Sukhraj Kaur; Harsimrat Kaur; Prati Pal Singh

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Induction of colony-stimulating factors by a 30-kDa secretory protein of Mycobacterium tuberculosis H37Rv

European Cytokine Network. Volume 15, Numéro 4, 327-38, December 2004, Research papers

Summary

Auteur(s) : Sukhraj Kaur, Harsimrat Kaur, Prati Pal Singh , National Institute of Pharmaceutical Education and Research, S. A. S. Nagar, India.

Illustrations

ARTICLE

Auteur(s) :, Sukhraj Kaur, Harsimrat Kaur, Prati Pal Singh^*

National Institute of Pharmaceutical Education and Research, S. A. S. Nagar, India

accepté le 1 Septembre 2004

Introduction

Tuberculosis (TB) is a worldwide health problem and the leading cause of deaths due to infectious diseases (5% of all deaths worldwide) [1]. Presently, about one-third of world’s population is infected with Mycobacterium tuberculosis (the main etiological agent of human TB), and every year nearly 54 million new cases including 6.8 million clinical cases, and about three million deaths occur [2]. The emergence of multidrug resistant (MDR) strains of M. tuberculosis has further compounded the problem, and in many countries >15% MDR TB cases occur [3]. However, despite the severity of TB, the molecular mechanisms of pathogenesis and protective immunity continue to remain elusive.

M. tuberculosis secretory antigens interact with the host immune system early in the infection, and thus constitute important potential targets of protective immunity in human TB [4, 5]. The 30-kDa antigen of M. tuberculosis (Mtb30) is a major secretory protein [6], and has also been designated independently as α-antigen [7], antigen a₂[8], antigen 6 [9], M. tuberculosis purified MPT-59 [10], the one having crossed-immuno electrophoresis No. 85B [11], and M. bovis purified MPB-59 [12]. In guinea pig [4] and mouse [13] models of TB, Mtb30 has been reported to be the most immunoprotective protein. Therefore, it is considered as one of the candidate, for subunit [5] and DNA [14] anti-TB vaccines. Recombinant BCG vaccine expressing Mtb30 induced greater protection than conventional BCG vaccine [15], and the immunogenecity of BCG vaccine has been reported to be improved by using granulocyte (G)-macrophage (M; MØ) colony-stimulating factor (GM-CSF) transgene-based adjunct formulations [16]. The Mtb30-induced production of Th1 cytokines such as tumor necrosis factor-α (TNF-α) [17], interleukin-12 (IL-12) [18] and interferon-γ (IFN-γ) [13], and of Th2 cytokines transforming growth factor-β (TGF-β) [19] and IL-10 [20] is now well known. However, the Mtb30-induced production of CSFs, an important group of proinflammatory and regulatory cytokines, has apparently not been reported.

The CSFs (mol. wt. 18-90 kDa) are glycoproteins that are characterized by their ability to induce the proliferation and differentiation of the myeloid hemopoietic progenitor cells, in vitro [21, 22]; in vivo they stimulate hematopoiesis [23]. The lineage specific CSFs i. e. G-CSF and M-CSF, stimulate G and M colony formation, respectively. GM-CSF, on the other hand, supports the formation of colonies consisting mainly of G, M and eosinophils, whereas multi-CSF (IL-3) induces colonies containing cells of different lineages. The genes for human and mouse CSFs have been cloned [24, 25], and large quantities of recombinant CSFs (rCSFs) can now be produced. Structurally, M-CSF is a homodimer, whereas G-, GM- and multi-CSFs consist of a single polypeptide chain. The CSFs are active at picomolar concentrations, and their constitutive levels are very low; however, during infections, the concentrations of CSFs are rapidly elevated [26]. Functionally, CSFs can enhance the effector functions of mature cells of myeloid lineage for example; human monocyte cytotoxicity can be activated by GM-CSF and IL-3 [27]. CSFs are also known to induce the synthesis and secretion of various cytokines such as IFN-γ, TNF-α [28] and IL-1 [29], and can indirectly augment the release of IL-2 by stimulating antigen-presenting cells [30]. Because of their immunopotentiating properties, CSFs are known to boost host defence against infections [31-33]. Treatment of human MØs with GM-CSF has been shown to enhance their antimycobacterial activity, in vitro [34, 35], and rGM-CSF has been reported to protect patients infected with M. kansasii [36]. GM-CSF therapy has also been observed to enhance the uptake and mycobactericidal activity of monocytes from AIDS patients with M. avium bacteremia [37, 38]. In another study, M. avium-M. intracellulare, an opportunistic pathogen, has been shown to stimulate human monocytes and large granular lymphocytes to produce CSFs [39]. Clinically, augmented production of monocytes and their efflux from bone marrow (BM) has also been observed in TB [40]. There is, however, apparently no report of the M. tuberculosis- or its component(s)-induced production of CSFs. Because CSFs play important role(s) in the pathogenesis and protection from TB [32], we considered it expedient to determine the CSF induction potential of Mtb30. Our results, apparently for the first time, demonstrate that purified Mtb30 can induce the synthesis and secretion of CSFs, both in vivo and in vitro.

