- Email: [email protected]
Intracellular signals triggered during association of Mycobacterium leprae and Mycobacterium bovis BCG with human monocytes
Article available online at http://www.idealibrary.com on
Microbial Pathogenesis 2001; 31: 37–45 doi:10.1006/mpat.2001.0444
MICROBIAL PATHOGENESIS
SHORT COMMUNICATION
Intracellular signals triggered during association of Mycobacterium leprae and Mycobacterium bovis BCG with human monocytes C. S. Lima, M. L. Ribeiro, L. A. Souza, A. B. Sardella, V. M. A. Wolf & M. C. V. Pessolani∗ Laborato´rio de Hansenı´ase, Instituto Oswaldo Cruz, Fundac¸a˜o Oswaldo Cruz (FIOCRUZ), Rio de Janeiro, RJ 21045-000, Brazil (Received January 17, 2001; accepted in revised form April 2, 2001)
To gain a better understanding of mycobacteria–host cell interaction, the present study compared the signal transduction events triggered during the interaction of Mycobacterium leprae (the causative agent of leprosy) and of Mycobacterium bovis BCG (an attenuated strain used as a vaccine against leprosy and tuberculosis) with human monocytes. The assays consisted of pretreating or not THP-1 cells (a human monocytic cell line) with different kinase inhibitors, followed by incubation with fluorescein-labelled bacteria and analysis of bacterial association via fluorescence microscopy. The specific tyrosine kinase (TK) inhibitor tyrphostin AG126 provided the highest rates of association inhibition (>90% for BCG and >65% for M. leprae). The early activation of TKs during mycobacteria–host cell interaction was confirmed by immunoblot analysis, demonstrating that in several host cell proteins mycobacteria stimulated tyrosine phosphorylation. The use of the drugs wortmannin and bisindolylmaleimide I which, respectively, inhibit phosphatidylinositide 3kinase (PI 3-kinase) and protein kinase C (PKC), produced lower but consistent results within a 35–60% association inhibition range for both bacteria. Dose response curves with these inhibitors were obtained. Similar results were obtained when primary human monocytes were used as host cells, strongly suggesting that TK, PKC and PI 3-kinase signals are activated during the interaction of human monocytes with both pathogenic and attenuated species of mycobacteria. 2001 Academic Press
Key words: Mycobacterium leprae, adherence, phagocytosis, human monocytes, signalling pathways, Mycobacterium bovis BCG.
∗ Author for correspondence. E-mail: [email protected]. fiocruz.br 0882–4010/01/070037+09 $35.00/0
2001 Academic Press
38
Introduction The knowledge accumulated to date in the field of microbial pathogenesis, in particular those aspects regarding the molecular mechanisms involved in microbial adherence to host cells, bacterial uptake and pathogen survival and replication inside mammalian cells, has increased dramatically in recent years and has pointed to the adoption of new strategies in the treatment of infectious diseases. In this regard, the interaction of the invading bacteria with the host cell has been shown to be very dynamic and characterized by the occurrence of an intense cross talk interplay between these cells. Upon the binding/internalization of bacterial pathogens, a multiplicity of signals are generated inside the host cell, leading to cytoskeletal reorganization and bacterial internalization and to the regulation of parallel events that often accompany phagocytosis, namely, cytokine release and the activation of microbicidal mechanisms. Since this interference in host cell metabolism ultimately favours the perpetuation of these bacterial pathogens inside the host, great efforts have been made to elucidate the pathways of phagocytic signal transduction generated at the moment of microbial internalization. The data accumulated in the literature so far has pointed to tyrosine phosphorylation as a common signal of bacterial internalization within mammalian cells. Actually, both opsonin-mediated and opsonin-independent phagocytoses in human phagocytes seem to be controlled by tyrosine kinases (TKs). Moreover, tyrosine phosphorylation accompanies binding and/or invasion of several pathogenic bacteria by human ‘‘nonprofessional’’ phagocytes. In addition to tyrosine phosphorylation in phagocytosis, an involvement has been established on the part of other cellular signalling elements, such as members of the superfamily of monomeric GPTases and of the protein kinase C (PKC) family, and more recently of phosphatidylinositide phosphate 3kinase (PI 3-K) [1–4]. Leprosy, a chronic infectious disease caused by the obligate intracellular bacterium Mycobacterium leprae, remains a major health care problem in several developing countries [5]. The capacity of M. leprae and other pathogenic mycobacteria like M. tuberculosis to successfully parasitize monocytes and macrophages appears to play an essential role in mycobacterial pathogenesis [6]. It is well known that monocytes and
C. S. Lima et al.
macrophages internalize mycobacteria by means of conventional phagocytosis, which in either the presence or absence of serum is predominantly mediated by the complement receptors (CR)1, CR3 and CR4 [7–9]. Additionally, experiments comparing attenuated vs virulent strains and live vs dead bacteria have revealed critical differences of both the final bacterial fate inside the cell as well as of the effects on host cell functions, which points to differences in the signal pathways generated early on during these interactions [10–13]. However, only limited information is available about the transmembrane signalling mechanisms responsible for regulating the phagocytosis of mycobacteria. A previous study by the present authors compared the level of TNF- secretion induced in THP-1 cell (a human monocytic cell line) after phagocytosis of M. leprae and Mycobacterium bovis BCG, which is an attenuated strain used as a vaccine against leprosy and tuberculosis (Oliveira MM, Charlab R, Pessolani MCV, unpublished data). The results demonstrated that BCG, but not M. leprae, induces TNF- secretion in THP-1 cells, thereby emphasizing the potential this in vitro model has to explore differences in signalling pathways triggered by vaccine vs the pathogenic species of mycobacteria. To continue investigating the matter, this study focused on investigating the signalling events that follow the encounter of human monocytes with M. leprae and comparing them with the events triggered upon incubation of these cells with BCG.
Results Kinase inhibitors decrease the degree of association of M. leprae and BCG with human monocytic cells To determine the precise involvement of TKs, PKC and PI 3-K during interaction between human monocytic cells and M. leprae and BCG, THP-1 cells were treated with specific inhibitors of these kinases prior to incubation with mycobacteria. Fluorescein-labelled bacteria were then added to the cells. Two h later, the phagocytosis mixture was stained with EB, and the preparations were observed under a fluorescent microscope. This fluorescent method was originally reported for its ability to distinguish between adherent and intracellular Listeria in
Signalling pathways during internalization of M. leprae
39
(a)
(b) M. leprae
Inhibition of control (%)
100
BCG
100
80
80
60
60
40
40
20
20
0
0 lm oly ind
Bis im
ale
im ale
in
nn
a rtm Wo
in ost
lm oly ind
rph
Ty
Bis
in
nn
a rtm Wo
in ost
rph
Ty
ide
ide
Figure 1. Kinase inhibitors block mycobacteria association to THP-1 cells. (a) Effect of tyrphostin on the association of M. leprae, as seen by fluorescence microscopy. Bacteria were pre-labelled with fluorescein and then added to the cells at a ratio of approximately 100 bacteria to one cell. After 2 h of incubation at 37°C, cells were washed and stained with EB for analysis under a fluorescent microscope. Representative M. leprae infected cultures in the presence or not of tyrphostin is shown (×800). (b) The inhibition of the degree of association of BCG and M. leprae with THP-1 cells was quantified by counting the number of cells binding bacteria in at least 100 cells. Results are expressed as the percentage of decrease of control cultures. Wortmannin and bisindolylmaleimide were added 20 min prior to infection. Tyrphostin was added 1 h prior to infecting the cells with mycobacteria. Data represent the mean±SEM of three to four experiments carried out in duplicate.
