the production of nitric oxide and tumor necrosis factor by murine macrophages infected with...

Microbiol. Immunol., 43(7), 637-644, 1999

The Production of Nitric Oxide and Tumor Necrosis

Factor by Murine Macrophages Infected with

Mycobacterial Strains Differing by Hemolytic Activity

Wiestawa Rudnicka*'1, Marzena Brzychcy1, Magdalena Klink2, Arley Gomez Lopez 3 ,

Pierre Alain Fonteyne3, Sabine Rlisch-Gerdes4, and Barbara Radalska 1

1 Deparment of Infectious Biology; Institute of Microbiology and Immunology , University of Lack, 90-237 Lodz, Banacha 12,

Poland, 2Department of immunology, Microbiology and Virology Centre, Polish Academy of Science, 93-232 LedZ, Lodowa 106, Poland, 3Department of Microbiology, Institute of Tropical Medicine, Nationale-Straat 155,2000 Antwerpen, Belgium, and 4National Reference Centre for Mycobacterium Forschungszentrum Borstel, Parkallee 18, 23 845 Borstel, Germany

Received December 25, 1998; in revised form, March 1, 1999. Accepted March 27, 1999

Abstract: In this study, we compared the secretion of nitric oxide (NO) and tumor necrosis factor (TNF-a) by murine macrophages infected in vitro with hemolytic or unhemolytic mycobacteria isolates. We observed that unhemolytic mycobacteria induced more intensive NO production by macrophages and were more susceptible to bactericidal effect of mononuclear phagocytes than hemolytic mycobacterial strains. In contrast, the high-virulence hemolytic isolates induced significantly stronger TNF-a production by infect-ed macrophages than the low-virulence unhemolytic bacilli.

Key words : Mycobacteria, Hemolysis, Macrophages, Nitric oxide, TNF

Contrary to expectations, a global rise in the inci-dence of tuberculosis (TB) has been reported recently

(14, 27). It is currently estimated that at least a third of the world's population has been infected with patho-

genic mycobacteria (27). Annually, about 3 million people die of TB. Partly, the rise in the incidence of TB is associated with human immunodeficiency virus (HIV) infections (27, 31).

Mycobacterium avium complex (MAC) organisms are common in many environmental sites including water, soil, animals, hot water systems, dental devices, and dust (21, 30, 49). They produce three major clinical syndromes: disseminated disease usually in patients with advanced HIV infection, pulmonary disease, and cervical lymphadenitis in individuals whose systemic immunity is intact (29). The incidence of all of these syndromes appears to be increasing. Moreover, the iso-lation of multi-drug-resistant mycobacteria has been associated with very high mortality rates (13).

MAC bacilli, like Mycobacterium tuberculosis com-

plex bacteria (MTB), are intracellular pathogens residing

within the mononuclear phagocytes of the host (31) .

The survival and replication or inhibition and killing of

any given microbe depend on both the nature of the

pathogen and the repertoire of activity induced in the host

cells. Recently, we have found that some MAC iso -

lates are hemolytic in vitro (10), which is a property

characteristic for all MTB strains (16, 32, 59). Moreover ,

hemolytic versus unhemolytic MAC isolates have been

found to be more resistant to the mycobactericidal effects

of murine macrophages, and they survived for a longer

time in the spleens of C57BL/6 mice (9, 10) which are

naturally susceptible to the mycobacterial infections

(26) .

In this study, we addressed the question as to whether

hemolytic M. avium bacilli- or unhemolytic M. avium

bacteria- induced macrophage response is the same as

regards nitric oxide (NO) and tumor necrosis factor

alpha (TNF-ƒ¿) production. The reactive nitrogen inter -

mediates play an important role in the killing of intra -

cellular mycobacteria (1, 15, 33) and a gene, Nramp 1 ,

* Address correspondence to Dr. Wieslawa Rudnicka, Depart -

ment of Infectious Biology, Institute of Microbiology and Immunology, University of Lodz, 90-237 Lodz, Banacha 12 , Poland. Fax: (48) 42 678 49 32 .

Abbreviations: CDH, contact-dependent hemolysin; MAC,

Mycobacterium avium complex; May, M. avium strains; MTB, M.

tuberculosis complex; Mtb, M. tuberculosis strains; NO, nitric

oxide; NO2-. nitrite; TB, tuberculosis; TNF, tumor necrosis fac-

tor.

