FEMS Microbiology Immunology 89 (1992) 91-96 © 1992 Federation of European Microbiological Societies 0920-8534/92/$05.00 Published by Elsevier

91

FEMSIM 00192

T N F - a failed to reverse the M. leprae-induced defective chemiluminescence response of human mononuclear cells Pascal Launois, M ' B a y a n g Niang, A l i o u n e Dieye and Jean-L oui s Sart hou lmmunologie cellulaire, Institut Pasteur de Dakar, Senegal Received 12 July 1991 Revision received 1 October 1991 Accepted 3 October 1991

Key words: TNF-a; Chemiluminescence; Respiratory burst; Mycobacterium leprae; Leprosy 1. SUMMARY The effect of phagocyte activation by TNF-a on the ability to trigger a chemiluminescence (CL) response, associated with the release of oxidizing species was evaluated in healthy human mononuclear cells in the presence of Mycobacterium leprae. Recombinant TNF-a (r-TNF-a) increased the CL response of unstimulated M. boris BCG- and PMA-stimulated cells but did not reverse the M. leprae defective activation of the human phagocyte oxidative burst. M. leprae was less well phagocytosed than M. boris BCG but phagocytosis of mycobacteria was not altered by addition of r-TNF-a. The failure of activation of oxygen-free radical production might have some relevance to the pathogenesis of leprosy.

2. INTRODUCTION Tumor Necrosis Factor-a (TNF-a) is a multipotent cytokine mainly secreted by activated

Correspondence to: P. Launois, Immunologie cellulaire, Institut Pasteur de Dakar, BP 220, Dakar, Senegal.

monocytes/macrophages. TNF-a may play a crucial role in host defense mechanisms against bacteria and parasites [1-5]. Its participation in resistance to some mycobacterial infections has been described in a mouse model [6,7] and in man [8,9]. In leprosy, a chronic disease caused by Mycobacterium leprae, the role of TNF-a in conferring resistance to M. leprae has been disputed. Indeed, in sera from lepromatous leprosy patients who failed to develop efficient cell-mediated immunity towards M. leprae, high [10,11] or normal titres [12] of TNF-a have been demonstrated. The presence of inhibitors of biological properties of TNF-a has been suggested [13]. Recently, T N F - a has been implicated in the immunopathology of leprosy particularly in reactional states [14]. However, no correlation between levels of TNF-a in sera and any particular clinical symptomatology has been shown. A study of the functional response of monocytes/macrophages to r-TNF-a in vitro would clearly be of interest. As TNF-a enhances the respiratory burst of mononuclear cells, we studied in healthy subjects the activation by r-TNF-a of peripheral monocytes infected with M. boris BCG and M. leprae by measuring the native

92 chemiluminescence (CL) associated with the release of oxygen free radicals.

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

3.1. Cell isolation Peripheral blood was obtained from healthy adults attending the Pasteur Institute of D a k a r by venepuncture using heparinised ' V a c u t a i n e r ' tubes ( B e c t o n - D i c k i n s o n , R u t h e r f o r d , N J). Mononuclear cells were isolated on a Ficoll-Hypaque gradient (Sigma, St Louis, MO). Percentages of lymphocytes and monocytes were evaluated on May-Griinwald Giemsa smears.

3.2. Mycobacteria Irradiated M. leprae (batch CD 128), extracted from armadillo tissues, was provided by Dr R.J.W. Rees, Mill Hill, U.K. through the W H O Immunology of Leprosy Programme. M. boris BCG IP D a k a r was obtained in lyophilised samples from the Service du BCG (Institut Pasteur, Dakar). Viable M. boris B C G IP Dakar was killed by heating for 120 min at 70 o C.

3.3. Reagents L u m i n o l (5-amino-2,3,-dihydro- 1,4-phthalazinedione) (Sigma, St Louis, MO) was dissolved in dimethylsulphoxide (DMSO; Sigma) and used at a final concentration of 10 - 4 M in phosphatebuffered saline solution. Phorbol myristate acetate (PMA, Prolabo, Paris, France) was used at 1.5 ~ g / m l . F I T C (fluorescein isothiocyanate)was purchased from Sigma. R e c o m b i n a n t - T N F - a (rT N F - a ) was purchased from A m e r s h a m (Buckinghamshire, U.K.).

at 37 ° C under constant agitation and 200 /zl of luminol solution was subsequently added. Then it was immediately introduced into a p h o t o m e t e r counting chamber (A = 425 nm). A m e a s u r e m e n t mode was available for continuous recording during 150 min. Results were expressed as light intensity in mV at the peak of the course for 104 monocytes.

