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Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. Bibliography

The protection against Mycobacterium tuberculosis infection is mediated by T helper type-1 (Th1) cells. Infection of BALB/c mice with M. tuberculosis downregulates expression of a Th1-specific costimulatory molecule, M150, on the surface of infected macrophages. The proliferation of Th cells and Th1-cytokine production by these cells are higher in case of M. tuberculosis antigen presentation by uninfected macrophages than by infected macrophages. The difference in inducing interleukin(IL)-2 and interferon (IFN)-γ secretion is abolished by providing bystander costimulation through M150 on liposomes.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. Bibliography

Mycobacterium tuberculosis (Mtb), an obligatorily intramacrophagic bacterium, is the causative agent of tuberculosis [ 1]. The clinical features displayed in humans are also manifested in murine models [ 2]. A major hallmark of the disease is the suppression of cell-mediated immunity (CMI) shown in terms of tuberculin skin test anergy, proliferative block and decreased IL-2 secretion [ 2, 3]. An efficient antimycobacterial CMI is mounted by Th1 cells [ 2, 4] which secrete IL-2 and IFN-γ [ 5]. The function of Th1 cells is inhibited by a Th subset, Th2 [ 6], producing IL-4 and IL-10, which also deactivate the antimycobacterial [ 7] functions of macrophages. In a susceptible host, the Th1 subset loses its function resulting in predominance of Th2 subset while a resistance host continues to maintain Th1 function. Although the mechanism of the downregulation of the Th1 function during the progressive infection is proposed to be an inappropriate T-cell costimulation through B7-1 and ICAM-1 [ 8], none of these has been conclusively shown to be Th1 specific.

We discovered M150, a Th1 specific costimulatory molecule of 150 kDa, on the unstimulated macrophage surface but not on resting B cells [ 9, 10]. The molecule, when reconstituted on liposomes, costimulates anti-CD3 stimulated T cells to generate Th1 cytokines [ 9] which can be blocked by anti-M150 antibody [ 10]. We showed earlier that in an infection with a protozoan parasite, Leishmania donovani, where the suppression of the Th1 function is well documented, the expression of M150 on L. donovani-infected macrophages decreased and that the addition of liposomized M150 to the T-cell culture restored the Th1 function [ 11]. Therefore, we have designed experiments to check the expression of M150 on Mtb-infected macrophages, and to test whether or not providing the trans-costimulation through the liposomized M150 restores the Th1 function in tuberculosis.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. Bibliography

Animals and experimental infection with M. tuberculosis

BALB/c mice, 8–10-weeks-old, were initially procured from the National Institute of Immunology, New Delhi, and were subsequently reared and supplied from the Central Animal Facilities of Institute of Microbial Technology, Chandigarh. M. tuberculosis, H37Rv (a kind gift of Dr G.K. Khullar, PGIMER, Chandigarh, India), grown in Sauton's medium, harvested, washed three times with 0.15 m phosphate-buffered saline (PBS) (pH 7.2), and were injected intraperitoneally with a dose of 1 × 107 colony-forming units (CFU)/mouse [ 8]. Five to seven mice were used for each experimental group and age- and sex-matched control group.

Preparation of crude soluble antigen (CSA) of M. tuberculosis and immunization of mice

The Mycobacterium cultures were harvested, washed three times in PBS (pH 7.4), frozen and thawed, sonicated and clarified by microfugation for 30 min at 4 °C. The supernatant is considered as CSA as described earlier [ 8]. BALB/c mice were immunized with CSA (100 μg/mouse) in IFA (Gibco, Grand Island, NY, USA) subcutaneously in the foot-pad.

