu des indices tr~s bas. L'indice de r~duction du nitrobleu de tetrazolium (NBT) dans des PMNLs caprins, a 6t6 &udi~ apr6s phagocytose de diff6rentes fractions de B. melitensis. Les valeurs obtenues ~taient tr6s basses par rapport au contr61e et aucune influence de l'infection ou de la vaccination n'a 6t6 d6montr6e. Finalement nous avons estim~ l'action bactericide du s~rum de chaque animal sur B. melitensis sans trouver d'effet. Mots-clefs: Phagocytose, bact6ricidie, Brucella melitensis, phagocytes polymorphonucl6aires, ch6vre, nitrobleu de tetrazolium.

INTRODUCTION

Phagocytic cells constitute an effective line of defense against most invading microbial pathogens. In contrast to extracellular parasites, facultative intracellular parasites survive by mechanisms which enable them to evade, inhibit, or resist intraleukocytic killing systems. 59

60

M . C . GALLEC,O and M . A . LAPE~A

Brucella is a facultative intracellular pathogen that can remain within phagocytic cells of the host and is apparently resistant to the normal mechanisms of bacterial killing. In a series of studies, Mackaness [1,2] demostrated the importance of mononuclear phagocytes in resistance to intracellular bacteria such as Brucella. The mechanisms and virulence factors responsible for evasion of the host's phagocytic system are not yet well understood. Kreutzer et al. [3] and Riley and Robertson [4] compared the ability of human and bovine polymorphonuclear leukocytes (PMNLs) to ingest and kill smooth and rough strains of Brucella abortus. Both bacterial strains were readily ingested by either human or bovine phagocytes. Both smooth and rough strains were resistant to killing by human and bovine PMNLs; however, the smooth strain was more resistant than the rough strain. The chemical composition of cell walls of both strains has been examined with respect to total lipids, proteins, peptidoglycan content, and composition, as well as lipopolysaccharides (LPS) [5, 6]. The only significant difference was in the LPS composition; that is, when two strains were extracted by the Westphal procedure, LPS was found in the phenolic and aqueous phases of B. abortus strain 45/0, but only in the aqueous phase of B. abortus strain 45/20. The purpose of this study was to determine the effects of virulent B. melitensis (16-M) on specific aspects of caprine PMNLs function. In vitro assays were conducted in the presence of B. melitensis (or fractions of the organism) to determine the ability of PMNLs to carry out ingestion, intracellular killing and the metabolic oxidative burst.

MATERIALS AND METHODS Bacteria Brucella melitensis (Biovar 1, strain 16-M) was cultured on Trypticase soy agar (TSA) (Difco Laboratories, Detroit) at 37°C, and maintained on smooth phase as described by Alton et al. [7]. Forty-eight-hour growth cultures were washed three times in 0.85% NaCI, resuspended in Hanks' balanced solution (HBSS) (pH 7.2) containing 0.1% gelatin (gelatin-HBSS), and adjusted spectrophotometrically to 1-2 x 10 7 cfu/ml. Leukocytes

PMNLs were purified from heparinized (100U/ml) goat's peripheral venous blood by Person's technique [8]. The cells were washed 3 times in siliconized glass tubes, resuspended in 2.2 ml of gelatin-HBSS solution, and adjusted by haemocytometer to 1-2 × 107pMNLs/ml. Purified suspensions contained less than 1% erythrocytes and 90-95% PMNLs. Viability (greater than 95%) was confirmed by exclusion of trypan blue dye. In all, 88 samples were studied, which were grouped as follows: --Group I (control group): 17 clinically healthy animals, unvaccinated and negative to the serological reactions of slow agglutination, rapid agglutination and complement fixation tests. --Group II: 11 animals studied 8 months after vacccination with the living vaccine Rev-1, with various agglutination and complement fixation titres. --Group III: 6 animals vaccinated and studied for 6 weeks postvaccination (once a weekj.