3.5. Mycobacterial phagocytosis assay 1 mg of heat-inactivated BCG and M. leprae were conjugated by suspending in 1 ml of carbonate-bicarbonate buffer (pH 9.5) with 0.05% Tween 80 and 0.03% F I T C for 60 min at room temperature as previously described [15]. The bacteria were pelleted by centrifugation and resuspended in Hanks' solution without Phenol red and then sonicated 30 s to disrupt clumps. Aliquots of 100 /xg of bacteria were frozen at - 2 0 ° C until use. Phagocytosis was measured by incubating 1 ml of a suspension of mononuclear cells adjusted to 105 m o n o c y t e s / m l with bacteria at a ratio of 50 per cell. After gentle agitation for 4 h at 37 ° C, the tubes were centrifuged twice at 300 x g and supernatants were discarded. The pellets were resuspended in 200 /zl of Hanks' solution and examined with a Leitz fluorescence microscope with a 100 x water immersion lens. Three to four hundred cells were counted and results are expressed as percentage of cells with associated mycobacteria.

3.6. Statistics Student's t-test was used to compare the different experimental groups.

4. R E S U L T S

3.4. Measurement of CL We used an automatic thermostated photometer and dispenser (1251 Luminometer and 1291 dispenser, L B K Wallac, Turku, Finland) for measuring CL. Briefly, 100 /xl of a suspension of mononuclear cells adjusted to 10 s monocytes per ml was transferred into a counting tube previously filled with 1 ml of a suspension of bacteria diluted in phosphate-buffered solution to a ratio of 50 per cell. The tube was placed in a dry bath

4.1. Effects of r-TNF-a on BCG and M. leprae-induced chemiluminescence response of mononuclear cells First we analysed the optimal conditions for r-TNF-a to stimulate a maximal response in the chemiluminescence (CL) assay in the presence of BCG or M. leprae. Normal mononuclear cells were stimulated with varying concentrations of r - T N F - a in the absence or presence of killed

93 Table 1

Table 2

Chemiluminescence response to r-TNF-a and mycobacteria of mononuclear cells from healthy subjects

Phagocytosis of mycobacteria by monocytes from healthy subjects

Stimuli

Lymphokine

CL response a

Bacteria

Lymphokine

None None

None r-TNF-a None r-TNF-a None r-TNF-a None r-TNF-a

5.52 + 8.54+ 6.06 + 7.80 + 23.2 + 39.9 + 55.95+ 66.86+

% Monocytes associated with mycobacteria

BCG BCG

None r-TNF-a None r-TNF-ot

77.75_+ 5.85 a 72.58 +_ 10.35 59.90_+ 8.77 b 61.06_+ 9.31 b

M. leprae M. leprae BCG BCG PMA PMA

1.94 2.94 0.59 2.68 5.09 10.47 3.47 8.25

c

M. leprae M. leprae b,c c b,c

a Mean_+ SD (n = 5). b Significantly different from BCG ( P < 0.05, unpaired t-test).

a m V / 1 0 4 monocytes (Mean ± S D , n = 5). b Significantly different from control without bacillus ( P < 0.01, unpaired t-test). c Significantly different from control without lymphokines ( P < 0.05, unpaired t-test).

tration of 100 I U / m l of r-TNF-a and a preincubation of 2 h. As previously described [16,17], M. leprae failed to trigger any response in mononuclear cells from healthy subjects. Conversely, M. boris BCG elicited a strong response (Fig. 1 and Table 1). r-TNF-ot increased the CL response of unstimulated M. boris BCG and PMA stimulated cells. However, the presence of M. leprae did not influence the r-TNF-a CL response. Fig. 2 shows a representative mycobacteria-stimulated CL assay of monocytes from a healthy subject in the presence of tumor necrosis factor over time.

mycobacteria. Dose response curve analysis indicated that maximal response to 10 /xg/ml of mycobacteria was obtained with 100 I U / m l of r-TNF-a (data not shown). Preincubation of mononuclear cells with r-TNF-a for 2 h at 37 ° C increased the CL response compared to that obtained when r-TNF-a and mycobacteria were introduced simultaneously (Fig. 1). Thus, further studies were performed with a constant concen60

50

40

¢ E

[]

None

[]

TNF BCG + TNF

30

M. leprae M. leprae + TNF

20

l°r

¢ ¢ ¢

o

2

16

time of preincubation (in hours) Fig. 1. Effect of preincubation of mononuclear cells in the presence of 100 I U / m l of r-TNF-~ on unstimulated or mycobacteria and P M A stimulated CL response from five healthy subjects. Values are expressed in m V / 1 0 4 monocytes (SD did not exceed 15%).

94 50

40 [] • A •

30

E

BCG BCG+TNF M. leprae M. leprae+TNF

20

10

0 0

I

I

I

I

I

30

60

90

120

150

rain Fig. 2. A representative mycobacteria stimulated-CL profile of monocytes from a healthy subject in the presence of 100 I U / m l of r-TNF-a. Values are expressed in mV/104 monocytes.