Materials and methods

Mice and mycobacteria

Male Swiss mice (18-20 g), obtained from the Central Animal Facility of the institute, were maintained at 22-24 °C with food and water provided ad libitum. All studies were carried out in accordance with the guidelines for Care and Use of Animals in Scientific Research, Indian National Science Academy, New Delhi, India, as adapted and promulgated by the Institutional Animal Ethics Committee. M. tuberculosis H37Rv, obtained from Tuberculosis Research Centre, Chennai, India, was cultured at 37 °C in dispersed form in Middlebrook 7H9 broth (Difco, Sparks, MO, USA) supplemented with 1% glycerol, 0.05% Tween 80 (Sigma Aldrich, St. Louis, MO, USA) and 10% Middlebrook OADC enrichment (Difco). Culture aliquots in 1 mL volumes were stored at -70 °C until used.

Production of M. tuberculosis culture filtrate proteins and purification of Mtb30

Culture filtrate proteins were purified from the pooled cultures of M. tuberculosis H37Rv grown in 7H9 broth (pH 6.7) prepared with glycerol but without albumin and Tween. The bacteria were cultured at 37 °C for three weeks from an initial OD₅₄₀ of 0.05 to a final OD₅₄₀ of 0.5. The cells were removed by centrifugation (16,270 g; 20 min; 4 °C) and the supernatant was filtered by first using a 0.45 and then a 0.22 μm membrane filter. The sterile culture filtrate was concentrated (x 100) by ultrafiltration using an Amicon PM10 membrane (Amicon, Bedford, MA, USA). The concentrated proteins were brought to 50% ammonium sulphate saturation at 4 °C. The precipitate was dialysed against water, dried by lyophilization and designated culture filtrate, and its protein content was determined by Lowry’s method [41] using bovine serum albumin as a standard. The major proteins from the culture filtrate were purified as described [10]. Briefly, the culture filtrate protein concentrate (100 mg) was loaded onto a DEAE-Sepharose CL-6B (Sigma) anion exchange column equilibrated with 30 mM Tris-hydrochloride buffer (pH 8.7) containing 3% (v/v) methyl cellosolve. The column was eluted using a sodium chloride linear gradient from 50 – 300 mM. Each fraction was read at 280 nm and the absorbency was plotted. The resultant fractions obtained were pooled and concentrated separately by ultrafiltration using a YM-3 membrane (Amicon). The concentrated fractions were further purified to homogeneity by applying the pooled fraction serially to columns of DEAE-Sepharose CL-6B (containing 10% ethylene glycol/3M urea), phenyl Sepharose CL-4B and sephacryl-S-200 HR (Sigma). Finally, the pooled fractions were analysed by reduced 12% sodium dodecyl sulphate polyacrylamide gel electrophoresis, and stained with Coomassie Blue to determine the apparent molecular mass of proteins (( figure 1 )A). The fractions containing the Mtb30 were pooled, dialysed against water and identified by immunoblotting with the 30-kDa antigen specific monoclonal antibody TB-c-27 [42]. The CSF-inducing capability of Mtb30 was characterized by heat, enzymatic and sodium periodate treatments. For heat stability testing, Mtb30 was heated at 70 °C for 1 h at pH 7.0. The effects of proteases were tested by incubating Mtb30 with pronase E (2.5 mg/mL; pH 7.5; Merck, Darmstadt, Germany) and trypsin (25 μg/mL; pH 8.0; Merck). The stability of Mtb30 (10 μL) in the presence of periodate was determined by treatment with 5 μL of 0.6 M sodium periodate (pH 7.2; Sigma) overnight at room temperature. Excess of periodate was eliminated by the addition of 5 μL of 0.4 M sodium metabisulfite. The endotoxin content of Mtb30 was determined by Limulus amoebocyte lysate (LAL) assay and was <20 ng/mg Mtb30. Lipopolysaccharide at this concentration did not induce CSF production by mouse peritoneal MØs (PMs). The purified Mtb30 was filter (0.22 μm) sterilized, and stored at -20 °C until used. A single preparation of Mtb30 was used in these studies.

