Natural resistance to mycobacteria: Antimicrobial activity and reactive oxygen intermediate releasing functions of murine macrophages

Ann. Immunol. (Inst. Pasteur) 1984, 135 D, 25-37

NATURAL RESISTANCE TO MYCOBACTERIA : ANTIMICROBIAL ACTIVITY AND REACTIVE OXYGEN I N T E R M E D I A T E RELEASING FUNCTIONS OF MURINE MACROPHAGES by J. L. Stach ~, (0, G. Delgado (0, V. Tchibozo Q), M. Strobel Q) and P. H. Lagrange (0 (1) Laboraloire d'Immunologie cellulaire, Inslilu! Pasteur de Dakar, Dakar, and (0 Laboraloire d'Immunobiologie du BCG, Unitd d'Immunophysiologie cellulaire, Ddparlement de Physiopalhologie expdrimenlale, Inslitul Pasleur, 73724 Paris Cedex 15

SUMMARY After infection with Mycobaclerium lepraemurium or with M. boris strain BCG (bacillus Calmette-Gu6rin), splenic macrophages from mice naturally resistant (NR) to these pathogens (C3H and A/J) spontaneously produced H20~, whereas splenic macrophages from naturally susceptible (NS) mice (C57BL/6 and Swiss) did not. None of them produced superoxide anion 0~-. In addition, NR macrophages had higher levels of superoxide dismutase (SOD) than did NS macrophages. In vivo treatments thought to enhance H20~ metabolism (phorbol myristate acetate, SOD, Zymosan) decreased M. lepraemurium survival, whereas treatment with diethyl-dithiocarbamate, a potent inhibitor of SOD, had the converse effect. These results favour the hypothesis of a link between natural resistance to BCG (and M. lepraemurium) and H~O~ metabolism, with higher producers being naturally resistant. KEY-WORDS: Macrophage, Hydrogen peroxidel BCG, Mycobacterium lepraemurium; Superoxide anion, Natural resistance, Mouse.

INTRODUCTION One of the most important features of human leprosy is the wide variety of individual behaviours after infection by Mycobacterium leprae. Some Manuscrit re~u le 27 octobre 1983, accept6 le 28 avril 1984.

26

J.L.

STACH AND COL[..

patients (with lepromatous leprosy) do not resist the bacilli, which spread and grow freely in the organs, whereas others (tuberculoid leprosy) inhibit multiplication and dissemination into the body. Development of the leprom a t o u s or tuberculoid form of leprosy appears to be genetically determined [10, 33, 34] but the gene(s) involved have not been defined, even t h o u g h a link between the tuberculoid form and some H L A - D R haplotypes is well documented [9]. An analogous p a t t e r n has been described in experim e n t a l mouse infection with M. lepraemurium. Genetic studies of mouse susceptibility to M. lepraemurium started with t h e work by Kawaguchi [16]. This author and others after him [7, 17, 19] described some inbred strains of mice (e. g. C3H) as being susceptible to M. lepraemurium, and other strains (e. g. C57BL/6) as being resistant. Nevertheless, the strain distribution of resistance can be confusing depending on the criteria used to define resistance (mortality, spreading of bacilli, i m m u n e response), on the dose of inoculated bacilli and on the mode of injection (i. v., i. p., s. c.) [20]. Some insight into t h e field was provided by the discovery of the BCG gene [12, 13]. This single, dominant, autosomal gene mapped to chromosome 1 exists in two allelic forms: BCG s (susceptibility) and BCG r (resistance). Susceptibility is defined as the capacity of a low dose of BEG (2.5 • 10~/ mouse) to grow vigorously in the spleen within g weeks: E57BL/6, B A L B / c and B10-A strains of mice are BEG s. In the BEG r strains (A/J, C 3 H / H e J and DBA/2J), there is a restricted growth of bacilli. The very early phenot y p i c expression of t h e gene (the difference between BEG s and BEG r is detectable as early as 48 h after infection) rules out a role for the acquired i m m u n e response and clearly defines it as a gene of (( natural resistance ,~. Moreover, a tight linkage was found for genes which determine resistance to BCG, Leishmania donovani (Lsh) and Salmonella typhimurium (Ity). Finally, the BCG gene is also involved in natural resistance to M. lepraemurium [5]. We will follow t h e classification proposed by Gros el al. [13]. As t h e y fit the above criteria, we consider C3H as naturally resistant (NR) to BCG and BALB/c and C57BL/6 or Swiss as naturally susceptible (NS). We have recently shown t h a t the macrophage is the cell which actually expresses the BCG gene [35]. This report will provide evidence t h a t the phenotypic expression of natural resistance might be related to H20~ metabolism in the infected macrophage.