that, in this protocol, intracellular bacteria retain their green colouration, while the fluorescence of the extracellular bacteria changes to a reddish–
orange colour due to their EN staining [14]. However, in this study’s preparations, most mycobacteria, including the unbound, extracellular
C. S. Lima et al.
Inhibition of control (%)
40 100
50
80
40
60
30
40
20
20
10
0
1
10 100 Tyrphostin (µM)
0
10 100 Wortmannin (nM)
Figure 2. Tyrphostin and wortmannin inhibit BCG association with human blood-derived monocytes in a dose dependent manner. Monolayers enriched in monocytes were prepared from PBMC of healthy individuals. Bacterial association assays were then performed as described for THP-1 cells. Results are reported as the mean±SEM of three experiments carried out in duplicate.
bacteria, remained green in colour, which made it impossible to precisely quantify either the adherent or intracellular bacteria. Thus, in this study, the quantification of monocyte-associated (adherent and ingested) bacteria was performed. M. leprae and BCG were able to associate efficiently and in equal measure with control THP1 cells, and between 35–45% of all cells had at least one mycobacterium associated to them. When tyrphostin, a specific TK inhibitor, was added to the cells, a pronounced decrease in the number of cells binding mycobacteria was observed. The cultures shown in Fig. 1(a) are representative of THP-1 cells pre-incubated or not with tyrphostin and then treated with M. leprae. The effect of these inhibitors was quantified by determining the percentage of cells binding bacteria, expressed as a percentage of decrease from control cultures [Fig. 1(b)]. The results obtained were similar for both mycobacteria used. However, lower overall inhibition levels were obtained with the M. leprae treated cells. Tyrphostin was the most effective suppressor drug of M. leprae and BCG association with monocytes by up to 67.4±5.0% and 91.6±2.8%, respectively [Fig. 1(b)]. Treating cells with wortmannin and bisindolylmaleimide generated lower, though consistent, inhibition levels. Wortmannin, a specific inhibitor of the lipid kinase PI 3-K, blocked M. leprae and BCG association with THP-1 by 44.7±7.0% and 59.1±6.8%, respectively. Bisindolylmaleimide, a PKC inhibitor, evoked a decrease in M. leprae and BCG association of 34.0±2.5% and 44.2±6.0%, respectively. The results shown in Fig. 2 are representative
of blood-derived monocytes infected with BCG. Similarly to THP-1, tyrphostin was more effective than wortmannin in decreasing adherence/phagocytosis. However, both compounds blocked bacterial association in a dosedependent manner. The results obtained with primary cells were similar to those seen with THP-1. Tyrphostin at concentrations 1, 10 and 100 M inhibited association of BCG in 22.0±2.0%, 36.9±4.6% and 84.3±1.2%, respectively, while wortmannin at concentrations of 10 and 100 nM blocked bacterial binding/phagocytosis in 30.0±4.6% and 43.0±2.1%, respectively. Exposure to the kinase inhibitors at any of the different concentration levels used had no effect on monocyte viability. Similar results were also obtained when comparing M. leprae-infected primary monocytes and THP-1 cells (data not shown).
M. leprae and BCG stimulated tyrosine phosphorylation in human monocytic cells To further demonstrate the involvement of TK during mycobacteria interaction with human macrophages, immunoblot analysis of phosphotyrosine proteins in total cell lysates of control and treated THP-1 cells was performed. Typical results for these experiments are shown in Fig. 3. Incubation of THP-1 cells with M. leprae and BCG caused increased tyrosine phosphorylation of several host cell proteins, while no differences in the tyrosine phosphorylation pattern between cells treated with M. leprae and BCG were observed. Tyrosine phosphorylation
20
60
+
10
B
C
G
(6
0
m
in
)
+ M (m . le in pr ) a
FB %
r. au
10 +
st +
C
kDa
on
tr
ol
S
e
Signalling pathways during internalization of M. leprae
98
67
36
Figure 3. Detection of phosphotyrosine proteins in THP-1 cells treated with M. leprae and BCG. Serum starved cells were treated with staurosporin (20 min), 10% FBS (20 min) or mycobacteria at times indicated. Cell lysates were then prepared, resolved by SDS 8.5% PAGE, and immunoblotted with mouse antiphosphotyrosine antibody (4G10). The secondary antibody used was a goat anti-mouse IgG-peroxidase conjugate. Membranes were developed using a chemiluminescent substrate. The values on the left are molecular weights.
was monitored during a consecutive 60 min period, and an increase in tyrosine phosphorylation was detected as soon as 5 min after treatment with mycobacteria. Cells treated with staurosporine, a broad-spectrum protein kinase inhibitor, lacked most of the phosphoproteins found in unstimulated cells, whereas cells stimulated with 10% FBS showed a pronounced increase in the tyrosine phosphorylation of several protein bands, similarly to what was seen in cells treated with mycobacteria.
Discussion Interaction of bacterial pathogens with their host cells triggers signal transduction pathways that, in turn, lead to a variety of cellular responses which ultimately favour their perpetuation in the host. Among these responses are those
41
giving rise to an extensive reorganization of the cytoskeleton, which results in morphological changes, and the secretion of cytokines into the medium. Although this interference in host cell metabolism by bacteria represents a central feature of their pathogenesis, these events are poorly understood in mononuclear phagocytes infected with mycobacteria and have only recently received attention of a few investigators [15–17]. In the present study, the stimulation of three classes of kinases (TKs, PKCs and PI 3Ks) during interaction of M. leprae with human monocytes has been investigated. The effects evoked by M. leprae were compared with those by BCG. This study has demonstrated that tyrosine phosphorylation represents a major signal generated during the association of M. leprae and BCG with human monocytes. This conclusion was based on the inhibition studies showing that the association of M. leprae and BCG is blocked by over 65% by tyrphostin, a specific TK inhibitor, and the immunoblot analysis showing that mycobacteria stimulated tyrosine phosphorylation of several host cell proteins. Besides TKs, inhibition assays also suggested that PKC and PI 3-K are activated during the encounter of mycobacteria with human monocytes. Similar results were obtained when THP-1 cells and/or primary human monocytes were used as host cells. However, no qualitative differences in the signals triggered by either M. leprae or BCG were detected. The association of M. leprae and BCG with the human monocytic cells employed here is probably mediated by the complement receptors expressed on the surface of these cells. Although other receptors may also participate [18–20], it has been demonstrated that the complement receptors, and especially CR3, are the most important ones involved in the uptake by human monocytes of both pathogenic and non-pathogenic mycobacteria [7–9]. In spite of the fact that THP-1 cells are immature monocytes, they have been shown to express CR1 and CR3, although at lower levels than monocytes [21]. Likewise, the similar results obtained here with both the in vitro models support the hypothesis of identical receptors mediating the uptake of mycobacteria by THP-1 and primary monocytes, regardless of their degree of differentiation. These results reinforce previous data that indicate THP-1 as being a useful model to investigate the nature of mycobacteria/monocyte interactions [22]. The activation of TKs during the binding/ phagocytosis of M. leprae and BCG is not a
42
surprising finding. Data have regularly been reported in the literature pointing to tyrosine phosphorylation as a common signal for bacterial internalization into mammalian cells [1–4]. Furthermore, previous studies have shown that, in macrophages under the influence of TK inhibitors, the phagocytosis mediated by CR3 decreased, indicating that the tyrosine phosphorylation of proteins is also required for the efficient internalization of complement-opsonized particle [2]. In this context, it has been recently shown that TKs are involved in the activation of phospholipase D, which plays a key role in the CR3-mediated phagocytosis of either Erdman or H37Ra strains of M. tuberculosis by human macrophages [15]. In the same study, prominent tyrosine phosphorylation was noted in the proteins of >150, 95, 72, 56 and 42 kDa induced by M. tuberculosis, a finding resembling those observed in M. leprae and BCG-treated THP-1 cells (Fig. 3). The involvement of TKs and PKC has also been demonstrated in CR3mediated phagocytosis of Legionella pneumophila by human monocytes [23]. The same study also showed that the CR-3 mediated entry of either virulent or avirulent strains as well as of zymosan and Escherichia coli caused the tyrosine phosphorylation of similar proteins and was not a virulence-associated event. This is in agreement with the results of the present study, in which no differences in the signals triggered by M. leprae or BCG were observed. The results are also in agreement with a recent study showing the involvement of TKs during the uptake of M. leprae by murine macrophages [17]. The partial inhibition of BCG and M. leprae association with human monocytes by bisindolylmaleimide and wortmannin also suggests the activation of PKC and PI 3-K, respectively, during interaction of mycobacteria with human monocytes. These results are in agreement with recent findings indicating the involvement of PKC in CR3-mediated phagocytosis [2, 23]. With respect to PI 3-K, this kinase belongs to a family of enzymes that phosphorylates the 3′-hydroxyl group on the inositol ring of phosphoinositides [24]. These phospholipids are involved in a large array of signal transduction pathways controlling mitogenic responses, differentiation, apoptosis, cytoskeletal organization and membrane flow along the secretery and endocytic pathways [25]. A growing body of evidence has implicated this family of lipid kinases in receptor-mediated phagocytosis as well as in the bacterial invasion of nonphagocytic cells [2, 26].