637

638 W. RUDNICKA ET AL

for natural resistance-associated macrophage protein 1, has been postulated to regulate the mycobactericidal

capacity of macrophages (42, 58) and their ability to

present mycobacterial antigens to T lymphocytes (60). On the other hand, Medina and North (36) observed no linkeage between the Nramp l gene and the resistance of

mice to M. tuberculosis infection. Recently, Bellamy et

al (2) have shown a polymorphism of the human homo-

logue of the murine Nramp 1 gene which seems to be

associated with susceptibility to tuberculosis. The microbicidal activity of macrophages against

MTB and other mycobacteria has also been shown to be

regulated by the TNF-oc alone or in synergy with other

cytokines (5, 6, 47). Furthermore, TNF-sa production in vivo may lead to granuloma formation, which limits

bacterial dissemination within the host (52). On the

other hand, TNF-oc is involved in the pathology of

mycobacterial infections (38, 44, 53, 54). In the present study, the Mycobacteria-driven response

of macrophages was examined in naturally susceptible to

mycobacterial infection C57BL/6 mice, both immu-

nized with M. bovis BCG vaccine and nonimmunized.

Previously, we found that hemolytic M. avium and M.

tuberculosis strains were more virulent for C57BL / 6 mice than unhemolytic M. avium or M. bovis BCG bacil-

li (10).

Materials and Methods

Bacteria. Mycobacterium tuberculosis (Mtb-1) and

four M. avium clinical strains (Mav-2-5) were isolated in

the Institute for Tuberculosis and Lung Diseases in Warsaw, Poland (the Tuberculosis Reference Laboratory).

M. avium isolates were identified on the basis of their

16SrRNA analysis sequences (25) and by serotyping

(46). In addition, M. tuberculosis H37Rv (NCTC 7416) and M. bovis BCG (Brasiliane strain) were included in the study. The bacteria were grown in Middlebrook

7H9 medium with ADC Enrichment (Difco, Detroit,

Mich., U.S.A.) for 4-6 weeks at 37 C.

Hemolytic activity. The production of contact-depen-dent hemolysin (CDH) by mycobacterial strains was

estimated by the method of King et al (32) as described

earlier (9, 10).

Mice. C57BL/6 mice were obtained from the Labo-ratory of Experimental Animals, Velaz, Czech Republic,

and grown under conventional conditions. They were

used when 8-10 weeks old. The experiments were per-

formed according to the ethical guidelines of the Uni-

versity of LOdi. In some experiments, mice were intraperitoneally immunized with 2 X 106 M. bovis BCG

bacteria for 16 weeks before using.

Macrophages. A peritoneal exudate was induced by

intraperitoneal (i.p.) injection of 2 ml 10% proteose

peptone (Difco) to the nonimmunized or M. bovis BCG-immunized mice. Three days later, cells from the peri-toneal exudate were suspended (5 X 10' cells/ml) in RPMI-1640 medium supplemented with 10% heat-inac-tivated fetal calf serum (FCS) and 2 mm L-glutamine

(complete medium) supplemented with antibiotics (peni-cillin and streptomycin). The cells were incubated in tis-sue culture plates (Nunc, Kamstrup, Denmark) for 1.5 hr at 37 C in an atmosphere with 5% CO, to permit the adherence of macrophages (45). The adherent cells were resuspended in complete medium. The suspension of adherent cells contained about 95% macrophages as determined in smears stained with Giemsa; the viability of the cells was 96% as estimated by trypan blue exclusion.

Measurement of nitrite production. The macrophages

(5 X 10' cells) were cultured with a mycobacteria strain (1 X 107 cells) in 24-well culture plates (Nunc, Roskilde, Denmark) in 1 ml of RPMI- 1640 medium without phe-nol red, supplemented with 10% FCS, at 37 C and 5% CO,. To some wells, 10 ill of recombinant IFN-y

(RandD) was added. The supernatants were harvested 24 or 48 hr after initiation of the cultures. Nitric oxide,

quantified by the accumulation of nitrite (as a stable end product) in the supernatants, was determined accord-ing to Ding et al (18) and Hibbs et al (28). One-hundred microliter aliquots of the supernatants were mixed in

96-well plates with an equal volume of 0.5% sulfanil-amide dihydrochloride and 0.05% naphthylethylenedi-amide dihydrochloride in 2.5% phosphoric acid, and allowed to react for 10 min at room temperature. The colored products were quantified with a plate reader

(Nunc Intermed NY-200) at 550 nm. The concentration of the nitrite in the samples was determined as [tm NO, / 5 X 105 macrophages using a standard curve for nitrite which was included in each experiment.