AFB CuSOD DDTC FCS HBSS H~O~ i.p. i.v. MnSOD

= = = = = = = = =

acid-fast bacteria. copper-containing SOD. diethyl-dithiocarbamate. foetal c a l f s e r u m . Hank's balanced salt solution. hydrogen peroxide. intraperitoneal(ly). intravenous(ly). manganese-containing SOD.

NR NS O2OD PBS PMA ROI s.e. SOD

= = = = = = = = =

naturally resistant. naturally susceptible, superoxide anion. optical density. p h o s p h a t e - b u f f e r e d saline. phorbol myristate acetate. reactive oxygen intermediate. subcutaneous(ly). superoxide dismutase.

N A T U R A L RESISTANCE TO MYCOBACTERIA

MATERIALS

AND

27

METHODS

1) Animals. Specific t;athogen-free female mice, 8-10 weeks old, were housed in plastic cages and fed with sterilized food and water ad libitum. Inbred C 3 H / H e J mice were purchased from the Jackson Laboratory. Inbred C57BL/6, B A L B / c and some CgH/HeJ mice were obtained from the CNBS laboratory (Orleans, La Source). Outbred Swiss mice were bred in our laboratory. They were classified as naturally susceptible to BCG according to the in vivo criteria of Gros el al. [13]. 2) Mgcobacleria.

M. lepraemurium ceils were harvested from outbred albino rats after repeated passages every six months, as described previously [18], In brief, the bacilli were purified from local non-ulcerative lepromas by differential centrifugation, washed and, after determiimtion of acid-fast bacteria (AFB), aliquots containing 1 • 108 AFB/ml were stored at --80 ~ C until use. The same lot of M. lepraemurium was used for all in vivo and in vitro experiments. M. boris strain BCG was cultured in vitro in liquid Sauton medium and harvested during the exponential phase. Single bacteria suspensions were prepared by sedimentation at 400 g for 5 min. 3) Reagenls.

Supeloxide dismutase (SOD). - - Bovine SOD obtained from Sigma Chemical Co. (St Louis, M0) was sterilized by Millipore filtration (0.22 b~m) after dissolving 30,000 U/ml in Hank's balanced salt solution (HBSS) and kept in aliquots at --80 ~ C. Dielhyl-dithiocarbamate (DDTC). - - DDTC (60 mM) was dissolved in HBSS, sterilized by Millipore filtration (0.22 ~m) and kept at 4 ~ C until use. Phorbol myrislate acetate (PMA). - - Three mg of PMA (Sigma Chemical Co.) were dissolved in 1 ml of ethanol and kept in small aliquots at --80 ~ C until use, when they were diluted in HBSS. Zymosan. - - Zymosan (Sigma) was suspended in HBSS at l mg/ml and kept in small aliquots at --20 ~ C after opsonization by incubation with normal human serum. 4) In vitro production o/superoxide anion (02-) and hydrogen peroxide (H~02) by macrophages. 02- and H20~ production by in vitro explanted macrophages was measured on monolayers plated on 30-ram diameter plastic Petri dishes (tissue culture-treated) containing approximately 106 adherent cells. Peritoneal macrophage monolayers were prepared from washing out of the peritoneal cavity with 5 ml HBSS. After one wash in HBSS and differential counting (Wright staining), cells were adjusted to l0 s macrophages per ml of RPMI medium supplemented with glutamine (2 mM final) and 10% FCS, then incubated for 2 h at 37 o C, 5~o COs (1.5 ml/Petci dish). Non-adherent cells were removed by three vigm ous washes with prewarmed HBSS (without phenol red) before performing O3- and H~O2 production measurements. Spleen macrophages were obtained from a single cell suspension of spleen leucocytes. Red blood cells were lysed with Gey's haemolytic solution [24]. Remaining leucocytes were washed three times in culture medium (RPMI supplemented

28

J.L.