C. S. Lima et al.
Precisely how PI 3-K participates in phagocytosis is still not known, but a recent study has suggested that it is involved in co-ordinating exocytic membrane insertion and pseudopod extension [27]. In conclusion, in the present study, three classes of signal transduction elements, namely, TKs, PKC and PI 3-K, have been shown to be activated as a result of interaction between M. leprae and human monocytes. These signals probably represent universal signals generated by human monocytes early on during phagocytosis and are not associated with bacterial virulence. A strong possibility is that these kinases control cytoskeleton reorganization, pseudopod extension and phagosomal closure that are fundamental events accompanying phagocytosis. The molecular mechanisms responsible for the different effects that virulent and avirulent mycobacteria exert on host cell functions most certainly require a more detailed dissection of those signalling pathways activated at the moment of phagocytosis.
Materials and Methods Source of reagents RPMI 1640 medium, penicillin/streptomycin and glutamax-I were obtained from Gibco BRL (Rocksville, MD, U.S.A.). Phorbol-12-myristate13-acetate (PMA), staurosporin and tyrphostin AG 126 were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Wortmannin and bisindolylmaleimide were purchased from Calbiochem (San Diego, CA, U.S.A.). Middlebrook 7H9 broth was purchased from Difco Laboratories (Detroit, MI, U.S.A.). Anti-phosphotyrosine monoclonal antibody (4G10) was obtained from Upstate Biotechnology Inc. (Lake Placid, NY, U.S.A.).
Bacteria Armadillo-derived M. leprae was kindly donated by Dr Patrick Brennan (Colorado State University, Fort Collins) through NIAID contract NO155262. M. bovis BCG Pasteur 1173P2 WHO was grown at 37°C in Middlebrook 7H9 broth supplemented with 0.05% Tween 80, under constant agitation on a magnetic plate. Cultures were harvested in mid-log phase, aliquotted in
Signalling pathways during internalization of M. leprae
43
Eppendorf tubes and kept frozen at −80°C until use.
(100 nM) were added to the corresponding wells 20 min before the infection, and tyrphostin AG126 (100 M) was added 1 h prior to the addition of mycobacteria to the cells. Treatment with kinase inhibitors was omitted in the control wells and all of the assays were carried out in duplicate wells. Association assays were performed using a fluorescence method to distinguish between internalized and adherent bacteria [14]. BCG Pasteur and M. leprae were pre-labelled with fluorescein isothiocyanate (FITC) at a concentration of 0.1 mg/ml for 1 h at room temperature. The cells received enough mycobacteria to achieve a ratio of approximately 100 bacteria to 1 cell. The plates were then centrifuged at 500×g for 10 min and were returned to the incubator at 37°C with 5% CO2. After 2 h, the cultures were removed from the wells and cells were recovered by centrifugation at 325×g for 5 min. Cells were resuspended in 1 ml of PBS and washed four more times for the removal of unbound extracellular bacteria. The last pellet was resuspended in 100 l of PBS followed by staining with ethidium bromide (EB) at a final concentration of 50 g/ml for 10 min. The cells were then citospun at 30×g for 10 min and the glass slides fixed with cold methanol for 1 min. The glass slides were mounted with 90% glycerol and 10% PBS 10× containing 1 mg/ml p-phenylenediamine. For the blood-derived monocytes, at 2 h the wells were washed five times with PBS at room temperature followed by staining with EB for 10 min and fixation with cold methanol for 1 min. The glass coverslips were then removed from each well and mounted on glass slides with entellan glue and 90% glycerol and 10% PBS 10× containing 1 mg/ml p-phenylenediamine. The glass slides were viewed using a Zeiss fluorescent microscope and the percentage of cells having at least one mycobacterium associated with them was determined by counting a minimum of 100 cells for each glass slide. The cells were tested for viability by being stained with a solution of EB (4 g/ml) and fluorescein diacetate (20 g/ml) for 10 min at room temperature and at a ratio of 2:1 (cell culture: staining solution).
THP-1 cells The THP-1 monocytic cell line was obtained from the American Type Culture Collection (ATCC) (Rockville, MD, U.S.A.). The cells were propagated by means of weekly passages in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 10 mM HEPES, 2 mM glutamax-I, 100 U/ml penicillin, 100 g/ml streptomycin and incubated at 37°C with 5% CO2. Before being plated, the cell culture was centrifuged at 500×g for 10 min and the pellet was resuspended in a phosphate buffered saline (PBS) solution (10 mM phosphate buffer pH 7.2, 0.15 M NaCl). This wash was repeated twice more and the last pellet was resuspended in fresh supplemented RPMI. The cells were counted and their concentration was adjusted to 5×105 cells in 0.5 ml. Each well of a 24 well microtitre plate then received 0.5 ml of the cell culture and the plate returned to the incubator at 37°C and 5% CO2.
Human blood-derived monocytes Normal human peripheral blood mononuclear cells (PBMC) were separated by Ficoll–Hypaque density gradients from sterile heparinized blood. The cells from the interface were washed three times in PBS supplemented with 1% fetal bovine serum (FBS) with no calcium or magnesium. They were seeded at a density of 1×106/well/ 1 ml of RPMI supplemented with 10% FBS, 100 U/ml penicillin, 100 g/ml streptomycin and 2 mM/l glutamine, in 24 well microtitre plates containing glass coverslips. The cells were incubated at 37°C with 5% CO2 and allowed to settle for 24 h. Then, the non-adherent cells were removed with three washes of PBS at room temperature. To the adherent cells, at about 2×105/well, 0.5 ml of fresh supplemented RPMI was added before the infection and the inhibition assays.