Bioassay for TNF-a determination. TNF-oc activity in macrophage culture supernatants was assayed colori-metrically as cytotoxicity for an L 929/73 murine fibro-blastoid cell line, as previously described (8). The macrophages (5 X 10' cells) were mixed with a mycobac-teria strain (1 X 106 cells) on 24-well culture plates in 1 ml of RPMI-1640 medium with 10% FCS and incubat-ed at 37 C and 5% CO,. At each culture period, culture supernatants were harvested. To measure cytotoxicity, 2 X 104 L 929/73 cells in 100 Ill of RPMI-1640 with 10% FCS and penicillin/streptomycin were seeded in each well of 96-well culture plates (Nunc, Roskilde, Denmark) and incubated for 8 hr at 37 C and 5% CO,. Then the 100 Ill aliquots of the culture supernatants and the 50 Ill aliquots of actinomycin D (20 lig/m1) were added to the wells. The plates were incubated for 24 hr

MYCOBACTERIA - INDUCED NO AND TNF 639

at 37 C and 5% CO,. After incubation, the plates were emptied and 100 of 0.5% crystal violet (Merck) in PBS with 20% methanol was added to each well. After 15 min at room temperature, the cell monolayers were washed intensively with distilled water, and then 100 ill aliquots of SOrensen's buffer (6.1 ml 0.1 M sodi-um citrate+ 3.9 ml 0.1 N HC1+ 10 ml 95% ethanol) were added to the wells. After 5 min at room tempera-ture, the absorbance was measured at 590 nm (ELISA reader, DHN, Poland). Levels of TNF-a in the super-natants were calculated by extrapolation from a recom-binant TNF-a, (Genzyme) (0.10-30 U/ml) standard curve

prepared for each experiment. Killing of mycobacteria. The bactericidal effect of

macrophages from nonimmunized and M. bovis BCG-immunized mice towards mycobacteria strains was deter-mined as described earlier (10). Briefly, a 100 Ill aliquot of bacterial suspension (1 X 106/m1) was added to 100 ul of macrophage suspension (1 X 106/m1) and the phago-cytes were allowed to ingest the bacilli for 1 hr at 37 C. The extracellular bacilli were washed away. To esti-mate the bactericidal activity of macrophages, the num-ber of live macrophage-associated bacilli was deter-mined just after removing the uningested bacteria (time 0) and after the incubation of the cells for 24 hr at 37 C and 5% CO, (time 24 hr). To determine the number of macrophage-associated bacilli, a phagocyte suspension was mixed with 0.5% saponin for 5 min, diluted with PBS and spread on LOwenstein-Jensen plates. Mycobac-teria colony-forming units were determined after 6-8 weeks at 37 C. The killing activity of macrophages was calculated as: killing% = 100 - (number of bacteria at time 24 hr X 100/number of bacteria at time 0).

Statistics. All samples and standards were assayed in triplicate. The results are expressed as the mean values from three independent experiments ± S.D. Statistical significance was defined as PC 0.05.

Results

The M. avium isolates selected for the study differed by the hemolysin production (10). The Mav-2 and Mav-3 isolates, similarly to M. tuberculosis strain H37Rv and Mtb-1 isolate, expressed contact-dependent hemolyt-ic activity, which was observed during the 3-hr coincu-bation of the bacilli with erythrocyets from various species. The Mav-4 and Mav-5 isolates, similarly to M. bovis BCG bacilli, were unhemolytic (10).

Data in Fig. 1 demonstrate the mycobacteria-stimu-lated NO production by macrophages from unimmu-nized mice (Fig. 1 A) and from the animals immunized with M. bovis BCG, 16 weeks earlier (Fig. 1 B). In the culture media of macrophages from nonimmunized mice

A

B

infected in vitro with hemolytic or unhemolytic mycobac-

teria strains, similar levels of nitrite were demonstrated

(Fig. 1A). In these cultures, we observed the survival of all mycobacteria strains excluding the Mav-5 isolate.

The number of Mav-5 bacilli decreased by 25% during

Fig. 1. The relation between the NO production by peritoneal macrophages from nonimmunized (A) and M. bovis BCG-immu-

nized (B) mice infected in vitro with mycobacteria strains and the susceptibility of those bacilli to the bactericidal activity of phago-cytes. The mycobacteria-infected macrophages were cultured in

medium without IFN-y, for 24 or 48 hr. Nitrite concentration

(ium/5 X 10' macrophages) in the supernatants was determined using the Griess reagent. The mean values + S.D. from three inde-

pendent experiments are shown.