STACH AND COLL.

as above) and, after differential counting to appreciate the relative number of macrophages, adjusted to l0 s macrophages per ml in culture medium; 1.5 ml of cell suspension was incubated overnight at 37 ~ C, 5~o COs in 30-mm diameter plastic Petri dishes (tissue culture-treated). Non-adherent cells were removed by three washes with prewarmed HBSS (without phenol red). Superoxide anion (02-) production was determined according to Johnston el al. [15]. Briefly, the macrophage monolayer was overlaid with 1 ml Krebs-Ringer phosphate solution containing 1 g/l glucose and cytochrome c (type III, Sigma; final concentration 80 ~M). PMA (0.5 2g/ml final concentration) or opsonized zymosan (0.5 mg/ml) was added when we measured stimulated production. They were omitted when studying spontaneous production. Dishes were incubated for 1 h at 37 ~ C; afterwards, supernatants were removed, rapidly centrifuged (Eppendorf centrifuge, 10,000 rpm, 2 min) and 0D taken up at 550 nm. The amount of reduced cytochrome c was calcultated by using an extinction coefficient of 2.1 • 104 M-~ cm-1. H~O~ production was measured according to Pick [28]. A f t e r 05- or H~O2 measurements, dishes were washed with HBSS (without phenol red) and cell monolayers were lysed by 1 ml of 0.5 NaOH. Cellular protein content was determined using the Bradford method (Biorad reagents). 5) SOD levels. The radioimmunoassay used here was able to distinguish copper SOD (CuSOD) from manganese SOD (MnSOD). H u m a n CuSOD was labelled with 125I using the chloramine-T method; human MnSOD was also labelled with 1-~5I but using the Bolton and Hunter method. These reagents were obtained from Amersham (UK). Specific antibodies against these two human SOD were obtained from hyperimmune rabbits and cross-reacted with murine SOD. Cell aliquots were dissolved in 0.5 N of NaOH to determine the protein content. The remainder of the cell suspension was diluted with Triton-X100 (final concentration 10%, v/v) and used for the radioimmunoassay as described by Baret eI al. [1, 2]. Results were expressed in ~g/mg of protein. The chemical assay of SOD used here was described by Beauchamp and Fridovitch [3] using the inhibition of nitroblue-tetrazolium reduction by superoxide anion obtained from riboflavin photoreduction. In brief, cells in 2 ml PBS were sonicated three times at 4 ~ C for 45 s at a 50-w output. Five ~l of the sonicate were used for the determination of protein content; SOD was then extracted by mixing, at 4 ~ C, 2 ml of sonicate with 1 ml of a chloroform/ethanol solution (3/5, vol/vol), the precipitate was centrifuged at 10,000 g for 20 min, at 4 ~ C, and supernatant SOD activity measured. Results were expressed in units/mg of protein. 6) M. lepraemurium: in/eclion and treatment. Separate groups of mice were inoculated i. p. with 1 x l0 s M. lepraemurium. At predetermined intervals after infection, the mice were killed by cervical dislocation and mycobacteria in spleen determined as described [18]. Briefly, spleens were homogenized using a sterile pestle in 5 ml saline containing 1% saponin; 5 ~1 of the suspension were then smeared on a 5-mm diameter surface, fixed with hot methanol and stained by the Ziehl-Neelsen method. The mean number of AFB per ml was calculated from 4 countings by different investigators. Separate groups of infected mice were treated daily from 1 day before to 5 days after M . lepraernurium inoculation with one i. p. injection of 0.5 ml of HBSS containing either 100 U of bovine SOD, 3 t~g of PMA, 1 mg of zymosan or 50 mg/kg DDTC. Five mice per group were killed on day 6 and the five remaining mice on day 18 after infection. Two other groups were treated either with two i. p. injections of 100 U bovine SOD on day 0 and on day 5 (and killed on the same days as the former) or with four injections of 100 U human CuSOD (donated by A. Baret,

NATURAL RESISTANCE TO MYCOBACTERIA

29

Marseille) given on days 0, 7, 14 and 21 after inoculation of M. lepraemurium. These mice were killed on day 23 post-infection. Control mice received the same number of i. p. injections of HBSS and were killed on the same day as the homologous treated groups. RESULTS