Brucella melitensis 16-M and caprine PMNLs

61

--Group IV: 4 animals inoculated intravenously with a 48-h growth culture of B. melitensis, Biovar 1, studied for 6 weeks postinfection (once a week). Serum

The following sera were used as opsonising factors in each test for phagocytosis and intracellular killing: (A) inactivated serum (30 min at 56°C) from the animal itself; (B) serum from the animal itself; (C) inactivated positive control serum; (D) inactivated negative control serum; and (E) negative control serum. Phagocytosis

The reaction mixture, containing: PMNLs suspension (1 ml), bacteria suspension (1 ml) and appropriate serum (0.2 ml), were mixed in siliconized glass tubes and incubated at 37°C under continuous rotation. At 0, 30 and 60 min, aliquots (0.5 ml) of the reaction mixture were removed, diluted with 1.5 ml of ice-cold HBSS solution, and centrifuged (for 4 min at 110 g and 4°C). The supernatant was serially diluted in saline and plated on TSA plates. The number of viable bacteria was calculated from the means of the colony counts of duplicated plates of the two highest dilutions showing isolated colonies. The phagocytic index after t min was: F ( t ) = log N o - log Nt, where No = the initial number of viable bacteria, and Nt = the number of viable extracellular bacteria after t min [8]. Intracellular killing

After an incubation period of 15 min, phagocytosis was stopped by placing the reaction mixture tubes in ice-cold water for 1 min. The bacterial-cell suspension was then centrifuged (for 4 min at 110g and 4°C) and the supernatant removed. The sedimented PMNLs were washed twice with gelatin-HBSS solution, resuspended in HBSS with 10% serum, and incubated at 37°C under continuous rotation. At 0, 30 and 60 min, aliquots (0.5 ml) were removed, diluted with 1.5 ml of ice-cold HBSS solution and centrifuged (for 4 min at 110g and 4°C). The supernatant was discarded and the sedimented PMNLs were lysed by adding 2 ml ice-cold distilled water with 1% bovine serum albumin and then pipetting vigorously for 30 s [9]. The suspension was serially diluted in saline and plated on TSA plates. The index of intracellular killing after t min was: K(t) = log No - log Nt, where No = the initial number of viable intraceilular bacteria, and Nt = the number of viable intracellular bacteria after t min [8]. Nitroblue tetrazolium (NBT) reduction test

Reduction of NBT (Sigma, St Louis) by PMNLs was performed as described by Segal and Peters [10], using the following stimulant factors: (1) HBSS as control. (2) Lipopolysaccharide (LPS) of Escherichia coli (Sigma, St Louis). (3) LPS of B. melitensis extracted as described by Redfearn [11]. (4) Proteic fraction of B. melitensis obtained as described by Bhongbhibhat et al. [12]. (5) Polysaccharide B (Poly-B) of B. melitensis extracted as described by Diaz et al. [13]. (6) B. melitensis subjected to ultrasonic disruption (ultrasonic disruptor ARTER Mod. 300) and (7) B. melitensis whole cells. In siliconized glass tubes, the heparinized blood samples (1.5 ml) were mixed with 15 gl of appropriate stimulant factor, and 0.3 ml of NBT solution (0.15%). After incubation for 10 min at 37°C, the reaction was stopped by the addition of 0.5 ml of 0.1 N CIH. The mixture was transferred through a nylon column (Wool-Leukopak, Travenol, Deerfield) and erythrocytes lysed by addition of distilled water. Purple formazan, formed as a product

62

M . C . GALLEGO and M. A. LAPENA

of reduction of NBT, was extracted with 5 ml of dioxan at 70°C for 20 min, and the optical density at 520 nm determined in a spectrophotometer. The results are expressed in ~g of formazan/107 cells x min ~ [9]. Bactericidal activity o f serum

The reaction mixture, containing: bacteria suspension (1 ml) and appropriate serum (0.2 ml) of all samples, were incubated at 37°C under continuous rotation. At 60 rain (final time of the experiments), aliquots were removed and plated, serving as a rough check on bactericidal effects of serum factors [14]. RESULTS