24 hr in the cultures. When the media from the 48-hr

cultures of mycobacteria-infected macrophages from M. bovis BCG-immunized mice were examined, the

NO, levels were significantly higher in the cultures of

macrophages infected with unhemolytic mycobacteria

than those infected with hemolytic mycobacteria. Unhemolytic mycobacterial strains Mav-4, Mav-5 and M.

bovis BCG stimulated very strong NO production by

the macrophages and, at the same time, they were very

susceptible to the bactericidal activity of macrophages from immunized mice. In comparison, hemolytic

mycobacteria strains M. tuberculosis H37Rv, Mtb-1,

Mav-2 and Mav-3 stimulated much weaker NO pro-

duction, and most were not killed during 24-hr incubation

of infected macrophages from immunized animals (Fig. 1 B). The difference between hemolytic and unhemolyt-

ic mycobacteria strains, in terms of the stimulation of NO

production, disappeared when mycobacteria - infected macrophages from M. bovis BCG-immunized mice were

cultured in the medium containing murine IFN-y (Fig. 2). Data in Fig. 3 show the kinetics of mycobacteria - driv -

en TNF-a production in the 24-48 hr cultures of

macrophages from nonimmunized (Fig. 3A) and M.

bovis BCG-immunized mice (Fig. 3B). All hemolytic mycobacteria strains, both M. tuberculosis and M. avium,

stimulated vigorous TNF-oc production by the

macrophages from nonimmunized and immunized mice.

In contrast, unhemolytic mycobacteria practically did not

stimulate murine macrophages to TNF-cc production. A negative correlation was observed between the sus-

ceptibility of mycobacteria to bactericidal effect of

macrophages and their ability to stimulate TNF-cc, pro-

duction (Fig. 4).

Discussion

In 1993, the production of contact - dependent hemolysin (CDH) by M. tuberculosis was reported by

King et al (32). Later, the hemolytic activity of some

MAC strains (9, 10, 17) and M. haemophilum bacilli

A

B

Fig. 2. Lack of any significant differences between the nitrite con-

centrations (tM/5 X 105 macrophages) in the supernatants from the 24- and 48-hr cultures of macrophages from M. bovis BCG-

immunized mice infected in vitro with mycobacteria strains and cultured in the presence of IFN-y (10 itig/m1). The mean val-ues + S.D. from three independent experiments are shown.

Fig. 3. TNF-a production by peritoneal macrophages from non-immunized (A) and M. bovis BCG-immunized (B) mice, infect-

ed in vitro with mycobacteria strains. TNF activity was measured in the supernatants by the L 929 bioassay. The mean values from three independent experiments are shown.


(23) was demonstrated. The CDH isolated from M. tuberculosis H37Rv bacilli was found to be cytotoxic for

lung cells also (16). Recently, we showed that two

unhemolytic M. avium strains, similarly to M. bovis BCG bacteria, were eliminated from the spleens of

infected C57BL/6 mice more rapidly than two hemolyt-

ic isolates, M. avium or M. tuberculosis (10). The longer

survival of hemolytic May strains in the tissues of infect-ed animals was related to their resistance to the bacteri-

cidal effect of macrophages. In this study, we observed

that the NO production assessed by NO, measurement

was significantly less potent in the cultures of macro-

phages from M. bovis BCG-immunized C57BL/6 mice, infected with hemolytic M. avium or M. tuberculosis

strains, than those infected with unhemolytic M. avium

or M. bovis BCG bacilli (Fig. 1B). There was a cor-

relation between the stimulation of intensive NO

production by unhemolytic mycobacteria and their sus-ceptibility to the bactericidal effect of macrophages.

The hemolytic bacilli stimulated less intensive NO pro-

duction in infected macrophages, and they were practi-

cally resistant to macrophage killing. The importance of reactive nitrogen intermediates for intracellular killing of

mycobacteria has been demonstrated in various models

(1, 11, 33). However, other studies showed that NO

production was not related to the killing of MAC strains (4, 56). Moreover, some authors (19, 41) have suggest-ed that the overproduction of NO may be more important

for the immunopathology than for host resistance. This

discrepancy could be caused by investigating different

macrophage populations at various bacilli-phagocytes

ratios, and also by differences in mycobacterial isolates; for example, their NO resistance (20). As we have

shown, the differences in NO production by macrophages

stimulated with unhemolytic and hemolytic mycobacte-

ria organisms could not be observed if an excess of IFN-y was added at the start of the cultures. This could

suggest that weak NO production by macrophages infect-

ed with hemolytic mycobacteria was caused by some

hemolysin-derived alternations in IFN-y production. An

augmentation of NO secretion by IFN-y was observed in the cultures of murine macrophages stimulated with M.

avium (22) and M. bovis BCG bacilli (43) or with Leish-

mania parasites (34), as well as human leukocytes stim-

ulated with E. coli LPS (55). Recently, IFN-y secretion

by murine bone marrow-derived macrophages has been

postulated (39). However, we should not exclude the possibility that, in this study, some NK cells and T cells which were present in small proportion (<5% of lym-

phocytes) in the suspensions of inflammatory peritoneal macrophages could possibly be a source of INF-y.