Generation of rabbit anti-Mtb30 serum

Rabbit anti-Mtb30 serum was generated by injection of 400 μg of Mtb30 in complete Freund’s adjuvant (FA; 1:1) at four subcutaneous (s.c.) sites followed by a booster of 200 μg of Mtb30 in incomplete FA, s.c., four weeks later. The rabbit was boosted once again, one week later, with 100 μg of Mtb30, intravenously (i. v.), and was finally bled for antiserum, three days after the last boost. The antiserum was heat-inactivated (HI; 56 °C, 30 min) and stored at -20 °C. The specificity of the antiserum was determined by Western blot analysis, and was found to be specific (( figure 1 )B). The antiserum had an antibody titre 1:2048 as determined by enzyme-linked immunosorbent assay (ELISA).

Macrophages

For PMs, thioglycollate-injected (4% wt/vol; 0.5 mL/mouse; 96 h) normal mice peritoneal exudates cell suspension was centrifuged (700 x g; 7 min; 4 °C), and the cell pellet was resuspended (1x10⁶cells/mL) in 10 ml antibiotic-free Dulbeccos’ modified Eagles Medium (DMEM; Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS, Gibco), 2 mM L-glutamine (Gibco), 0.01 M HEPES (Gibco), and 1x10^-4 M 2-mercaptoethanol (2-ME, Sigma; CDMEM). The splenic MØs were obtained from splenocyte single cell suspension made by using a 20 μm nylon sieve. The cells were then washed by centrifugation (700 x g; 12 min; 4 °C). Erythrocytes were lysed with rbc-lysing buffer (Sigma-Aldrich), and the cells were resuspended in CDMEM. For bone marrow (BM)-derived MØs, mouse femurs were flushed with chilled DMEM using a 24G needle, and the BM cells were washed (x2) and suspended in CDMEM. The adherent MØs from these three cell suspensions were harvested, separately, by allowing them to attach to the plastic surface of T-25 culture flasks at 37 °C for three hours in 5% CO₂-air atmosphere, and were then further incubated for 30 min in an equal volume of CDMEM containing 2 μg/mL indomethacin (Sigma). The MØs were detached using a sterile rubber scraper, washed (x3) and resuspended in 5 mL chilled Hanks’ balanced salt solution (HBSS). T-cells from these MØs were eliminated by rabbit anti-mouse T-cell serum (1:20) treatment for 1 h at 4 °C followed by a HBSS wash (x1), and incubation with rabbit complement (1 h; 37° C). The DMEM and HBSS contained <0.1 ng/mL endotoxin as determined by LAL assay. MØs were >96% pure as determined by morphologic, phagocytic and non-specific esterase staining criteria, and were >98% viable as judged by trypan blue exclusion.

Generation of CSFs

For serum CSFs, a single injection of Mtb30 (0.1, 0.5, 1, 5 and 10 mg/kg) was administered to mice (i. v.; 0.1 mL; six mice/dose), and their blood samples were collected aseptically after six, 12, 24, 48 and 72 h. Sera from these blood samples were separated and pooled for each time-point, separately. Pooled sera from mice injected with muramyl dipeptide (MDP; 25 μg/kg), HI-Mtb30 (70 °C; one h; pH 7.0; 10 mg/kg) or sterile normal saline (vehicle) served as controls. In vitro, the cultured MØs (5x10⁵ cells/mL; 2 mL/well) were exposed to different concentrations of Mtb30 (1, 5, 10, 25 and 50 μg/mL) for six, 12, 24, 48 and 72 h. For CSFs induced by whole mycobacteria, the cultured PMs (1x10⁵ cells; 1.4 mL) were exposed to live (multiplicity-of-infection, MOI; 1:1 and 1:10) and heat-killed (80 °C, 20 min; MOI; 1:10) M. tuberculosis for 4 h at 37 °C in 5% CO₂-air atmosphere, washed (x5) with warm DMEM to remove the extracellular bacteria, and then further cultured for six days in the presence or absence of 100% neutralizing concentration (25 μg/mL) of rabbit anti-Mtb30 antibody, and 100 μL of fresh medium containing antibody was added daily for the next six days. The conditioned media (CM) of the MØs were then collected aseptically, centrifuged (1000 x g; 10 min; 4 °C) and filter-sterilized (0.2 μ). For controls, CM of MØs treated with MDP (1 μg/mL), HI-Mtb30, normal rabbit serum and CDMEM only were used. All the sera and CM were stored at -20 °C until used.