AND

DISCUSSION

In Fig. 1, the phagocytic indices for the four animal groups are shown according to the opsonising factor used. In the phagocytosis of B. melitensis using inactivated serum from the animal itself (opsonising factor A) we found very low indices in all groups. There are only small differences between groups I and II probably due to the vaccinal antibodies of the group II [Fig. I(A)]. The same evolution, but with higher indices (probably due to the opsonising action of the complement) appear when the serum from the animal itself (opsonising factor B) was used. The phagocytosis percentages of the control group (equivalent to 80%) are comparable to those obtained by Kreutzer et al. [3] with human peripheral PMNLs when using a smooth strain (B. abortus 45/0), and slightly higher when dealing with a rough strain B. abortus 45/20). No significant difference was found between the four animal groups for the opsonising factors C and D, when we used inactivated serum [Figs I(C, D)]. Nevertheless, in presence of negative control serum (opsonising factor E) [Fig. I(E)], the phagocytic indices obtained represents a 82% reduction in the bacterial population, similar to that reported by Young 0.8

(A)

(B)

-

(C)

_ (D)

(E)

0.6 o

._u ~, 0.4 ~n o rl

0.2

I I I I 1 2:5456

I

I 1 2 5 4 5 6

I I I I I 1 2 5 4 5 6

I 1 2 5 4 5 6

I I I I I 1 2 5 4 5 6

Weeks

Fig. I. Phagocytic indices of Brucella melitensis by goats' PMNLs. (A) Inactivated serum from the animal itself. (B) Serum from the animal itself. (C) Inactivated control serum. (D) Inactivated negative control serum. (E) Negative control serum. G r o u p I at 30 ( O ) and 60 ( O ) min. Group II at 30 ( × ) and 60 ( * ) m i n . The evolution of indices of G r o u p III at 30 (11) and 60 (E])rain, and G r o u p IV at 30 ( V ) and 60 ( V ) min were studied during 6 weeks. Coincidence of two or more data are represented as (o).

I

Brucella melitensis 16-M and caprine PMNLs

63

et al. [15] with B. abortus and B. melitens& in human PMNLs and to that obtained by Gallego et al. [16] with B. melitensis Rev-1 and goat's PMNLs. The absence of ingestion that we found, coincided with Young et al. [15], who used human PMNLs and 4 Brucella

strains with normal inactivated serum. In contrast, phagocytosis is almost complete 20 min after incubation when the 4 strains are preopsonised with 10% normal serum. Likewise, Gay et al. [17] obtain similar results in their experiments with Brucella suis both in vivo and in vitro when infecting mouse peritoneal macrophages. Our apparent conclusion is that termolabile factors of serum are the most active in the phagocytosis of B. melitensis by goat's PMNLs. Although in vitro phagocytosis of B. melitensis is rapid, this situation becomes different in the case of intracellular killing. Ultrastructural analysis of postphagocytic events were made by transmission electronic microscopy using B. melitensis and human PMNLs by Young et al. [15]. These authors confirmed that no more than 50% of B. melitensis phagocyted showed any degree of morphological alteration. The mechanisms by which virulent B. melitensis evades killing by PMNLs remains unknown. Kreutzer et al. [3] reported that rough strains, but not smooth strains of B. abortus were killed by human PMNLs. They found that the metabolic burst was not triggered by either rough or smooth strains, nor was killing accomplished by lysates of neutrophil granules. Our results indicate that the intracellular killing of B. melitensis by goat's PMNLs was scarce in all cases (Fig. 2). Intracellular killing indices (no higher than 0.13) were similar between groups, in coincidence with the survival of B. abortus in bovine PMNLs [18], bovine macrophages [19] and human PMNLs [20], and B. suis in mouse peritoneal macrophages [17, 21]. Oxidative metabolism by the PMNLs is an important prerequisite for oxygen-dependent bactericidal activities. When a PMNLs is stimulated, the oxidase enzyme on the surface of the plasma membrane catalyzes the conversion of oxygen to superoxide anion, which is then spontaneously converted to H202. NBT is directly reduced by superoxide anion 0.8

-

(A)

-

(B)

-

(C)

(E)

(D)

0.6

0.4

"~ 0.2

m

m

I ~._,_. i I I I i I

IIII1~

1

1 2 3 4 . 5 6

2:54'56

t'l 1

2 3 4 . 5 6

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ill

1234.556

Weeks

Fig. 2. Intracellular killing indices of Brucella melitensis by goats' PMNLs. (A) Inactivated serum from the animal itself. (B) Serum from the animal itself. (C) Inactivated positive control serum. (D) Inactivated negative control serum. (E) Negative control serum. Group I at 30 (O) and 60 (O) min. Group II at 30 ( x ) and 60 ( , ) m i n . The evolution of indices o f Group Ill at 30 ( i ) and 60 (E]) min, and Group IV at 30 (V) and 60 ( ~ ) min were studied during 6 weeks. Coincidence of two or more data are represented as ( , ) .