Isolation and reconstitution of the M150 molecule in liposomes

M150 was purified from the surface of peritoneal macrophages as described elsewhere [ 9]. Briefly, peritoneal exudate cells from BALB/c mice were harvested, lysed and homogenized in a buffer (pH 7.4) containing 0.25 m sucrose, 20 m m Tris-HCl, 1 m m EDTA and protease-inhibitor cocktail. After removing the debris, the supernatant was centrifuged at 110 000 × g for 2 h. The pellet was solubilized in 1% tritonX-100, 20% glycerol, 20 m m Tris-HCl (pH 7.5), spun at 100 000 × g for 1 h, and the proteins from the supernatant were separated by SDS-PAGE. The 150 kDa band was eluted with 0.1% SDS, 0.05 m Tris-HCl (pH 8.0), 0.1 m m EDTA and 0.15 m NaCl. The sample was dialyzed against 0.01% SDS, 0.01 m Tris-HCl, 0.01 m m EDTA, and 0.1 m NaCl with at least four changes in buffer. The protein was quantitated and used for reconstitution into liposome as described below. Liposomal vesicles were prepared by dissolving l-α-phosphatidyl choline (PC; Sigma Chemical Co., St. Louis, MO, USA) in a 1 : 1 ratio of chloroform and methanol. Solvents were evaporated under nitrogen while rotating the vial to deposit a thin film. The lipids were subsequently dried under vacuum for 2 h and were dissolved in 10 m m Tris-HCl (pH 8.0), 0.5% Lubrol-Px, 140 m m NaCl and 0.1 m m EDTA. This was followed by sonication at 4 °C in a bath type sonicator (Branson Ultrasonics Corporation, CT, USA) for 30 min. M150 and vesicles were mixed at a ratio of 500 : 1 and vortexed to acquire a homogeneous solution. The contents were dialyzed at 4 °C for 4 days against 10 m m Tris-HCl(pH 8.0), 140 m m NaCl and 0.1 m m EDTA with eight changes of buffer. The dialyzed material was then centrifuged for 2 h at 4 °C at 178 000 × g. The pellet was resuspended in normal saline. The liposomized M150 were purified and sterile filtered as described earlier [ 9].

Preparation of antigen-specific Th cells

Antigen-specific Th cells were prepared as described earlier [ 9]. Briefly, CD4+ T cells were purified from lymph nodes of CSA-primed BALB/c mice and cultured with mitomycin C-treated feeder cells in the presence of 100 μg/ml of CSA. The cells were harvested on the fourth day, run on Ficoll–Hypaque (Sigma Chemical Co.) to separate the live cells at the interface and re-cultured with mitomycin C-treated syngeneic spleen cells. The antigen-specific T cells were enriched for four such cycles of activation and were assayed by increased stimulation index (data not shown). These cells did not show any skewing towards Th1 or Th2 cells as assessed by cytokine profiles (data not shown).

Isolation of M. tuberculosis-infected macrophages

The infected macrophages were isolated from the splenocytes of Mtb-infected BALB/c mice as described earlier [ 12]. Briefly, the cell suspension was loaded on a discontinuous percoll gradient (75–63–55–40–30%) and centrifuged at 1600 × g at 4 °C for 30 min. Cells between the 30–40% interface containing 95–97% infected macrophages were taken out, washed with PBS-5% fetal calf serum (FCS), and stained for M150 expression or treated with paraformaldehyde (1% at 37 °C,30 min) to use as antigen-presenting cells (APC).

M150 expression on macrophages

Syrian hamsters were immunized with M150 purified from mouse in incomplete Freund's adjuvant intraperitoneally (50 μg/hamster). The hamsters were given a boost 3 weeks after priming. The sera collected from unprimed hamsters or preimmune animals were compared with sera from the primed hamsters for M150 staining on Western blots of murine macrophage membrane antigens [ 9]. The sera were also used for staining macrophages for the M150 molecule. The unprimed or preimmune hamster sera did not stain any band on Western blots. Analysis of M150 expression was performed as described earlier [ 10, 11].