Measurement of CSF activity

CSFs were measured in terms of their colony-stimulating activity [43, 44]. Briefly, normal mouse femur BM mononuclear cells were washed (x3) and suspended (5x10⁶ cells/mL) in CDMEM. After removal of non-adherent cells by adherence/depletion processes, the adherent cells were resuspended (2x10⁴ cells/mL) in CDMEM without FBS but containing 30% HI (56 °C, 30 min)-horse serum (Gibco), 0.9% methylcellulose (Sigma), 0.9% deionized bovine serum albumin (Sigma) and 1x10^-4 M 2-ME. The test and control serum (5%), and CM (10%) samples were then added to this cell suspension. One mL cultures of this cell suspension were established in 35 mm plastic dishes and incubated at 37 °C in humid 5% CO₂-air atmosphere for 14 days. The number of colonies with 50 or more cells were then counted under a dark-field inverted microscope (40x magnification) or fixed on the glass slides and stained with May-Grünwald-Giemsa solution for identification.

Quantification of GM-CSF, M-CSF and G-CSF in serum and CM

Sandwich-ELISAs were performed to quantify mouse GM-CSF, M-CSF and G-CSF secretion in serum and CM. The following pairs of capture and detection antibodies were used to quantify mouse CSFs, respectively: monoclonal antibody (MAb) pairs anti-mouse GM-CSF (clone MP1-22E9) and biotinylated anti-mouse GM-CSF (clone MP1-31G6; PharMingen, San Diego, CA, USA), polyclonal pairs anti-mouse M-CSF and biotinylated anti-mouse M-CSF (R & D Systems Inc.; Minneapolis, MN, USA), and monoclonal anti-mouse G-CSF (clone 67604.111) and polyclonal biotinylated anti-mouse G-CSF (R & D Systems). Capture antibodies were coated on the MaxiSorp (Nunc, GIBCO BRL, Paisely, UK) plates at the concentration of 15 μg/mL and the biotinylated antibodies were added at the final concentration of 10 μg/mL, before adding streptavidin-labelled horseradish peroxidase. CSFs were quantified by extrapolation from a standard curve constructed by determining absorbencies using limiting dilutions of recombinant mouse GM-CSF (rmGM-CSF) and M-CSF (PharMingen), and G-CSF (R & D Systems). The results were expressed as mean ng/mL, and the sensitivities of the assays were 50 pg/mL for GM-CSF, 90 pg/mL for M-CSF and 30 pg/mL for G-CSF.

GM colony-forming units (CFU-GM) assay

The number of CFU-GM in single cell suspensions prepared from the spleens and BM of Mtb30-treated mice were determined in colony-forming assays, performed in semi-solid cultures [45, 46]. Briefly, spleens were minced and passed through a sterile, nylon mesh (20 μm) to obtain single-cell suspensions which were resuspended in 15 mL of DMEM containing 10% FBS, 2% HEPES, and 40 μg/mL gentamycin, and centrifuged (700 g; 12 min; 4 °C). Erythrocytes were lysed with rbc-lysis buffer, the cells were washed with DMEM, and erythrocyte ghosts were removed by filtering cell suspensions through sterile gauze. BM cells were flushed out from the mouse femurs with 1 mL of cold Iscove’s modified Dulbeccos’ medium (IMDM; Gibco) supplemented with 5% FBS, 40 μg/mL gentamycin and 2 mM L-glutamine. The spleen and BM cell suspensions were washed (x3) in IMDM. Total, viable cell counts were obtained by using 0.1% trypan blue exclusion, and finally suspended at a concentration of 4 x 10⁶ cells/mL in the same medium. The CFU-GM medium consisted of 0.8% methylcellulose, 30% FBS, 10% pokeweed mitogen-stimulated spleen cell-CM, 2 mM L-glutamine and 5 x 10^-5 M 2-ME. The spleen and BM cells were resuspended in CFU-GM medium and plated in 35 mm petri dishes at densities of 2 x 10⁵ and 3 x 10⁵ cells, respectively. CFU-GM counts were determined according to colony morphology after seven days of incubation in humidified 5% CO₂-air atmosphere at 37 °C. Based on the total spleen or BM cell counts, the final CFU-GM numbers were expressed/spleen or femur.