Association assay Before the addition of mycobacteria, the cells were treated with protein kinase inhibitors. Bisindolylmaleimide (1 M) and wortmannin
Immunoblotting analysis of cellular tyrosine phosphorylation For the analysis of global tyrosine phosphorylation, THP-1 cells (1.5×106 cells) were
44
incubated in serum-free RPMI medium. The next day cultures were treated or not with M. leprae and BCG for 0–60 min. Cells were then washed twice with PBS solution and lysed in ice-cold RIPA buffer containing a cocktail of protease and phosphatase inhibitors (50 mM Tris buffer, 1% Nodinet P40, 0.25% sodium deoxycolate, 0.1% SDS, 1 mM EGTA, 1 mM NaF, 1 mM sodium orthovanadate, 1 mM phenylmethylsulphonylfluoride (PMSF), 10 g/ml leupeptin, 10 g/ml aprotinin and 2 g/ml pepstatin). Lysates were centrifuged in microcentrifuge at the maximum speed for 20 min at 4°C, and protein assay was estimated in the supernatants by BCA kit (Pierce, Rockford, IL, U.S.A.). Equal amounts of proteins (100 g protein) were resolved on 8.5% SDS-PAGE [28], followed by transfer to nitrocellulose membranes [29] and blots were blocked with 3% BSA TBS/T for 2 h/RT. Membranes were incubated with 4G10 antiphosphotyrosine monoclonal antibody in TBS/ T-3% BSA for 2 h at room temperature. Immunoblots were developed by incubation with a goat anti-mouse horseradish proxidase-conjugated antibody (Sigma Chemical Co.) and use of the chemiluminescent Supersignal Substrate (Pierce).
Acknowledgements This study was supported by The Heiser Program for Research in Leprosy and Tuberculosis, CNPq (Brazil), and PAPES/FIOCRUZ.
References 1 Finlay BB, Cossart P. Exploitation of mammalian host cell functions by pathogens. Science 1997; 276: 718–25. 2 Kwiatkowska K, Sobota A. Signaling pathways in phagocytosis. BioEssays 1999; 21: 422–31. 3 Ireton K, Cossart P. Interaction of invasive bacteria with host signaling pathways. Curr Op Cell Biol 1998; 10: 276–83. 4 Greenberg S. Modular components of phagocytosis. J Leukoc Biol 1999; 66: 712–7. 5 WHO. Global leprosy situation, September 1999. Wkly Epidemiol Rec 1999; 74: 313–6. 6 Fenton MJ, Vermeulen MW. Immunopathology of tuberculosis: roles of macrophages and monocytes. Infect Immun 1996; 64: 683–690. 7 Schlesinger LS, Horwitz MA. Phagocytosis of leprosy bacilli is mediated by complement receptors CR1 and CR3 on human monocytes and complement component C3 in serum. J Clin Invest 1990; 85: 1304–14.
C. S. Lima et al. 8 Schlesinger LS, Bellinger-Kawahara CG, Payne NR, Horwitz MA. Phagocytosis of Mycobacterium tuberculosis is mediated by human monocyte complement receptors and complement component C3. J Immunol 1990; 144: 2771–80. 9 Launois P, Niang M, Dieye A, Sarthou JL, Rivier F, Millan J. Human phagocyte respiratory burst by Mycobacterium bovis BCG and M. leprae: functional activation by BCG is mediated by complement and its receptors on monocytes. Int J Lepr Other Mycobact Dis 1992; 60: 225–33. 10 Holzer TJ, Kizlaitis L, Vachula M, Weaver CW, Andersen BR. Human phagocytic cell responses to Mycobacterium leprae and Mycobacterium bovis Bacillus CalmetteGue´rin. J Immunol 1988; 141: 1701–8. 11 Suzuki K, Fukutomi Y, Matsuoka M et al. Differential production of interleukin 1 (IL-1), IL-6, tumor necrosis factor, and IL-1 receptor antagonist by human monocytes stimulated with Mycobacterium leprae and M. bovis BCG. Int J Lepr Other Mycobact Dis 1993; 61: 609–18. 12 Barker LP, George KM, Falkow S, Small PLC. Differential trafficking of live and dead Mycobacterium marinum organisms in macrophages. Infect Immun 1997; 65: 1497–1504. 13 Ferrari G, Langen H, Naito M, Pieters J. A coat protein on phagosomes involved in the intracellular survival of Mycobacteria. Cell 1999; 97: 435–47. 14 Drevets DA, Campbell PA. Macrophage phagocytosis: use of fluorescence microscopy to distinguish between extracellular and intracellular bacteria. J Immunol Meth 1991; 142: 31–8. 15 Kusner DJ, Hall CF, Schlesinger LS. Activation of phospholipase D is tightly coupled to the phagocytosis of Mycobacterium tuberculosis or opsonized zymosan by human macrophages. J Exp Med 1996; 184: 585–85. 16 Malik ZA, Denning GM, Kusner DJ. Inhibition of Ca2+ signaling by Mycobacterium tuberculosis is associated with reduced phagosome–lysosome fusion and increased survival within human macrophages. J Exp Med 2000; 191: 287–302. 17 Prabhakaran K, Harris EB, Randhawa B. Regulation by protein kinase of phagocytosis of Mycobacterium leprae by macrophages. J Med Microbiol 2000; 49: 339–42. 18 Schlesinger LS. Macrophage phagocytosis of virulent but not attenuated strains of Mycobacterium tuberculosis is mediated by the mannose receptors in addition to complement receptors. J Immunol 1993; 150: 2920–30. 19 Bermudez LE, Young LS, Enkel H. Interaction of Mycobacterium avium complex with human macrophages: roles of membrane receptors and serum proteins. Infect Immun 1991; 59: 1697–702. 20 Rao SP, Ogata K, Catanzaro A. Mycobacterium avium–M. intracellulare binds to the integrin receptor alpha v beta 3 on human monocytes and monocyte-derived macrophages. Infect Immun 1993; 61: 663–70. 21 Thieblemont N, Haffner-Cavaillon N, Ledur A, L’AgeStehr J, Ziegler-Heitbrock HW, Kazatchkine MD. CR1 (CD35) and CR3 (CD11b/CD18) mediate infection of human monocytes and monocytic cell lines with complement-opsonized HIV independently of CD4. Clin Exp Immunol 1993; 92: 106–13. 22 Stokes RW, Doxsee D. The receptor-mediated uptake, survival, replication, and drug sensitivity of Mycobacterium tuberculosis within the macrophages-like cell
Signalling pathways during internalization of M. leprae
45
line. THP-1: a comparison with human monocyte-derived macrophages. Cell Immunol 1999; 197: 1–9. 23 Coxon PY, Summersgill JT, Ramirez JA, Miller RD. Signal transduction during Legionella pneumophila entry into human monocytes. Infec Immun 1998; 66: 2905–13. 24 Vanhaesebroeck B, Leevers SJ, Panayotou G, Waterfield MD. Phosphoinositide 3-kinases: a conserved family of signal transducers. Trends Biochem Sci 1997; 22: 267–72. 25 Martin TJF. Phosphoinositide lipids as signaling molecules: common themes for signal transduction, cytoskeletal regulation, and membrane trafficking. Annu Ver Cell Dev Biol 1998; 14: 231–64.
26 Ireton K, Payrastre B, Chap H et al. A role for phosphoinositide 3-kinase in bacterial invasion. Science 1996; 274: 780–2. 27 Cox D, Tseng C-C, Bjekic G, Greenberg S. A requirement for phosphatidylinositol 3-kinase in pseudopod extension. J Biol Chem 1999; 274: 1240–7. 28 Laemmli UK. Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680–5. 29 Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrilamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 1979; 76: 4350–4.