T-cell proliferation assay

T-cell proliferation assay was performed using a standard 3H-TdR incorporation assay. 3 × 104 antigen-specific resting CD4+ T cells were cultured in 200 μl of RPMI 1640 complete medium (10% FCS, 2 m ml-glutamine, 5 × 10−5 M 2-ME, 10 m m HEPES, 10 m m sodium bicarbonate, penicillin (100 μg/ml), streptomycin (75 μg/ml) in 96-well plates (Costar, Cambridge, MA, USA) in the presence of plate-bound anti-CD3 (clone 145.2C11; 2 μg/ml; a kind gift of C.A. Janeway, Jr. Yale University, New Haven, CT, USA) for 3 days at 37 °C. A second signal was provided by either uninfected or infected macrophages. In some experiments, antibody against M150 or liposomized M150 was added to block or reconstitute the costimulatory signal through M150.

Lymphokine assays

The 24 h culture supernatants from the experimental and control wells as described above were collected and assayed for cytokine content. IL-2 and IL-4 were estimated by their ability to induce the proliferation of HT-2 cells as reported earlier [ 9] and IFN-γ was measured by its inability to inhibit the proliferation of WEHI-279 cells as described earlier [ 9]. 1 × 104 HT-2 cells were cultured in 96-well plates with medium or various concentrations of the supernatants of the experimental and control cultures or recombinant cytokines. While assaying for IL-2, neutralizing antibody to IL-4 (11B11, 600 ng/ml) was used to inhibit the IL-4 mediated proliferation of HT-2 cells. While assaying IL-4, antibodies against IL-2 and IL-2 receptor were used. The cells were cultured for 16 h at 37 °C containing 5% CO2. The cultures were pulsed with 1μCi of 3H-TdR for 8 h and then harvested. The 3H-thymidine incorporation was measured by liquid scintillation and expressed as units/ml as deduced from standard graph. On the other hand, WEHI-279 cells are sensitive to IFN-γ so that the extent of WEHI-279 growth inhibition is dependent on the concentration of IFN-γ added to the culture. Therefore, WEHI-279 cells were cultured in 96-well plate at a density of 1 × 105 cells/ml with different concentration of test supernatants for 24 h. Cells were pulsed with 1μCi of 3H-TdR for the last 8 h and harvested. The tritiated thymidine incorporation was measured by liquid scintillation and presented as percent inhibition. Specificity of the assay was ensured by neutralizing anti-IFN-γ antibody at a concentration of 4 μg/ml [ 9].

RESULTS AND DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. Bibliography

Because the susceptibility to M. tuberculosis infection is associated with the loss of Th1 function [ 2, 3, 8], and a Th1-specific costimulation is delivered by M150 [ 9–11], we have tested the possible role of M150 in depressed antimycobacterial Th1 function. The Mtb-infected splenic macrophages express approximately 50-fold less M150 than the uninfected macrophages ( Fig. 1A) suggesting a role for M150 in the disease. In order to investigate its role in the T-cell function, mycobacterial antigen-specific CD4+ T cells were stimulated with anti-CD3 in the presence of uninfected or Mtb-infected macrophages. It was registered that T-cell proliferation significantly less (P < 0.001) in the culture where infected macrophages were used as APC ( Fig. 1B) suggesting that infected macrophages are deficient in providing costimulation. While addition of liposom-M150 restored the T-cell proliferation significantly (P < 0.001), anti-M150 antibody inhibited the T-cell proliferation in the presence of normal macrophages. Liposomes alone do not augment the anti-CD3 induced T-cell proliferation in the presence of infected or uninfected macrophages (data not shown). These observations together indicate that downregulation of M150 on Mtb-infected macrophages may cause decreased T-cell expansion in vivo.

image

Figure . 1. (A) M150 expression on M. tuberculosis infected splenic macrophages. Compared to normal macrophages (normal), expression of M150 is decreased in M. tuberculosis-infected BALB/c derived splenic macrophages (infected). Unprimed hamster sera was used as control primary antibody (control). The secondary antibody used was goat antihamster-fluorescein isothiocyanate (FITC). (B) Role of M150 in T-cell costimulation. Ability of uninfected and percoll-separated, paraformaldehyde-fixed M. tuberculosis infected splenic macrophages to augment anti-CD3 induced T-cell proliferation is assessed in presence of anti-M150 antibody (hatched bar in left panel) or liposomized M150 (hatched bar in right panel). The data presented here is from one of three individual experiments.