4.2. Effects of r-TNF-a on mycobacteria phagocytosis Percentage of monocytes in mononuclear cells suspension was evaluated on May Griinwald Giemsa smears. It was 17.9 + 3.6 (mean + SD, n = 5). Table 2 shows that the number of FITC-M. leprae associated with monocytes was significantly lower than that of FITC-conjugated BCG ( P < 0.05, unpaired t-test). Addition of r-TNF-o~ neither increased nor decreased phagocytosis of mycobacteria.

5. DISCUSSION TNF-a has been shown to activate human as well as murine phagocytic cells to inhibit the growth of intracellular pathogens [3-5,7]. However, regarding mycobacteria, discordant results have been reported. Indeed, T N F - a has bacteriostatic properties against M. avium [8], but it either reduced [18] or did not influence [19] the in vitro growth of M. tuberculosis within human monocytes/macrophages. In mouse macrophages TNF-a reduced in vitro growth of M. lepraemurium [7] but not the growth of M. tuberculosis [20]. In mice resistant to M. boris BCG, the formation of the granuloma was mediated by T N F - a secretion [6]. The role of T N F - a in anti-M. leprae bactericidal mechanisms is poorly under-

stood. It has been shown that in leprosy very high titres of TNF-a in sera correlate with an absence of function T cell response [10,11]. However, in vitro cultures of mononuclear cells from lepromatous leprosy patients did not produce TNF-a upon stimulation with endotoxin [12]. Phagocytes contain a variety of antimicrobial systems that use toxic oxygen metabolites formed by a phagocytosis-induced respiratory burst [21]. Mycobacteria such as M. leprae, M. boris BCG and M. tuberculosis are sensitive to oxygen metabolites in vitro [22], but it is not known if these components are effective in phagocytes. Indeed, Flesh and Kaufmann [23] have shown that killing of M. boris or M. tuberculosis in mouse macrophages was oxygen-independent and recently reactive nitrogen intermediates have been implicated in antimycobacterial activity towards M. avium [9] and M. leprae [24]. However, Mariola et al. demonstrated that M. leprae was sensitive in vitro to reactive oxygen metabolites in normal human macrophages [25]. As TNF-c~ might enhance the respiratory burst from mononuclear cells we investigated the capacity of r-TNF-a to induce a CL response from mononuclear cells in the presence of M. leprae, r-TNF-a could increase the M. bouis BCG but not the M. lepraeinduced CL response. M. leprae has been previously described to fail to produce oxygen-free radicals [16,17]. Phenolic glycolipid-1 unique to

95 M. leprae [26] and l i p o a r a b i n o m a n n a n [27] acting as a scavenger of reactive oxygen species, could explain in part this p h e n o m e n o n . PGL-1 inhibited the oxygen free radical p r o d u c t i o n triggered by various species of mycobacteria as well as P M A [28]. Moreover, H o l z e r et al. [15] have previously described that addition of M. leprae at high c o n c e n t r a t i o n slightly decreased the oxygen species p r o d u c t i o n of h u m a n m a c r o p h a g e s to BCG. In our hands, M. leprae, even at high ratio, decreased very slightly the C L response o f B C G and P M A (data not shown). T h e failure of M. leprae to trigger an efficient C L response of m o n o n u c l e a r ceils could also result from defective phagocytosis of the bacilli. Indeed, phagocytosis of M. leprae by h u m a n phagocytes was lower than B C G and was not altered by addition of r - T N F - a . In the present work, phagocytosis experiments were p e r f o r m e d without presence of antibody or complement. As no electron microscopic observations were available, we have really no evidence that phagocytosis rather than a t t a c h m e n t occurred. However, using electron microscopy it was previously shown that M. tuberculosis and M. leprae were effectively ingested by h u m a n m a c r o p h a g e s after 150 min of incubation [29,30]. T h u s we considered that in our phagocytosis assay, after 240 min of gentle agitation, the majority of the bacteria was internalised. In the present study we d e m o n s t r a t e that the defective activation of the phagocyte oxidative burst by M. leprae can not be reversed by r - T N F - a . B e r m u d e z et al. [31] had previously suggested that virulent strains of M. avium blocked the ability of m a c r o p h a g e s to r e s p o n d to activation to T N F - a . It is interesting to note that in n u d e mice, M. leprae-burdened m a c r o p h a g e s are refractory to activation by g a m m a - i n t e r f e r o n [32]. I n d e e d l i p o a r a b i n o m a n n a n has b e e n recently r e p o r t e d to inhibit the protein kinase activity, k n o w n to have a regulatory role in m a c r o p h a g e activation, and to be a p o t e n t inhibitor of the transcriptional activation of IFN-~/-inducible genes [27]. T h e r e fore, inhibition of cytokine-induced m a c r o p h a g e activation by virulent strains of mycobacteria could contribute to the pathogenesis of mycobacterial infections. I n d e e d in leprosy, M. leprae

might grow m o r e easily in defective cytokineactivated phagocytic cells.

ACKNOWLEDGEMENTS This work was supported in part by a grant from the Association R a o u l Follereau, Paris. T h e technical assistance of B. D i o u f is gratefully acknowledged.

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