Microbial Pathogenesis 2001; 31: 37–45 doi:10.1006/mpat.2001.0444
MICROBIAL PATHOGENESIS
SHORT COMMUNICATION
Intracellular signals triggered during association of Mycobacterium leprae and Mycobacterium bovis BCG with human monocytes C. S. Lima, M. L. Ribeiro, L. A. Souza, A. B. Sardella, V. M. A. Wolf & M. C. V. Pessolani∗ Laborato´rio de Hansenı´ase, Instituto Oswaldo Cruz, Fundac¸a˜o Oswaldo Cruz (FIOCRUZ), Rio de Janeiro, RJ 21045-000, Brazil (Received January 17, 2001; accepted in revised form April 2, 2001)
To gain a better understanding of mycobacteria–host cell interaction, the present study compared the signal transduction events triggered during the interaction of Mycobacterium leprae (the causative agent of leprosy) and of Mycobacterium bovis BCG (an attenuated strain used as a vaccine against leprosy and tuberculosis) with human monocytes. The assays consisted of pretreating or not THP-1 cells (a human monocytic cell line) with different kinase inhibitors, followed by incubation with fluorescein-labelled bacteria and analysis of bacterial association via fluorescence microscopy. The specific tyrosine kinase (TK) inhibitor tyrphostin AG126 provided the highest rates of association inhibition (>90% for BCG and >65% for M. leprae). The early activation of TKs during mycobacteria–host cell interaction was confirmed by immunoblot analysis, demonstrating that in several host cell proteins mycobacteria stimulated tyrosine phosphorylation. The use of the drugs wortmannin and bisindolylmaleimide I which, respectively, inhibit phosphatidylinositide 3kinase (PI 3-kinase) and protein kinase C (PKC), produced lower but consistent results within a 35–60% association inhibition range for both bacteria. Dose response curves with these inhibitors were obtained. Similar results were obtained when primary human monocytes were used as host cells, strongly suggesting that TK, PKC and PI 3-kinase signals are activated during the interaction of human monocytes with both pathogenic and attenuated species of mycobacteria. 2001 Academic Press
Key words: Mycobacterium leprae, adherence, phagocytosis, human monocytes, signalling pathways, Mycobacterium bovis BCG.
∗ Author for correspondence. E-mail: [email protected]. fiocruz.br 0882–4010/01/070037+09 $35.00/0
2001 Academic Press
38
Introduction The knowledge accumulated to date in the field of microbial pathogenesis, in particular those aspects regarding the molecular mechanisms involved in microbial adherence to host cells, bacterial uptake and pathogen survival and replication inside mammalian cells, has increased dramatically in recent years and has pointed to the adoption of new strategies in the treatment of infectious diseases. In this regard, the interaction of the invading bacteria with the host cell has been shown to be very dynamic and characterized by the occurrence of an intense cross talk interplay between these cells. Upon the binding/internalization of bacterial pathogens, a multiplicity of signals are generated inside the host cell, leading to cytoskeletal reorganization and bacterial internalization and to the regulation of parallel events that often accompany phagocytosis, namely, cytokine release and the activation of microbicidal mechanisms. Since this interference in host cell metabolism ultimately favours the perpetuation of these bacterial pathogens inside the host, great efforts have been made to elucidate the pathways of phagocytic signal transduction generated at the moment of microbial internalization. The data accumulated in the literature so far has pointed to tyrosine phosphorylation as a common signal of bacterial internalization within mammalian cells. Actually, both opsonin-mediated and opsonin-independent phagocytoses in human phagocytes seem to be controlled by tyrosine kinases (TKs). Moreover, tyrosine phosphorylation accompanies binding and/or invasion of several pathogenic bacteria by human ‘‘nonprofessional’’ phagocytes. In addition to tyrosine phosphorylation in phagocytosis, an involvement has been established on the part of other cellular signalling elements, such as members of the superfamily of monomeric GPTases and of the protein kinase C (PKC) family, and more recently of phosphatidylinositide phosphate 3kinase (PI 3-K) [1–4]. Leprosy, a chronic infectious disease caused by the obligate intracellular bacterium Mycobacterium leprae, remains a major health care problem in several developing countries [5]. The capacity of M. leprae and other pathogenic mycobacteria like M. tuberculosis to successfully parasitize monocytes and macrophages appears to play an essential role in mycobacterial pathogenesis [6]. It is well known that monocytes and
C. S. Lima et al.
macrophages internalize mycobacteria by means of conventional phagocytosis, which in either the presence or absence of serum is predominantly mediated by the complement receptors (CR)1, CR3 and CR4 [7–9]. Additionally, experiments comparing attenuated vs virulent strains and live vs dead bacteria have revealed critical differences of both the final bacterial fate inside the cell as well as of the effects on host cell functions, which points to differences in the signal pathways generated early on during these interactions [10–13]. However, only limited information is available about the transmembrane signalling mechanisms responsible for regulating the phagocytosis of mycobacteria. A previous study by the present authors compared the level of TNF- secretion induced in THP-1 cell (a human monocytic cell line) after phagocytosis of M. leprae and Mycobacterium bovis BCG, which is an attenuated strain used as a vaccine against leprosy and tuberculosis (Oliveira MM, Charlab R, Pessolani MCV, unpublished data). The results demonstrated that BCG, but not M. leprae, induces TNF- secretion in THP-1 cells, thereby emphasizing the potential this in vitro model has to explore differences in signalling pathways triggered by vaccine vs the pathogenic species of mycobacteria. To continue investigating the matter, this study focused on investigating the signalling events that follow the encounter of human monocytes with M. leprae and comparing them with the events triggered upon incubation of these cells with BCG.
Results Kinase inhibitors decrease the degree of association of M. leprae and BCG with human monocytic cells To determine the precise involvement of TKs, PKC and PI 3-K during interaction between human monocytic cells and M. leprae and BCG, THP-1 cells were treated with specific inhibitors of these kinases prior to incubation with mycobacteria. Fluorescein-labelled bacteria were then added to the cells. Two h later, the phagocytosis mixture was stained with EB, and the preparations were observed under a fluorescent microscope. This fluorescent method was originally reported for its ability to distinguish between adherent and intracellular Listeria in
Signalling pathways during internalization of M. leprae
39
(a)
(b) M. leprae
Inhibition of control (%)
100
BCG
100
80
80
60
60
40
40
20
20
0
0 lm oly ind
Bis im
ale
im ale
in
nn
a rtm Wo
in ost
lm oly ind
rph
Ty
Bis
in
nn
a rtm Wo
in ost
rph
Ty
ide
ide
Figure 1. Kinase inhibitors block mycobacteria association to THP-1 cells. (a) Effect of tyrphostin on the association of M. leprae, as seen by fluorescence microscopy. Bacteria were pre-labelled with fluorescein and then added to the cells at a ratio of approximately 100 bacteria to one cell. After 2 h of incubation at 37°C, cells were washed and stained with EB for analysis under a fluorescent microscope. Representative M. leprae infected cultures in the presence or not of tyrphostin is shown (×800). (b) The inhibition of the degree of association of BCG and M. leprae with THP-1 cells was quantified by counting the number of cells binding bacteria in at least 100 cells. Results are expressed as the percentage of decrease of control cultures. Wortmannin and bisindolylmaleimide were added 20 min prior to infection. Tyrphostin was added 1 h prior to infecting the cells with mycobacteria. Data represent the mean±SEM of three to four experiments carried out in duplicate.