1) Production of 03- and H303. In a first step of experiments, measurements of 02- and H303 production by peritoneal macrophages from NR and NS mice were performed. These studies concerned resident peritoneal macrophages, thioglycolate-elicited macrophages (harvested 3 days after an i. p. injection of 1 ml of thioglycolate broth) and M. lepraemurium-elicited peritoneal macrophages (harvested 4 days after an i. p. injection of 2 x 108 M. lepraemurium/mouse). 03- and H203 were measured 60 min after triggering a metabolic burst with PMA or zymosan or without stimulation (spontaneous production). 02- and H303 production by those populations of macrophages were found to show no statistical difference in C3H, BALB/c, C57BL and Swiss mice (results not shown). We then studied splenic macrophages and found a striking difference between NR and NS mice. Macrophages were isolated from spleens of mice previously injected with either 1 X 106 BCG i. v. (24 h before) or 2 x l03 M. lepraemurium i. p. (4 days before) and tested for spontaneous and stimulated 02- and H303 release. The results of several experiments (table I) showed t h a t NR and NS splenic infected macrophages had no spontaneous 0 3 - production and the same low level of PMA-induced 03- release. But the results of H~02 production were different: NR splenic macrophages (C3H, A/J) spontaneously produced statistically significant amounts of H30~, while NS did not. Similarly, PMA-induced H303 production was also higher in NR t h a n in NS splenic macrophages. The most striking difference between NR and NS splenic macrophages thus appeared in spontaneous H303 release. Moreover, this spontaneous H30~ production by NR macrophages was very stable: two tests performed at 2-4 h intervals with the same monolayer gave identical results. (( Spontaneous ,, means that no triggering agent such as PMA or zymosan was used. However, it should be noted t h a t most of the splenic macrophages used in the experiments described in table I contained AFB. Splenic macrophages from non-infected mice (of either strain) did not spontaneously produce 03- or H30~ (results not shown). As H20~ production was found to be dissociated from 03- production, the next experiment was performed to study SOD, the enzyme known to be involved in the dismutation of 03-. 2) SOD levels in macrophages. CuSOD levels (MnSOD is almost undetectible in mice) were measured with a radioimmunoassay described by Baret et al. [1] in adherent spleen

TABLE I. - - Spontaneous and stimulated production of 02- and H~02 by splenic macrophages from mice infected 4 days previously with 2>< l 0 s (( M. lepraemurium ~ i. p. or 10 ~ dispersed BCG i. v.

O2

H202 ( n m c l e s / h / m g protein)

( n m o l e s / h / m g protein)

Strain of mice

C 3 H / H e J (NR) Swiss (NS) A/,J ( N R ) C57BL/6 (NS) C3H (NR) C57BL/6 (NS)

Treatment 2 x 1 0 s 3llm i . p . 4 (lays b e f o r e test ,) )~ )~ )) )~ )~ 106 B C G i . v. 24 h before test ,, ,,

Sporttaneous

Stimulated PMA (0.5 a g / m l )

Spontaneous

Stimulated PMA (0.5 ~g/ml)

0

12•

16•

187•

0 0 0 0

11 • NT NT 15•

0.4• 13 • 0.3 • 19•

106• NT NT 191•

0

13 • 7

0.5 •

(*)

104 •

Spleen macrophages, obtained as indicated in (, Materials and Methods )), were cultivated overn i g h t in 30-ram plastic Petri dishes, then washed v i g o r o u s l y before performing t h e test. R e s u l t s from five similar experiments. (*) p < 0.01. 3lira = M. lepraemurium. N T = not tested.

4r

.g 0

O~

E t_.l

0 o

9 I

0

FIa. 1. - - Levels o[ copper superoxide dismutase ( C u S O D ) in adheren (Z-h) spleen cells harvested in control mice (horizontal line • 2 S E M ) two days (black bars) and 21 days (hatched bars) after i. v. in]ection of 2 • 108 M. l e p r a e m u r i u m in C.57BL/6, C3tI and Swiss mice. CuSOD determinations were performed w i t h a r a d i o i m m u n o a s s a y as described i n , Materials and Methods ,,. Mean of 5 experiments • SEM.