Our results demonstrate intensive TNF-a activity in

the cultures of inflammatory macrophages infected with

more virulent hemolytic M. avium or M. tuberculosis

strains, which was hardly seen in the cultures infected with less virulent unhemolytic M. avium or M. bovis

BCG bacilli (Fig. 3). On the contrary, Sarmento and

Appelberg (47) and Eriks and Emerson (22) found that

the low-virulence M. avium strains induced more inten-sive TNF-a production than the high-virulence M. avium

bacilli did. However, they were using resident peri-

toneal or bone marrow-derived macrophages, respec-

tively. Also, lipoarabinomannan (LMA) from the avir-

ulent M. tuberculosis H37Ra strain was found to be 100-fold more potent at inducing TNF-oc secretion than

mannosylated LMA from the virulent M. tuberculosis

(Erdman) strain (12). In contrast, the last reports by Silver et al (50) showed more intensive TNF-a produc-

tion by human monocytes infected with virulent M. tuberculosis H37Rv than by those infected with avirulent

M. tuberculosis H37Ra or M. bovis BCG bacilli. Simi-

lar to ourselves, these authors estimated TNF-a activity

in the macrophages stimulated with live bacteria.

Surprisingly, the intensive TNF-a activity in the cul-tures of macrophages infected with hemolytic mycobac-

terial strains was associated with slightly, if any, intra-

cellular killing and weak NO production. The inhibition

of nitrite production in the resident peritoneal macrophages infected with some M. avium strains was

reported by Sarmento and Appelberg (48). However,

several authors demonstrated an association between

the TNF-a activity and killing of mycobacteria by

Fig. 4. The relation between the TNF-a. production by peri-

toneal macrophages from M. bovis BCG-immunized mice infect-

ed in vitro with mycobacteria strains and the susceptibility of those

bacilli to bactericidal activity of phagocytes. The mycobacteria-

infected macrophages were cultured for 12 hr. TNF activity

was measured in the supernatants by the L 929 bioassay. The

mean values + S.D. from three independent experiments are

shown, P< 0.001.


macrophages (3, 15, 22, 24). The stimulation of peri -

toneal macrophages (5) or macrophages cell line (7)

with rTNF-ƒ¿, especially in the presence of IFN - ƒÁ,

increased the intracellular killing of M. avium and M .

bovis BCG bacilli. On the other hand, some authors

could see no effect of TNF - ƒ¿ on the replication of viru -

lent mycobacteria, in vitro or in vivo (47, 51. 52). In this

study, the activity of TNF - ƒ¿ was estimated only by the

biological method, in cultures of macrophages infected

with a high number of mycobacteria. It is worth men -

tioning that much higher levels of TNF - ƒ¿ were detected

in the sera from C57BL / 6 mice infected with hemolytic

M. tuberculosis or M. avium strains than with unhemolyt -

ic M. avium or M. bovis BCG bacilli (data not shown) .

The mechanisms of the hemolytic activity of

mycobacteria remain unknown. Could mycobacteria ,

like L. monocytogenes, use hemolysins to escape from a

phagosome into the cytosol? In fact, the presence of vir -

ulent but not avirulent mycobacteria in the cytoplasm of

macrophages has been recorded by some researchers

(35, 37, 40). In light of these reports, we can speculate

that hemolytic, more virulent M. tuberculosis and M .

avium bacilli, in contrast to unhemolytic, less virulent, M .

avium and M. bovis BCG strains, could spread from a

phagosome into the cytosol. It is possible, that mycobac -

teria located in the cytoplasm could activate

macrophages differently from mycobacteria remain -

ing in the phagosomes. If so, hemolysins could limit the

NO production, and by this, they could limit the bacte -

ricidal capacity of macrophages and eradication of the

bacilli in the infected host. On the other hand, the over -

production of TNF - ƒ¿ by macrophages infected with

hemolytic mycobacteria could intensify the

immunopathological processes in mycobacterial infec -

tions. Such inhibitors of TNF - ƒ¿ synthesis, as thalido -

mide (57) and pentoxiphylline (8), diminished the patho -

logical effects of mycobacteria and listeria, respectively .

Financial support for this work was provided by the Polish Research Commitee (KBN No. 4PO5D 037 12, No. 4PO5D 060 16) .

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