that, in this protocol, intracellular bacteria retain their green colouration, while the fluorescence of the extracellular bacteria changes to a reddish–
orange colour due to their EN staining [14]. However, in this study’s preparations, most mycobacteria, including the unbound, extracellular
C. S. Lima et al.
Inhibition of control (%)
40 100
50
80
40
60
30
40
20
20
10
0
1
10 100 Tyrphostin (µM)
0
10 100 Wortmannin (nM)
Figure 2. Tyrphostin and wortmannin inhibit BCG association with human blood-derived monocytes in a dose dependent manner. Monolayers enriched in monocytes were prepared from PBMC of healthy individuals. Bacterial association assays were then performed as described for THP-1 cells. Results are reported as the mean±SEM of three experiments carried out in duplicate.
bacteria, remained green in colour, which made it impossible to precisely quantify either the adherent or intracellular bacteria. Thus, in this study, the quantification of monocyte-associated (adherent and ingested) bacteria was performed. M. leprae and BCG were able to associate efficiently and in equal measure with control THP1 cells, and between 35–45% of all cells had at least one mycobacterium associated to them. When tyrphostin, a specific TK inhibitor, was added to the cells, a pronounced decrease in the number of cells binding mycobacteria was observed. The cultures shown in Fig. 1(a) are representative of THP-1 cells pre-incubated or not with tyrphostin and then treated with M. leprae. The effect of these inhibitors was quantified by determining the percentage of cells binding bacteria, expressed as a percentage of decrease from control cultures [Fig. 1(b)]. The results obtained were similar for both mycobacteria used. However, lower overall inhibition levels were obtained with the M. leprae treated cells. Tyrphostin was the most effective suppressor drug of M. leprae and BCG association with monocytes by up to 67.4±5.0% and 91.6±2.8%, respectively [Fig. 1(b)]. Treating cells with wortmannin and bisindolylmaleimide generated lower, though consistent, inhibition levels. Wortmannin, a specific inhibitor of the lipid kinase PI 3-K, blocked M. leprae and BCG association with THP-1 by 44.7±7.0% and 59.1±6.8%, respectively. Bisindolylmaleimide, a PKC inhibitor, evoked a decrease in M. leprae and BCG association of 34.0±2.5% and 44.2±6.0%, respectively. The results shown in Fig. 2 are representative
of blood-derived monocytes infected with BCG. Similarly to THP-1, tyrphostin was more effective than wortmannin in decreasing adherence/phagocytosis. However, both compounds blocked bacterial association in a dosedependent manner. The results obtained with primary cells were similar to those seen with THP-1. Tyrphostin at concentrations 1, 10 and 100 M inhibited association of BCG in 22.0±2.0%, 36.9±4.6% and 84.3±1.2%, respectively, while wortmannin at concentrations of 10 and 100 nM blocked bacterial binding/phagocytosis in 30.0±4.6% and 43.0±2.1%, respectively. Exposure to the kinase inhibitors at any of the different concentration levels used had no effect on monocyte viability. Similar results were also obtained when comparing M. leprae-infected primary monocytes and THP-1 cells (data not shown).
M. leprae and BCG stimulated tyrosine phosphorylation in human monocytic cells To further demonstrate the involvement of TK during mycobacteria interaction with human macrophages, immunoblot analysis of phosphotyrosine proteins in total cell lysates of control and treated THP-1 cells was performed. Typical results for these experiments are shown in Fig. 3. Incubation of THP-1 cells with M. leprae and BCG caused increased tyrosine phosphorylation of several host cell proteins, while no differences in the tyrosine phosphorylation pattern between cells treated with M. leprae and BCG were observed. Tyrosine phosphorylation
20
60
+
10
B
C
G
(6
0
m
in
)
+ M (m . le in pr ) a
FB %
r. au
10 +
st +
C
kDa
on
tr
ol
S
e
Signalling pathways during internalization of M. leprae
98
67
36
Figure 3. Detection of phosphotyrosine proteins in THP-1 cells treated with M. leprae and BCG. Serum starved cells were treated with staurosporin (20 min), 10% FBS (20 min) or mycobacteria at times indicated. Cell lysates were then prepared, resolved by SDS 8.5% PAGE, and immunoblotted with mouse antiphosphotyrosine antibody (4G10). The secondary antibody used was a goat anti-mouse IgG-peroxidase conjugate. Membranes were developed using a chemiluminescent substrate. The values on the left are molecular weights.
was monitored during a consecutive 60 min period, and an increase in tyrosine phosphorylation was detected as soon as 5 min after treatment with mycobacteria. Cells treated with staurosporine, a broad-spectrum protein kinase inhibitor, lacked most of the phosphoproteins found in unstimulated cells, whereas cells stimulated with 10% FBS showed a pronounced increase in the tyrosine phosphorylation of several protein bands, similarly to what was seen in cells treated with mycobacteria.
Discussion Interaction of bacterial pathogens with their host cells triggers signal transduction pathways that, in turn, lead to a variety of cellular responses which ultimately favour their perpetuation in the host. Among these responses are those
41
giving rise to an extensive reorganization of the cytoskeleton, which results in morphological changes, and the secretion of cytokines into the medium. Although this interference in host cell metabolism by bacteria represents a central feature of their pathogenesis, these events are poorly understood in mononuclear phagocytes infected with mycobacteria and have only recently received attention of a few investigators [15–17]. In the present study, the stimulation of three classes of kinases (TKs, PKCs and PI 3Ks) during interaction of M. leprae with human monocytes has been investigated. The effects evoked by M. leprae were compared with those by BCG. This study has demonstrated that tyrosine phosphorylation represents a major signal generated during the association of M. leprae and BCG with human monocytes. This conclusion was based on the inhibition studies showing that the association of M. leprae and BCG is blocked by over 65% by tyrphostin, a specific TK inhibitor, and the immunoblot analysis showing that mycobacteria stimulated tyrosine phosphorylation of several host cell proteins. Besides TKs, inhibition assays also suggested that PKC and PI 3-K are activated during the encounter of mycobacteria with human monocytes. Similar results were obtained when THP-1 cells and/or primary human monocytes were used as host cells. However, no qualitative differences in the signals triggered by either M. leprae or BCG were detected. The association of M. leprae and BCG with the human monocytic cells employed here is probably mediated by the complement receptors expressed on the surface of these cells. Although other receptors may also participate [18–20], it has been demonstrated that the complement receptors, and especially CR3, are the most important ones involved in the uptake by human monocytes of both pathogenic and non-pathogenic mycobacteria [7–9]. In spite of the fact that THP-1 cells are immature monocytes, they have been shown to express CR1 and CR3, although at lower levels than monocytes [21]. Likewise, the similar results obtained here with both the in vitro models support the hypothesis of identical receptors mediating the uptake of mycobacteria by THP-1 and primary monocytes, regardless of their degree of differentiation. These results reinforce previous data that indicate THP-1 as being a useful model to investigate the nature of mycobacteria/monocyte interactions [22]. The activation of TKs during the binding/ phagocytosis of M. leprae and BCG is not a
42
surprising finding. Data have regularly been reported in the literature pointing to tyrosine phosphorylation as a common signal for bacterial internalization into mammalian cells [1–4]. Furthermore, previous studies have shown that, in macrophages under the influence of TK inhibitors, the phagocytosis mediated by CR3 decreased, indicating that the tyrosine phosphorylation of proteins is also required for the efficient internalization of complement-opsonized particle [2]. In this context, it has been recently shown that TKs are involved in the activation of phospholipase D, which plays a key role in the CR3-mediated phagocytosis of either Erdman or H37Ra strains of M. tuberculosis by human macrophages [15]. In the same study, prominent tyrosine phosphorylation was noted in the proteins of >150, 95, 72, 56 and 42 kDa induced by M. tuberculosis, a finding resembling those observed in M. leprae and BCG-treated THP-1 cells (Fig. 3). The involvement of TKs and PKC has also been demonstrated in CR3mediated phagocytosis of Legionella pneumophila by human monocytes [23]. The same study also showed that the CR-3 mediated entry of either virulent or avirulent strains as well as of zymosan and Escherichia coli caused the tyrosine phosphorylation of similar proteins and was not a virulence-associated event. This is in agreement with the results of the present study, in which no differences in the signals triggered by M. leprae or BCG were observed. The results are also in agreement with a recent study showing the involvement of TKs during the uptake of M. leprae by murine macrophages [17]. The partial inhibition of BCG and M. leprae association with human monocytes by bisindolylmaleimide and wortmannin also suggests the activation of PKC and PI 3-K, respectively, during interaction of mycobacteria with human monocytes. These results are in agreement with recent findings indicating the involvement of PKC in CR3-mediated phagocytosis [2, 23]. With respect to PI 3-K, this kinase belongs to a family of enzymes that phosphorylates the 3′-hydroxyl group on the inositol ring of phosphoinositides [24]. These phospholipids are involved in a large array of signal transduction pathways controlling mitogenic responses, differentiation, apoptosis, cytoskeletal organization and membrane flow along the secretery and endocytic pathways [25]. A growing body of evidence has implicated this family of lipid kinases in receptor-mediated phagocytosis as well as in the bacterial invasion of nonphagocytic cells [2, 26].