NATURAL RESISTANCE TO MYCOBACTERIA

31

cells 48 h and 21 days after an i. v. injection of 2 x 108 M. lepraemurium. It is clear from t h e results shown in figure 1 t h a t M. lepraemurium-infected N R maerophages from C3H mice contained significantly higher levels of SOD t h a n normal (non-infected) control macrophages. No such increase occurred in M. lepraemurium-infected N S macrophages from C57BL/6 or Swiss mice. The enhanced SOD levels observed in infected C3H mice were not due s bacterial enzyme, since levels measured in a n u m b e r of bacteria analogous to t h a t found in the spleen at day 20 after infection were below the sensitiv i t y threshold of t h e assay. The enzyme detected b y the previous assay was, in fact, a functional SOD, since chemical dosage of enzyme a c t i v i t y performed on cells prepared in the same w a y gave identical results (table II).

TABLE II. - - SOD levels in spleen macrophages from n o r m a l and ~( M. lepraemurium ~- or BCG-infected C3H and Swiss mice.

Mice Swiss C3H/HeJ

Infection BCG Mira Control BCG Mlm Control

SOD (1) (U/mg protein) 14.0 2_3.7 16.6zk4.0 12.5 4-2.5 33.2 i7.0 (3) 22.5 • (3) 9.5 :kl.5

S e p a r a t e g r o u p s of 30 C 3 H / H e J and 20 Swiss mi c e were i n j e c t e d i. p. w i t h saline c o n t a i n i n g e i t h e r 7 x 10 ~ BCG or 2 x 108 M l m 20 d a y s before sacrifice. (1) For each d e t e r m i n a t i o n of SOD, p o o l e d cells of each g r o u p were e x a m i n e d . Mean f r o m 5 e x p e r i m e n t s • (2) p < 0 . 0 1 . Mira = M. lepraemurium.

To determine w h e t h e r such a rise in maerophage SOD levels could explain the previously described enhancement of H~02 production in N R macrophages, the next experiment was designed to observe in vitro the potential effect of addition of SOD on O~- or H~O~ production in a macrophage monolayer from normal Swiss mice. As shown in table III, the addition of 1,500 U / m l of bovine SOD for 2 h was able to boost the production of H~O~ induced b y 0.5 ~g/ml of PMA. However, no such effect was observed when 02- production was measured. This result indicated t h a t the effect observed was not due to some SOD remaining in t h e s u p e r n a t a n t (cells were washed thoroughly after SOD treatment); otherwise 0~- would have disappeared from the medium. On the other hand, when DDTC, a p o t e n t inhibitor of SOD [22], was added to the monolayer for 2 h before adding PMA, total inhibition of H~O~ production was observed for a dose of DDTC as low as 6 ~M. In contrast, DDTC was not able to significantly inhibit O~- production induced b y PMA. Ann. lmmunoL (Inst. Pasteur), 135 D, n ~ 1, 1984.

3

32

J.L.

STACH

AND

COLL.

TABLE III. - - Effect of bovine SOD and various concentrations of DDTC on the production of 02- and H202 by resident peritoneal macrophages of Swiss mice after stimulation with PMA for 60 min. O s-

HsO s

( n m o l e s / m g protein)

( n m o l e s / m g protein)

25 -4-5

33 4.11

Control SOD

(1,500 U / m l )

DDTC

25 • 11

6 mM 600 tzM 60 ~tM 6 tzM 600 nM 60 nM

102 •

NT NT NT 14 • 26• 194-6

0 0 0 0 7 2:4 29•

Peritoneal m a c r o p h a g e s were harvested b y w a s h - o u t of t h e peritoneal c a v i t y of n o r m a l Swiss mice. After 1-h i n c u b a t i o n at 37 ~ C, t h e y were w a s h e d twice and SOD or D D T C added for 2-h i n c u b a t i o n at 37 ~ C in 5 % COs. T h e y were t h e n w a s h e d vigorously three times and, after addition of PMA (0.5 tzg/ml), were tested for p r o d u c t i o n of O~- and H~O s after 60 rain. Mean f r o m 5 experiments i SEM. N T = n o t tested.

TABLE I V . - -

Experiment

G r o w t h o f (( M. l e p r a e m u r i u m ~ i n normal mice or mice treated with SOD, PMA, z y m o s a n and DDTC.