I

64

M.C. GALLEGOand M. A. LAPE/~A Table 1. Reductionof NBT by PMNLs in the absenceand presenceof different stimulantfactors Stimulantfactors X + SD SE I: HBSS 0.003 ± 0.001 0.0007 2: LPS of E. coli 1.380+ 0.040 0.0040 3: LPS of B. melitensis 0.120+ 0.040 0.0050 4: Proteic fraction of B. melitensis 0.370+ 0.020 0.0020 5: Poly-Bof B. melitensis 0.390+ 0.020 0.0020 6: B. melitensis (disrupted) 0.590+ 0.030 0.0030 7: B. melitensis (wholecells) 0.120 + 0.040 0.0040 Valuesare expressedin ,ngof formazan/107cells× min z.The variations observed in all groups were not significant(P > 0.05). (1) Hanks' solution. (2) Lipopolysaccharideof Escherichia coli. (3) Lipopolysaccharide of Brucella melitensis. (4) Proteic fraction of Brucella rnelitensis. (5) Polysaccharide B of Brucella melitensis. (6) Brucella melitensis subjected to ultrasonicdisruption.(7) Brucella melitensis whole cells. X, mean. SD, standard deviation.SE, standard error.

to insoluble purple f o r m a z a n . R e d u c t i o n o f N B T is therefore a measure of p r o d u c t i o n of superoxide a n i o n by the P M N L s [22]. The results of the assay of N B T reduction are presented in T a b l e 1. All the different fractions o f B. m e l i t e n s i s inhibit p r o d u c t i o n of superoxide a n i o n by caprine P M N L s in c o m p a r i s o n with LPS of E. coli ( s t i m u l a n t factor no. 2). O u r data indicate that the s t i m u l a n t factors of the N B T reduction could be classified as weak s t i m u l a n t factors (LPS of B. m e l i t e n s i s a n d B. m e l i t e n s i s whole cells), m e d i u m s t i m u l a n t factors (proteic fraction of B. m e l i t e n s i s , poly-B of B. m e l i t e n s i s a n d B. m e l i t e n s i s disrupted), a n d strong s t i m u l a n t factors (LPS of E. coli), by his different capacity of i n h i b i t i n g the p r i m a r y m e t a b o l i c b u r s t associated with the metabolic processes which generate the bactericidal power of the cell. This scarce s t i m u l a t i o n s h o w n by B r u c e l l a strains has been explained by the p r o d u c t i o n o f low m o l e c u l a r weight c o m p o n e n t s , characterized as nucleotide-like substances, that i n h i b i t the m y e l o p e r o x i d a s e - h y d r o g e n peroxide-halide antibacterial system of P M N L s [13]. It will be very interesting to get more data a b o u t the biochemical differences between LPS o f E. coli a n d LPS o f B. m e l i t e n s i s a n d between B. m e l i t e n s i s disrupted a n d whole cell, as s o o n as his physiopathological implications. At the end, we f o u n d n o bactericidal capacity in the serum towards B. m e l i t e n s i s , which could c o n t r i b u t e to the ineffectivity of the defence m e c h a n i s m s against this germ [15]. S a n s a n o e t al. [23] attributes this resistance, as s h o w n by other gram-negative bacteria, to the S-LPS o f the external m e m b r a n e . However, the role of the LPS in resisting the serum is n o t very clear. It r e m a i n s to be d e t e r m i n e d if the LPS plays a role in intracellular survival, a n d the precise m e c h a n i s m s by which B. m e l i t e n s i s m a y escape intracellular killing by P M N L s . Acknowledgements--The authors wish to thank Dr R. Caravano INSERM, U 65, Institut de Biologie,

Montpellier, France) for helpful criticism of the manuscript.