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Because T cells are anaergized by T-cell receptor (TCR)-mediated signal in absence of an appropriate second signal [ 13], we tested if downregulation of M150 on Mycobacterium-infected macrophages caused T-cell anergy. Antigen-specific T cells were stimulated with anti-CD3 in presence of either uninfected macrophages with or without anti-M150 antibody or infected macrophages with or without liposomized M150 for 24 h. In order to assess the extent of anergy, interacted T cells were purified and rested for 2 days and restimulated with anti-CD3 in the presence of normal macrophages. It was found that the T cells which interacted with the infected macrophages first, proliferated significantly less than those which interacted initially with the normal macrophages (P < 0.01; Fig. 2). Inclusion of liposome-M150 in the infected macrophage culture significantly enhanced the T-cell proliferation (P < 0.01; Fig. 2) suggesting that the Mtb-specific T cells are anaergized owing to downregulation of M150 on infected macrophages.

image

Figure . 2. The cells were harvested 48 h after the initiation of the above said cultures ( Fig. 1B), run on Ficoll-Hypaque and washed. T cells were cultured in presence of plate-bound anti-CD3 and paraformaldehyde-treated normal splenic macrophages for 72 h and assayed for proliferation as described in MATERIALS AND METHODS. The data presented here is from one of three individual experiments.

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Because CD4+ effector T cells can be broadly categorized into two subsets — Th1 and Th2 — on the basis of the cytokines they secrete [ 5], we attempted to identify the subset undergoing anaergy by measuring the cytokines in the supernatant of the same culture which were assayed for proliferation. It was observed that CD4+ T cells cultured with normal macrophages and anti-CD3 produced IL-2, IL-4 and IFN-γ suggesting the absence of any bias to any of the Th subsets ( Fig. 3). On the other hand, production of IL-2 and IFN-γ but not IL-4 was reduced upon interaction with the infected macrophages and by blockade of M150-mediated costimulation suggesting a Th1-specific effect. The observation is supported by the finding that the M150-mediated trans-costimulation in infected macrophage T-cell coculture induced IL-2 and IFN-γ secretion with no significant difference from the T-cell cultures presented by uninfected macrophages ( Fig. 3).

image

Figure . 3. Cytokine production by T cells in the above mentioned cultures ( Fig. 1B). Interleukin (IL)-2 and IL-4 were assayed by HT-2 bioassay in presence of either anti-IL-4 (11B11) or a combination of anti-IL-2 and anti-IL-2 receptor, respectively. Interferon(IFN)-γ was assayed by the extent of growth inhibition of WEHI-279 in presence of IFN-γ in recombinant IFN-γ or in test samples. The specificity of IFN-γ mediated growth inhibition [as described in 16]. The data presented here is from one of three individual experiments.

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Therefore, in conclusion, the observations suggest that infection of macrophages with Mtb causes lowered M150 expression eventuating in the decreased production of Th1 type of cytokines. Because M150 is a Th1 specific costimulatory molecule, it restored the Th1 cytokine level in the T-cell culture supernatant with concomitant decrease in IL-4, a pro-mycobacterial cytokine. Although the Mtb differs from Leishmania, a protozoan parasite, in many different aspects, the common feature of depressed CMI in both the infections is perhaps mediated by reduced cell surface expression of M150 on macrophages. However, as a finer tuning to this evasion strategy, whether Mtb exert any differential effect on M150 expression and other costimulatory molecules or whether M150 has interactions with the other costimulatory molecules remains an open question. Nevertheless, our observation that a bystander costimulation restores the normal T-cell response not only confirms the observation made by Ding et al. [ 14] but also shows its importance in treating the diseases caused by marked decrease in Th1 functions and Th subset re-polarization [ 15].

Bibliography

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. Bibliography
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