Mouse strain

1

Swiss (12/8)

2

C57BL/6 (6/4)

3

Swiss (3/3)

4

Swiss (3/3)

4

Swiss (3/3)

6

Swiss (3/3)

7

C 3 H / H e J (3/3)

Treatment

A F B / s p l e e n (2) ( • 10 -e)

Resistance Index

H u m a n SOD Q) Saline H u m a n SOD (1) Saline Bovine SOD 1 Saline Bovine SOD s Saline PMA Saline Zymosan Saline DDTC Saline

O.05SF 0.01 1.2 T 0.3 1.5T1 7.5~- 1.5 7.5:~ 1.5 17 T 4 4.4T 1 17 ~- 4 0.5-F 0.2 20-F 2 7 ~- 0.5 19T 6 12.3~ 0.7 4.8 • 1

+25 +5 +2.2 +3.9 +40 + 2.7 - 2.6

Separate g r o u p s of mice received i. p. 2 • 108 M. lepraemurium, and thereafter the A F B / s p l e e n were m e a s u r e d as described in ~ Materials and Methods 2. (1) Mice were treated w i t h 100 U of h u m a n SOD on days 0, 7, 14 and 21 post-infection and were saerifieed on day 23. (s) A r i t h m e t i c m e a n ~: SEM. Brackets indieate n u m b e r s of treated mice~numbers of eontrol mice. Resistanee index was calculated = A F B in e o n t r o l / A F B in treated mice ; for exp. 7, the reciprocal was ealculated and assigned a negative n u m b e r .

NATURAL RESISTANCE TO MYCOBACTERIA

33

It was thus interesting to test the in vivo effect of various modifiers of production of H~O~ by macrophages on the growth of M. lepraemurium. 3) In vivo modulation of M. lepraemurium growth. As shown in table IV, products such as PMA and zymosan, which are known to stimulate in vitro the production of O3- and H,O,, were able to decrease the n u m b e r of AFB measured in the spleen of infected NS mice (exp. 5 and 6). Repeated injections of SOD enhanced the resistance of Swiss and C57BL/6 mice. Injections of purified h u m a n SOD were more active; however, schedules of injection and timing after infection were different. When DDTC (exp. 7) was injected daily for 6 days, an increased number of AFB were observed in spleens of treated C3H mice compared to controls. From these results and the preceding findings, it can be concluded t h a t the high levels of SOD associated with increased H~O, production in phagocytes might be associated with a higher capacity to kill M. lepraemurium. Since the A F B counts of M. lepraemurium could represent both live M. lepraemurium and undigested killed M. lepraemurium which were still acid-fast, the efficacy of killing might be underestimated.

DISCUSSION We report here some experimental results favouring the hypothesis of a linkage between natural resistance to M. lepraemurium (or BCG) and H20~ production of the macrophage. Arguments are the following. In vitro explanted splenic macrophages from Nit mice (C3H, A/d) infected by M. lepraemurium or t3CG spontaneously produced H, O2, whereas the same cells from NS infected mice (C57BL/6, Swiss) did not. This spontaneous production by NR splenic macrophages was not very high, but was quite detectible and still statistically significant when compared to NS maerophage H20, production. It was not associated with any concomitant superoxide production, as usually occurs during the metabolic burst, and it was unexhaustible, since there was no decrease in production by the same monolayer tested at a 2-4 h interval. Thus, it differs from the classical metabolic burst in many characteristics. We tend to consider it more likely as an enhancement of one of the metabolic pathways, which also occurred in non-phagocytic cells, leading to H~O~ [6]. The observed dissociation of H~02 production from 03- production might be indirectly due to the rise in SOD levels observed in NR macrophages (but not in NS) after infection. This rise could favour H~O~ generation according to the dismutation reaction eatalysed by SOD: 0~-+02-+2

H + -+ H~0~+02

Whatever the case, it must be stressed t h a t the most i m p o r t a n t aspect of the phenomenon is its spontaneous production. In NR macrophages,

34

J.L.