Statistical analysis

All the experiments were run in triplicate, three-times, separately. For statistical analysis, Students’ t-test was used, and p<0.05 was considered significant.

Results

Induction of serum CSFs by Mtb30

Mtb30 (0.1–10 mg/kg) induced dose-dependent production of serum CSFs in mice (( figure 2 )a). Whereas, as low as 0.1 mg/kg Mtb30 could induce detectable increase in serum CSF activity, a 1 mg/kg dose induced maximum response (142 ± 16 colonies); at further higher doses, the response was a plateau. The Mtb30-induced increased serum CSF production was observed as early as six hours after stimulation, reached its maximum after 24 h, and then gradually returned to normal levels by 72 h. No increased CSF production was observed in mice treated with HI-Mtb30 or vehicle.

Mtb30-induced production of CSFs by MØs, in vitro

PMs (5x10⁵ cells/well; 2 mL) incubated with Mtb30 (1–50 μg/mL), in a concentration-dependent manner, elaborated CSFs in the CM (( figure 2 )b). Although as low as 1 μg/mL of Mtb30 induced detectable amounts of CSFs in the CM, maximum CSF induction (97 ± 11 colonies) occurred at 25 μg/mL; at higher concentration the response was a plateau. CSFs were detectable in the CM six h after stimulation, peaked at 24 h and levelled-off to background levels by 72 h. HI-Mtb30 (50 μg/mL) did not induce the elaboration of CSFs, over and above the background levels. Similar results were obtained using splenic and BM-derived MØs (( figure 3 )). Furthermore, Mtb30 (1–50 μg/mL) did not directly induce the colony formation (data not shown). Polyclonal rabbit anti-Mtb30 antibody neutralization of Mtb30 significantly (p<0.05) diminished its CSF-inducing capability, in vitro (( figure 4 )). The Mtb30-induced CSFs in the serum and CM were tested for their functional/molecular similarities by examining the colony types they induced. The data (( figure 5 )) show that CSFs from both sources formed G, M and GM colonies in similar proportions; the GM colonies were maximum (>79%). IL-1 is known to induce the production of CSFs, in vitro. Therefore, we determined the role of IL-1 in Mtb30-induced CSF production, in vitro. Incubation of PMs with Mtb30 along with neutralizing (100%) rabbit anti-mouse IL-1 polyclonal antibody (Endogen Inc., Boston, MA, USA) did not block Mtb30-induced production of CSFs (( figure 6 )), thereby, excluding the role of IL-1 in CSF production, in vitro. To exclude the role of TNF-α that may have been produced by Mtb30-treated PMs [17], in CSF production, PMs were stimulated with Mtb30 in the presence of 100% neutralizing rabbit anti-mouse TNF-α polyclonal IgG (R & D Systems). Data in ( figure 6 ) show that anti-mouse TNF-α IgG only partially blocked the production of CSFs. Polymyxin B (25 μg/mL), an antibiotic that can neutralize the biological activities of LPS, did not inhibit the Mtb30-induced CSF production (data not shown). Further, polymyxin B had no effect on the basal production of CSFs by unstimulated PMs or on the responsiveness of the committed progenitor BM cells to CSFs (data not shown). The Mtb30-induced CSF production was de novo as it was completely inhibited by cycloheximide (50 μg/mL; ( figure 7 )). The CSF-inducing capability of Mtb30 was destroyed by proteases (pronase E and trypsin), and was unaffected by periodate treatment (( figure 8 )).

Effect of rabbit anti-Mtb30 polyclonal antibody on the CSF elaboration by M. tuberculosis-infected PMs

To demonstrate whether whole, mycobacteria-induced CSF elaboration was through an Mtb30-dependent process, PMs infected with live and killed mycobacteria were cultured in the presence or absence of rabbit anti-Mtb30 polyclonal antibody, and the CSFs elaborated in the CM were measured. Infected-PMs cultured without rabbit anti-Mtb30 antibody elaborated CSFs that could be detected as early as on day one, peaked on day +3 (182 ± 18 colonies) and then plateaued thereafter (( figure 9 )). Contrastingly, incubation of infected-PMs with rabbit anti-Mtb30 polyclonal antibody significantly (p<0.05) reduced the peak CSF (42 ± 6 colonies) elaboration on day +3 and at all the other time-points. Heat-killed mycobacteria induced significantly (p<0.05) less CSF production on day +2, which declined thereafter.