C. S. Lima et al.
Precisely how PI 3-K participates in phagocytosis is still not known, but a recent study has suggested that it is involved in co-ordinating exocytic membrane insertion and pseudopod extension [27]. In conclusion, in the present study, three classes of signal transduction elements, namely, TKs, PKC and PI 3-K, have been shown to be activated as a result of interaction between M. leprae and human monocytes. These signals probably represent universal signals generated by human monocytes early on during phagocytosis and are not associated with bacterial virulence. A strong possibility is that these kinases control cytoskeleton reorganization, pseudopod extension and phagosomal closure that are fundamental events accompanying phagocytosis. The molecular mechanisms responsible for the different effects that virulent and avirulent mycobacteria exert on host cell functions most certainly require a more detailed dissection of those signalling pathways activated at the moment of phagocytosis.
Materials and Methods Source of reagents RPMI 1640 medium, penicillin/streptomycin and glutamax-I were obtained from Gibco BRL (Rocksville, MD, U.S.A.). Phorbol-12-myristate13-acetate (PMA), staurosporin and tyrphostin AG 126 were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Wortmannin and bisindolylmaleimide were purchased from Calbiochem (San Diego, CA, U.S.A.). Middlebrook 7H9 broth was purchased from Difco Laboratories (Detroit, MI, U.S.A.). Anti-phosphotyrosine monoclonal antibody (4G10) was obtained from Upstate Biotechnology Inc. (Lake Placid, NY, U.S.A.).
Bacteria Armadillo-derived M. leprae was kindly donated by Dr Patrick Brennan (Colorado State University, Fort Collins) through NIAID contract NO155262. M. bovis BCG Pasteur 1173P2 WHO was grown at 37°C in Middlebrook 7H9 broth supplemented with 0.05% Tween 80, under constant agitation on a magnetic plate. Cultures were harvested in mid-log phase, aliquotted in
Signalling pathways during internalization of M. leprae
43
Eppendorf tubes and kept frozen at −80°C until use.
(100 nM) were added to the corresponding wells 20 min before the infection, and tyrphostin AG126 (100 M) was added 1 h prior to the addition of mycobacteria to the cells. Treatment with kinase inhibitors was omitted in the control wells and all of the assays were carried out in duplicate wells. Association assays were performed using a fluorescence method to distinguish between internalized and adherent bacteria [14]. BCG Pasteur and M. leprae were pre-labelled with fluorescein isothiocyanate (FITC) at a concentration of 0.1 mg/ml for 1 h at room temperature. The cells received enough mycobacteria to achieve a ratio of approximately 100 bacteria to 1 cell. The plates were then centrifuged at 500×g for 10 min and were returned to the incubator at 37°C with 5% CO2. After 2 h, the cultures were removed from the wells and cells were recovered by centrifugation at 325×g for 5 min. Cells were resuspended in 1 ml of PBS and washed four more times for the removal of unbound extracellular bacteria. The last pellet was resuspended in 100 l of PBS followed by staining with ethidium bromide (EB) at a final concentration of 50 g/ml for 10 min. The cells were then citospun at 30×g for 10 min and the glass slides fixed with cold methanol for 1 min. The glass slides were mounted with 90% glycerol and 10% PBS 10× containing 1 mg/ml p-phenylenediamine. For the blood-derived monocytes, at 2 h the wells were washed five times with PBS at room temperature followed by staining with EB for 10 min and fixation with cold methanol for 1 min. The glass coverslips were then removed from each well and mounted on glass slides with entellan glue and 90% glycerol and 10% PBS 10× containing 1 mg/ml p-phenylenediamine. The glass slides were viewed using a Zeiss fluorescent microscope and the percentage of cells having at least one mycobacterium associated with them was determined by counting a minimum of 100 cells for each glass slide. The cells were tested for viability by being stained with a solution of EB (4 g/ml) and fluorescein diacetate (20 g/ml) for 10 min at room temperature and at a ratio of 2:1 (cell culture: staining solution).
THP-1 cells The THP-1 monocytic cell line was obtained from the American Type Culture Collection (ATCC) (Rockville, MD, U.S.A.). The cells were propagated by means of weekly passages in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 10 mM HEPES, 2 mM glutamax-I, 100 U/ml penicillin, 100 g/ml streptomycin and incubated at 37°C with 5% CO2. Before being plated, the cell culture was centrifuged at 500×g for 10 min and the pellet was resuspended in a phosphate buffered saline (PBS) solution (10 mM phosphate buffer pH 7.2, 0.15 M NaCl). This wash was repeated twice more and the last pellet was resuspended in fresh supplemented RPMI. The cells were counted and their concentration was adjusted to 5×105 cells in 0.5 ml. Each well of a 24 well microtitre plate then received 0.5 ml of the cell culture and the plate returned to the incubator at 37°C and 5% CO2.
Human blood-derived monocytes Normal human peripheral blood mononuclear cells (PBMC) were separated by Ficoll–Hypaque density gradients from sterile heparinized blood. The cells from the interface were washed three times in PBS supplemented with 1% fetal bovine serum (FBS) with no calcium or magnesium. They were seeded at a density of 1×106/well/ 1 ml of RPMI supplemented with 10% FBS, 100 U/ml penicillin, 100 g/ml streptomycin and 2 mM/l glutamine, in 24 well microtitre plates containing glass coverslips. The cells were incubated at 37°C with 5% CO2 and allowed to settle for 24 h. Then, the non-adherent cells were removed with three washes of PBS at room temperature. To the adherent cells, at about 2×105/well, 0.5 ml of fresh supplemented RPMI was added before the infection and the inhibition assays.