STACH AND COLL.

mycobacterial infection activates a metabolic pathway, giving H202. In NS macrophages, this p a t h w a y remains dormant. We also observed an enhancement of H202 production (but not 02-) during the metabolic burst triggered by PMA in splenic macrophages from in vivo infected NR mice when compared to NS. However, enhancement concerned only H202 production (02- release was the same in NR and NS), simply reflecting the rise in SOD levels. To us this seems less significant t h a n the spontaneous H202 production. A genuine perturbation of the metabolic burst would have appeared in 02- as well as in H202 production. We have been unable to find such a mouse strain difference in H202 production by M. lepraemurium or thioglycolate-elicited peritoneal macrophages from NR and NS mice. Also, the metabolic burst triggered in these cells was identical in Nlq and NS mice. These results are in agreement with those of Blumenstock el al. [4, 37], who showed t h a t S. typhimurium elicited t h e same metabolic burst in peritoneal macrophages of NR and NS mice (it should be recalled t h a t the Ity and BCG gene might be identical). There is no clear explanation for the different behaviours of peritoneal and splenic macrophages. A rise in splenic macrophage SOD levels has been found during infection by BCG or M. lepraemurium. The rise occurs in the resistant C3H strain, but not in susceptible Swiss or C57BL/6 mice. It involves CuSOD, with MnSOD being almost undetectable in mice. T h a t such a rise could account for the observed enhancement of H202 production in splenic NR macrophages is also suggested (though not proven) by the observation t h a t in vitro treatment of macrophage monolayers with SOD, which is probably internalized in the cell through pinocytosis as described for catalase [26], resulted in enhanced production of H202, an effect previously described by Pick et al. [29]. DDTC, which is a potent inhibitor of SOD [22], has the opposite effect without impairing the viability of macrophages, since it does not affect 02- production. In agreement with our results, mycobacteria were shown to raise the levels of different enzymes in infected tissues of the host; for instance, glutathione peroxidase in rabbit alveolar macrophages after BCG infection [32], $-glucuronidase in armadillo infected with M. leprae [30] and [~-galactosidase in resident and inflammatory peritoneal macrophages from mice infected with BCG [31]. Moreover, Rhodes et al. [31] described this rise as strain-specific, since it occurred in C3H but not in C57BL/6 mice. H~02 involvement in resistance to M. lepraemurium is also probable. In vivo injection of H202 inducers (PMA, zymosan, SOD) resulted in a decrease of AFB in t h e spleen; DDTC used as an SOD inhibitor had the reverse effect. The half-life of SOD after i. v. injection has been shown to be short [23] but, despite its quick disappearance from circulation, SOD has been shown to remain pharmacologically active [14]. In conclusion, our results suggest a possible linkage between H202 production in macrophages and natural resistance to BCG and M. leprae-

NATURAL RESISTANCE TO MYCOBACTERIA

35

murium, with higher producers of H , 0 , being classified as naturally resistant. If we assume a link between enhancement of H,0~ production and natural resistance, this might also partially explain the defective immunity well documented in C3H mice, since m a n y reports have implicated H~0~ in the depression of immune response by macrophages [ 11, 36].

RI~SUMI~ RIZSISTANCE NATURELLE AUX MYCOBACTI~RIES : ACTIVIT]~ ANTIMICROBIENNE ET Mt~TABOLISME DU PEROXYDE D'HYDROGIZNE DES MACROPttAGES MURINS

Apr6s infection par Mgcobacterium lepraemurium ou par le BCG, les macrophages spl6niques de souris naturellement r6sistantes (NR) ~ c e s agents pathog~nes (C3H, A/J) produisent spontan6ment H~O~, alors que les macrophages spl6niques de souris naturellement sensibles (NS) (C57BL/6, Swiss) n'en produisent pas. Aucune lign6e de souris n'a produit spontan6m e n t d'ion superoxyde. De plus, les macrophages spl6niques de souris NR infect6es produisent une quantit6 de superoxyde-dismutase (SOD) plus grande que celle des macrophages de souris NS infect6es. In vivo, les traitements dont on a des raisons de croire qu'ils augmentent la production d'H~0~ (myristate-ac6tate de phorbol, SOD, zymosan) provoquent une diminution de la survie de M. lepraemurium, alors qu'un traitement par le di6thyl-dithiocarbamate, puissant inhibiteur de la SOD, a un effet contraire. Ces r6sultats renforcent l'hypoth~se d'un lien entre la r6sistance naturelle au BCG (et i~ M. lepraemurium) et la production de H~O~, les forts producteurs 6tant naturellement rdsistants. MOTS-CLt~S " Macrophage, Peroxyde d'hYdrog~ne, BCG, Mgcobacterium lepraemurium ; Souris, Anion superoxyde, R6sistance naturelle. ACKNOWLEDGEMENTS

We thank C. Maczuka for typing the manuscript. Part of this work was supported by DGRST grant n ~ 81.L.699.

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