Mtb30-induced GM-CSF, M-CSF and G-CSF production in serum and CM

Mtb30-treated mice (1 mg/kg) and PMs (25 μg/mL) showed significantly (p<0.05) high GM-CSF levels in serum (9 ± 1 ng/mL) and CM (7.5 ± 0.8 ng/mL), respectively, as compared to the controls, 24 h later (( figure 10 )). Similarly, the levels of M-CSF in both serum (4.3 ± 0.5 ng/mL) and CM (3.9 ± 0.4 ng/mL) were also significantly (p<0.05) higher than the controls, after 24 h of Mtb30-stimulation (( figure 11 )). However, Mtb30-treatment did not induce significant (p>0.05) G-CSF production in either serum or CM (data not shown).

Hematopoietic activity in the spleen and BM of Mtb30-treated mice

The spleen and BM of mice, 24 hours after Mtb30 (0.1–10 mg/kg) administration, showed a maximum of 2.23- and 2.36-fold increases in CFU-GM counts, respectively, with 1 mg/kg Mtb30, compared to those given vehicle only or HI-Mtb30 (table 1( Table 1 )).
Table 1 CFU-GM counts in the spleen and BM of Mtb30-treated mice ^a

Mtb30 (mg/kg)	CFU-GM/spleen ^b (no. x 10³)			CFU-GM/femur ^b (no. x 10³)
	0 h	24 h	48 h	0 h	24 h	48 h
Vehicle	1.2 ± 0.3	1.3± 0.2	1.1 ± 0.2	1.0 ± 0.2	1.1 ± 0.1	1.2 ± 0.1
0.1	1.1 ± 0.2	1.8 ± 0.6	1.3± 0.1	1.1 ± 0.2	1.5± 0.3	1.2± 0.2
0.5	1.2 ± 0.2	2.2 ± 0.4	1.4 ± 0.1	1.1 ± 0.3	2.0 ± 0.3	1.3 ± 0.1
1.0	1.3± 0.3	2.9 ± 0.6*	1.4± 0.1	1.1 ± 0.1	2.6 ± 0.5*	1.4 ± 0.4
5.0	1.0 ± 0.2	2.4 ± 0.8*	1.2 ± 0.2	1.0 ± 0.1	2.3 ± 0.8*	1.1 ± 0.1
10.0	1.0 ± 0.1	2.3 ± 0.6*	1.2 ± 0.1	1.1 ± 0.3	2.0 ± 0.6*	1.2 ± 0.2
HI-Mtb30 (10 mg/kg)	1.0 ± 0.2	1.1 ± 0.1	1.0 ± 0.1	1.0 ± 0.3	1.0 ± 0.1	1.1 ± 0.1

^aSpleen and BM cells (2x10⁵ and 3x10⁵ cells/dish, respectively) from Mtb30-treated mice were cultured in CFU-GM assay medium (1 mL) for 48 h at 37 °C in a 5% CO₂-air atmosphere. For controls, mice were treated with vehicle only or HI-Mtb30.

^bAfter seven days, CFU-GM colonies were counted. Data are mean number of colonies ± SD of three separate experiments, run in triplicate.

Discussion

The most important observation of this study was that Mtb30 can induce the synthesis and secretion of serum CSFs in mice, and can induce MØs to elaborate CSFs, in vitro. Both in vivo and in vitro, elaboration of GM-CSF appeared to be the major activity. Additionally, the Mtb30-treated mice showed increased hematopoietic activity both in the spleen and BM. Our observations, therefore, appear to be in line with earlier reports suggesting the induction of GM-CSF in mycobacteria-infected monocytes and large granular lymphocytes [39, 47]. Interestingly, our earlier reports, which demonstrate the plasmodial [44] and leishmanial [48] antigen-induced production of CSFs are also in consonance with our present observations. Because CSFs are an important group of pro-inflammatory cytokines, and are known to play definitive role(s) in host defense [49, 50], their production by M. tuberculosis secretory component(s) may have considerable bearing on the outcome of infection.