Association assay Before the addition of mycobacteria, the cells were treated with protein kinase inhibitors. Bisindolylmaleimide (1 M) and wortmannin
Immunoblotting analysis of cellular tyrosine phosphorylation For the analysis of global tyrosine phosphorylation, THP-1 cells (1.5×106 cells) were
44
incubated in serum-free RPMI medium. The next day cultures were treated or not with M. leprae and BCG for 0–60 min. Cells were then washed twice with PBS solution and lysed in ice-cold RIPA buffer containing a cocktail of protease and phosphatase inhibitors (50 mM Tris buffer, 1% Nodinet P40, 0.25% sodium deoxycolate, 0.1% SDS, 1 mM EGTA, 1 mM NaF, 1 mM sodium orthovanadate, 1 mM phenylmethylsulphonylfluoride (PMSF), 10 g/ml leupeptin, 10 g/ml aprotinin and 2 g/ml pepstatin). Lysates were centrifuged in microcentrifuge at the maximum speed for 20 min at 4°C, and protein assay was estimated in the supernatants by BCA kit (Pierce, Rockford, IL, U.S.A.). Equal amounts of proteins (100 g protein) were resolved on 8.5% SDS-PAGE [28], followed by transfer to nitrocellulose membranes [29] and blots were blocked with 3% BSA TBS/T for 2 h/RT. Membranes were incubated with 4G10 antiphosphotyrosine monoclonal antibody in TBS/ T-3% BSA for 2 h at room temperature. Immunoblots were developed by incubation with a goat anti-mouse horseradish proxidase-conjugated antibody (Sigma Chemical Co.) and use of the chemiluminescent Supersignal Substrate (Pierce).
Acknowledgements This study was supported by The Heiser Program for Research in Leprosy and Tuberculosis, CNPq (Brazil), and PAPES/FIOCRUZ.
References 1 Finlay BB, Cossart P. Exploitation of mammalian host cell functions by pathogens. Science 1997; 276: 718–25. 2 Kwiatkowska K, Sobota A. Signaling pathways in phagocytosis. BioEssays 1999; 21: 422–31. 3 Ireton K, Cossart P. Interaction of invasive bacteria with host signaling pathways. Curr Op Cell Biol 1998; 10: 276–83. 4 Greenberg S. Modular components of phagocytosis. J Leukoc Biol 1999; 66: 712–7. 5 WHO. Global leprosy situation, September 1999. Wkly Epidemiol Rec 1999; 74: 313–6. 6 Fenton MJ, Vermeulen MW. Immunopathology of tuberculosis: roles of macrophages and monocytes. Infect Immun 1996; 64: 683–690. 7 Schlesinger LS, Horwitz MA. Phagocytosis of leprosy bacilli is mediated by complement receptors CR1 and CR3 on human monocytes and complement component C3 in serum. J Clin Invest 1990; 85: 1304–14.
C. S. Lima et al. 8 Schlesinger LS, Bellinger-Kawahara CG, Payne NR, Horwitz MA. Phagocytosis of Mycobacterium tuberculosis is mediated by human monocyte complement receptors and complement component C3. J Immunol 1990; 144: 2771–80. 9 Launois P, Niang M, Dieye A, Sarthou JL, Rivier F, Millan J. Human phagocyte respiratory burst by Mycobacterium bovis BCG and M. leprae: functional activation by BCG is mediated by complement and its receptors on monocytes. Int J Lepr Other Mycobact Dis 1992; 60: 225–33. 10 Holzer TJ, Kizlaitis L, Vachula M, Weaver CW, Andersen BR. Human phagocytic cell responses to Mycobacterium leprae and Mycobacterium bovis Bacillus CalmetteGue´rin. J Immunol 1988; 141: 1701–8. 11 Suzuki K, Fukutomi Y, Matsuoka M et al. Differential production of interleukin 1 (IL-1), IL-6, tumor necrosis factor, and IL-1 receptor antagonist by human monocytes stimulated with Mycobacterium leprae and M. bovis BCG. Int J Lepr Other Mycobact Dis 1993; 61: 609–18. 12 Barker LP, George KM, Falkow S, Small PLC. Differential trafficking of live and dead Mycobacterium marinum organisms in macrophages. Infect Immun 1997; 65: 1497–1504. 13 Ferrari G, Langen H, Naito M, Pieters J. A coat protein on phagosomes involved in the intracellular survival of Mycobacteria. Cell 1999; 97: 435–47. 14 Drevets DA, Campbell PA. Macrophage phagocytosis: use of fluorescence microscopy to distinguish between extracellular and intracellular bacteria. J Immunol Meth 1991; 142: 31–8. 15 Kusner DJ, Hall CF, Schlesinger LS. Activation of phospholipase D is tightly coupled to the phagocytosis of Mycobacterium tuberculosis or opsonized zymosan by human macrophages. J Exp Med 1996; 184: 585–85. 16 Malik ZA, Denning GM, Kusner DJ. Inhibition of Ca2+ signaling by Mycobacterium tuberculosis is associated with reduced phagosome–lysosome fusion and increased survival within human macrophages. J Exp Med 2000; 191: 287–302. 17 Prabhakaran K, Harris EB, Randhawa B. Regulation by protein kinase of phagocytosis of Mycobacterium leprae by macrophages. J Med Microbiol 2000; 49: 339–42. 18 Schlesinger LS. Macrophage phagocytosis of virulent but not attenuated strains of Mycobacterium tuberculosis is mediated by the mannose receptors in addition to complement receptors. J Immunol 1993; 150: 2920–30. 19 Bermudez LE, Young LS, Enkel H. Interaction of Mycobacterium avium complex with human macrophages: roles of membrane receptors and serum proteins. Infect Immun 1991; 59: 1697–702. 20 Rao SP, Ogata K, Catanzaro A. Mycobacterium avium–M. intracellulare binds to the integrin receptor alpha v beta 3 on human monocytes and monocyte-derived macrophages. Infect Immun 1993; 61: 663–70. 21 Thieblemont N, Haffner-Cavaillon N, Ledur A, L’AgeStehr J, Ziegler-Heitbrock HW, Kazatchkine MD. CR1 (CD35) and CR3 (CD11b/CD18) mediate infection of human monocytes and monocytic cell lines with complement-opsonized HIV independently of CD4. Clin Exp Immunol 1993; 92: 106–13. 22 Stokes RW, Doxsee D. The receptor-mediated uptake, survival, replication, and drug sensitivity of Mycobacterium tuberculosis within the macrophages-like cell
Signalling pathways during internalization of M. leprae
45
line. THP-1: a comparison with human monocyte-derived macrophages. Cell Immunol 1999; 197: 1–9. 23 Coxon PY, Summersgill JT, Ramirez JA, Miller RD. Signal transduction during Legionella pneumophila entry into human monocytes. Infec Immun 1998; 66: 2905–13. 24 Vanhaesebroeck B, Leevers SJ, Panayotou G, Waterfield MD. Phosphoinositide 3-kinases: a conserved family of signal transducers. Trends Biochem Sci 1997; 22: 267–72. 25 Martin TJF. Phosphoinositide lipids as signaling molecules: common themes for signal transduction, cytoskeletal regulation, and membrane trafficking. Annu Ver Cell Dev Biol 1998; 14: 231–64.
26 Ireton K, Payrastre B, Chap H et al. A role for phosphoinositide 3-kinase in bacterial invasion. Science 1996; 274: 780–2. 27 Cox D, Tseng C-C, Bjekic G, Greenberg S. A requirement for phosphatidylinositol 3-kinase in pseudopod extension. J Biol Chem 1999; 274: 1240–7. 28 Laemmli UK. Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680–5. 29 Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrilamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 1979; 76: 4350–4.