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Brucella melitensis 16-M and caprine PMNLs

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5. Kreutzer D. L. and Robertson D. C. Surface macromolecules and virulence intracellular parasitism: comparison of cell envelope components of smooth and rough strains of Brucella abortus. Infect. lmmun. 23, 819-828 (1978). 6. Kreutzer D. L., Buller C. S. and Robertson D. C. Chemical characterization and biological properties of lipopolysaccharides isolated from smooth and rough strains of Brucella abortus. Infect. immun. 23, 811-818 (1978). 7. Alton G. G., Jones L. M. and Pietz D. E. Las trcnicas de laboratorio en la brucelosis. FAO/OMS, Ginebra (1976). 8. Person J. M. Test de phagocytose et bactericide. Cours International D'lmmunologie Clinique Animale. l~cole Veterianire D'Alfort, Paris (1983). 9. Stossel, T. P. Application of immunological methods: Method for quantitative phagocytic and NBT assay. In: Handbook of Experimental Immunology, 3rd edn. Blackwell, Oxford (1978). 10. Segal A. W. and Peters T. T. The nylon column dye test: a possible screening test of phagocyte function. Clin. Sci. Molec. Med. 49, 591-599 (1975). 11. Redfearn M. S. An immunochemical study of antigens of Brucella extracted by the Westphal technique. Thesis Doctoral, The University of Wisconsin (1960). 12. Bongbhibhat N., Elberg S. and Chen T. H. Characterization of Brucella skin-test antigens. J. Infect. Dis. 122, 67-78 (1970). 13. Canning P. C., Roth J. A., Tabatabai L. B. and Deyoe B. L. Isolation of components of Brucella abortus responsible for inhibition of function in bovine neutrophils. J. Infect. Dis. 152, 913-921 (1985). 14. Van Furth R., Theda U and Leijh P. C. J. In vitro determination of phagocytosis and intracellular killing by polymorphonuclear phagocytes. In: Handbook of Experimental Immunology, 3rd edn. Blackwell, Oxford (1978). 15. Young E. J., Botcher M., Kretzer F. L. and Musher D. M. Phagocytosis and killing of Brucella by human polymorphonuclear leucocytes. J. Infect. Dis. 151, 682-690 (1985). 16. Gallego M. C., Cuello F. and Bordallo L. Fagocitosis de Brucella melitensis: Estudio de las fases de opsonizaci6n-ingesti6n y muerte intracelular en cabras brucel6sicas. Med. Vet. 4, 401-404 (1987). 17. Gay B., Mauss H. and Sanchez-Teff S. Aspects ultrastructuraux de la phagocytose in vivo et in vitro de Brucella par les macrophages de peritoine de la souris. Ann. Immun. 132D, 299-313 (1981). 18. Smith H. and Fitzgeorge R. B. The chemical basis of virulence of Brucella abortus. V. The basis of intracellular survival and growth in bovine phagocytes. Br. J. exp. Path. 45, 174-179 (1984). 19. Fitzgeorge R. B., Solotorovsky M. and Smith H. The behaviour of Brucella abortus within macrophages separated from the blood of normal and immune cattle by adherence to glass. Br. J. exp. Pathol. 48, 522-525 (1967). 20. Morris J. A. The interaction of Brucella abortus 544 and neutrophil polymorphonuclear leucocytes. Ann. Sclavo. 19, 143-150 (1977). 21. Oberti J., Caravano R. and Roux J. Essai de drtermination quantitative de la fusion phagolisosomiale au cours de l'infection de macrophages murins par Brucella suis. Ann. lmmun. 132, 201-206 (1981). 22. Yost F. J. Jr and Fridovich I. Superoxide radical and phagocytosis. Arch. Biochem. Biophys. 161, 395-401 (1974). 23. Sansano M., Reynard A. M. and Cunningham R. K. Inhibition of serum bactericidal reaction by lipopolysaccharide. Infect. lmmun. 48, 759-762 (1985). 24. Diaz R., Garatea P., Jones L. M. and Moriyon I. Radial immunodiffusion test with a Brueella polysaccharide antigen for differentiating infected from vaccinated cattle. J. elin. Microbiol. 37, 41-49 (1979).