The Mtb30-induced CSF production, both in vivo and in vitro, appeared to be dose/concentration-dependent within the dose/concentration limits studied. Whereas, a single Mtb30 stimulus of as low as 0.1 mg/kg (in vivo) and 1 μg/mL (in vitro) could trigger the onset of the induction of CSF production activity, a maximum production occurred only at 1 mg/kg and 25 μg/mL, respectively. At higher doses/concentrations the response became static suggesting the saturation of the induction mechanism(s). Furthermore, the in vivo and in vitro kinetics of Mtb30-induced CSF production also appeared to be similar, with peak production occurring after 24 h and which levelled-off to background levels by 72 h, possibly due to the “turning-off” of the stimulatory signal through decay and/or the possible initiation of the IL-10-mediated “negative feed-back loop”, resulting in the cytokine inhibitory functions. Nevertheless, after another 48 h in culture in fresh medium, these seemingly unresponsive MØs were fully responsive to a fresh Mtb30 stimulus (data not shown). Further, MØs from three different anatomical sites were used in this study. Elicited PMs were used because TB is known to cause chronic inflammation that results in the activation of MØs. Because in TB the bulk of MØs that infiltrate the infected-foci are recently derived from the bone-marrow [40], BM-derived MØs were tested for their responsiveness to Mtb30. The spleen, on the other hand, is a highly immunocompetent organ in the body and a site where M. tuberculosis grows and multiplies, and hence the splenic MØs were also tested for their potential to elaborate Mtb30-induced CSFs. Curiously, all three types of MØs, irrespective of their anatomical origin and activation status, responded to Mtb30 similarly. The MØs used in this study were depleted of T-cells. Therefore, the CSFs elaborated in the CM, to a large extent, must have been released from the Mtb30-stimulated MØs only. On the other hand, purified T-cells from the spleen and blood of mice, following in vitro stimulation with Mtb30, did not elaborate CSFs (data not shown).

The mechanism(s) of Mtb30 interaction with MØs remains unclear. However, our results, which demonstrate that the Mtb30-induced CSF production was saturated by both dose/concentration and time, and was specifically inhibited by rabbit anti-Mtb30 polyclonal antibody suggest it to be a ligand-receptor interaction. Additionally, these observations clearly demonstrate that Mtb30 was a potent inducer of CSF production, and thus provide unequivocal evidence that this purified secretory component of M. tuberculosis can induce CSF production. This contention is further supported by our observations showing the rabbit anti-Mtb30 polyclonal antibody inhibition of the elaboration of CSFs by M. tuberculosis-infected PMs, in vitro, and thus demonstrate that mycobacteria induced CSF elaboration through a Mtb30-dependent process. The CSF-inducing capability of Mtb30 appeared to be proteinaceous in nature. Firstly, it was heat-labile (70 °C; 1 h). Secondly, proteolytic enzymes (pronase E and trypsin) abrogated CSF induction. Finally, sodium periodate treatment did not affect the production of CSFs. Nevertheless, besides Mtb30, several other proteins of M. tuberculosis may also induce the production of CSFs.

The functional and molecular properties of the CSFs are mirrored by the formation of the colony types they support. Thus, our data, which demonstrate the similar, if not identical, proportion of G, M, and GM colony formation under the influence of Mtb30-induced CSFs present both in the serum and CM, suggest that they were functionally and molecularly similar. Additionally, the GM-CSF appeared to be the major activity in CSFs from both of these sources as indicated by the maximum proportion of GM colonies formed in vitro, as well as by the increased GM-CSF production as determined by ELISA. Furthermore, our data also indicated that quantitatively, the extent of colony formation induced by CSFs from both serum and CM appeared to be same. Taken together, our results demonstrate a commonality in the underlying molecular mechanism(s) of CSF induction both in vivo and in vitro. Curiously, the serum from M. tuberculosis H37Rv-infected mice also induced the increased formation of colonies in similar proportions (data not shown).

The Mtb30-treated mice, as compared to the controls, showed up to 2.23- and 2.36-fold increases in the CFU-GM counts, in the spleen and BM, respectively. The M. tuberculosis secretory component(s)-induced local production of CSFs by MØs at these sites might be a strategy employed by this highly adapted pathogen to maintain a continuous supply of myeloid cells (“safe sanctuaries”) for its own survival and persistence. Additionally, the locally produced CSFs may also increase the terminal differentiation/maturation and activation of the infiltrating MØs.

Bronchoalveolar lavage cells of TB patients synthesize increased quantities of IL-1β and TNF-α [51]. The mycobacterial proteins have been reported to induce IL-1 and TNF in monocytes [52], and Mtb30 has been demonstrated to induce production of TNF-α by monocytes [17]. Furthermore, both M. bovis BCG-infected and uninfected bystander MØs have also been shown to elaborate TNF-α in their CM [53]. Because both IL-1 and TNF-α are known to induce the production of CSFs [54, 55], the CSF induction observed in this study can be attributed to them. Therefore, to gain insight into the possibility that IL-1 and TNF-α may mediate the Mtb30-induced CSF production, we generated evidence that demonstrates the lack of the effect of neutralizing (100%) concentrations of anti-mouse IL-1 antibody on the Mtb30-induced production of CSFs by MØs, in vitro; whereas, neutralizing (100%) concentrations of anti-mouse TNF-α IgG, only partially blocked it. Our results therefore, suggest that the Mtb30-induced production of CSFs was IL-1-independent, but was only partly attributable to TNF-α.

The physiological relevance of highly immunoprotective protein Mtb30 [4], is incompletely understood, and appears very complex. Mtb30 may modulate cytokine production by binding to fibronectin on the mycobacterial surface and thus play role in the immunopathogenesis of TB [56]. Mtb30 induces mononuclear phagocytes to secrete TNF-α [56], and this, in turn, is known to increase Mtb30 expression [57]. This reciprocal production of TNF-α-induced Mtb30 may be further enhanced by the infiltration by the T-cells at the sites of infection, as Mtb30 is a strong inducer of IFN-γ [58] that is known to increase TNF-α production [57]. Furthermore, in mycobacteria-infected mice, TNF-α is involved in microbicidal granuloma formation [59], and has been reported to both suppress [60, 61] and enhance the growth of mycobacteria [62]. In latent M. tuberculosis infection, neutralizing TNF-α therapy has been associated with reactivation [63]. The TNF-α-dependent apoptosis in M. tuberculosis-infected phagocytes is thought to be a protective mechanism [64]. Thus, whether TNF-α plays a protective or pathological role in TB, remains unclear. In vivo, TNF-α functions as a dose-dependent, double-edged sword, by mediating protection at low levels, whereas at high concentrations it provokes necrosis [65]. It should be noted here that, as compared to the less-virulent M. tuberculosis H37Ra strain, the highly virulent H37Rv strain induced greater expression of both Mtb30 and TNF-α mRNA [66]. Recently, Mtb30 has also been shown to induce macrophage deactivating cytokines TGF-β [19] and IL-10 [20]. Thus Mtb30 appears to influence the in vivo cytokine milieu, and the interaction and the final balance of the macrophage-activating and immunoenhancing, and macrophage deactivating and immunosuppressive cytokines most likely determines the final outcome of the infection. Because CSFs function as proinflammatory and regulatory cytokines, the results of this study, which unequivocally demonstrate Mtb30-induced production of CSFs, apparently support this contention. Thus, Mtb30 appears to play extended central role(s) in the immunoregulation and immunopathogenesis of TB, in addition to several other, yet-to-be defined functions.

The precise biological role(s) of CSFs in TB is not completely understood. GM-CSF has been reported to activate MØs to kill mycobacteria, in vitro [34, 35]. Treatment of M. avium-infected mice with rmG-CSF has been reported to exert a protective effect [67]. In beige mice, rmGM-CSF has also been reported to enhance the effects of antibiotics against M. avium complex infection [68]. GM-CSF therapy enhances the uptake and the mycobactericidal activity of monocytes from AIDS patients with M. avium bacteremia [37, 38]. The results of the first ever clinical trial of recombinant human GM-CSF in pulmonary TB patients indicate its promise as adjuvant therapy [69]. Our results, which demonstrate Mtb30-induced production of CSFs, may have two potential implications: that the induced CSFs may merely serve to increase the number of phagocytes, which are the host cells for the M. tuberculosis expansion, as well as for increasing the phagocytic uptake of the pathogen, and that the CSFs produced may increase the mycobactericidal activity of the MØs. Overall, it is conceivable that the control of TB infection is determined by the maintenance of a balance between these two seemingly paradoxical functions of CSFs. Therefore, CSFs appear to play important role(s) in the pathogenesis of TB, wherein the immune competence of the host is integral to disease state. Detailed studies along these lines should provide greater insights into the molecular mechanism(s) of the pathogenesis of this highly successful, ancient pathogen of humanity.

Acknowledgements

We are grateful to Dr. C. L. Kaul, Director, National Institute of Pharmaceutical Education and Research, for his help and continued encouragement. Mrs. Sukhraj Kaur is grateful to the Council of Scientific and Industrial Research, New Delhi, India, for the award of a Senior Research Fellowship (National Entrance Test). This work was partly funded by a research grant to Dr. Prati Pal Singh, by the Indian Council of Medical Research, New Delhi, India, out of which Ms. Harsimrat Kaur received a Junior Research Fellowship. The technical assistance provided by Mr. Vijay Kumar Misra is greatly acknowledged. “Communication No. 305”

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