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Keywords:

  • BCG;
  • cytokine mRNA;
  • guinea pig;
  • T cells;
  • TNF-α

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

Previous studies from our laboratory demonstrated that treatment in vitro with recombinant guinea pig tumour necrosis factor TNF (rgpTNF)-α-enhanced T cell and macrophage functions. Similarly, injection of Mycobacterium tuberculosis-infected guinea pigs with anti-TNF-α altered splenic granuloma organization and caused inflammatory changes and reduced the cell-associated mycobacteria in the tuberculous pluritis model. In this study, rgpTNF-α was injected into bacille Calmette–Guérin (BCG)-vaccinated guinea pigs to modulate immune functions in vivo. Guinea pigs were vaccinated intradermally with BCG, 2 × 103 colony-forming units (CFU) and injected intraperitoneally with either rgpTNF-α (25 µg/animal) or 1% bovine serum albumin (BSA) for a total of 12 injections given every other day. Treatment with rgpTNF-α significantly enhanced the skin test response to purified protein derivative (PPD), reduced the number of CFUs and increased the PPD-induced proliferation in the lymph nodes at 6 weeks after vaccination. The levels of interleukin (IL)-12 mRNA were increased in the lymph node and spleen cells stimulated with PPD. TNF-α treatment induced a decrease in TNF-α, IL-12p40 and IL-10 mRNA levels in peritoneal cells following PPD stimulation while live M. tuberculosis caused an increase in TNF-α mRNA and a decrease in the IL-10 mRNA expression. TNF-α injection also induced an increase in the infiltration of mononuclear cells and in the proportions of CD3+ T cells in the lymph nodes. These results indicate that rgpTNF-α enhances some aspects of T cell immunity and promotes control of mycobacteria in the tissues. Future studies will address the role of TNF-α in BCG-vaccinated guinea pigs following low-dose pulmonary challenge with virulent M. tuberculosis.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

Among the many cytokines that contribute to a protective immune response against Mycobacterium tuberculosis, tumour necrosis factor (TNF)-α is known to play an essential role in the formation and maintenance of granulomas [1,2]. Resistance against M. tuberculosis is mediated by T cells and macrophages [3–5]. Several cytokines, including interleukin (IL)-12, IL-17 and IL-23, contribute to the host-response to mycobacteria by enhancing the development of T helper type 1 (Th1) immunity [6,7]. Among the Th1 cytokines, interferon (IFN)-γ and TNF-α have been identified as the most important players in the cytokine cascade for anti-mycobacterial immunity because the formation as well as the maintenance of the granuloma are mediated by TNF-α, and it synergizes with IFN-γ in activating macrophages for the production of effector molecules [2,8]. It is known that susceptibility to tuberculosis occurs with defects in the type-1 cytokine pathway in humans [9,10]. The importance of IFN-γ has been well established in mouse models, as disruption of IFN-γ, the IFN-γ receptor gene or components of the IFN-γ receptor signal-transducing chain resulted in an exacerbation of disease after M. tuberculosis or M. bovis infection [9,11]. Neutralization of TNF-α in mice resulted in the reactivation of latent M. tuberculosis infection, disrupted granuloma formation and rapid death [12]. In another study, neutralization of TNF-α resulted in marked disorganization of granulomas and an increase in proinflammatory cytokine and chemokine expression in mice given an aerosol infection with M. tuberculosis[13]. Mice deficient for TNF-α or TNF-R1 showed disruption in granuloma formation and succumbed to infection with M. tuberculosis[14]. The importance of TNF-α in anti-mycobacterial immunity has been reinforced by reports that the use of TNF-α neutralizing antibody in the treatment of chronic inflammatory diseases resulted in the reactivation of latent tuberculosis in humans [15], [10]. Several reports also indicate that injection of mice with recombinant TNF-α or IFN-γ alone or in combination was associated with decreased microbial growth and increased survival after infection with disseminated M. avium complex or M. tuberculosis[16,17]. Injection with TNF-α caused the recruitment of neutrophils and dendritic cells, as well as T cells to lungs or spleen in mice [13,17–19].

We showed that in vitro treatment of spleen cells with recombinant guinea pig TNF-α (rgpTNF-α) and neutralizing anti-gpTNF-α anti-serum modulated antigen-specific T cell proliferation in guinea pigs [20,21]. Injection of anti-TNF antibody into bacille Calmette–Guérin (BCG)-vaccinated and non-vaccinated guinea pigs following low-dose aerosol challenge with virulent M. tuberculosis resulted in splenomegaly in the BCG-vaccinated guinea pigs, while it augmented splenic granuloma organization in the non-vaccinated guinea pigs [22]. Furthermore, direct intrapleural injection of anti-TNF antibody into guinea pigs with tuberculous pleuritis altered the inflammatory exudates by decreasing the proportions of macrophages and increasing the neutrophil and lymphocyte proportions [23].

The purpose of this study was to determine whether administration of rgpTNF-α into guinea pigs would mimic the effects as demonstrated in our in vitro studies and whether recombinant TNF-α would enhance immune responses induced by BCG vaccine. Our results indicate clearly that low doses of TNF-α, a major player in both innate and specific acquired immunity, could augment BCG vaccine-induced immunity in the guinea pig, a relevant model that mimics human tuberculosis in terms of tissue pathology, protection afforded by BCG vaccination and granuloma organization.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

Animals

Random-bred Hartley strain guinea pigs weighing 250–350 g obtained from Charles River Breeding Laboratories, Inc. (Wilmington, MA, USA) were used for this study. The animals were housed individually in polycarbonate cages in a temperature- and humidity-controlled environment with a 12-h light/12-h dark cycle. They were given commercial chow (Ralston Purina, St Louis, MO, USA) and tap water ad libitum. All procedures were reviewed and approved by the Texas A&M University Laboratory Animal Care Committee.

BCG vaccination and TNF-α injection

Two groups of guinea pigs were vaccinated intradermally with 1 × 103 colony-forming units (CFU) of M. bovis BCG (Danish 1331 strain; Statens Seruminstitut, Copenhagen, Denmark) each in the left and right inguinal regions. The lyophilized vaccine was reconstituted with Sauton's medium (Statens Seruminstitut) for injection. Beginning immediately after vaccination, the animals were injected intraperitoneally with either rgpTNF-α (25 µg/animal) or 1% bovine serum albumin (BSA) for a total of 12 injections given every other day. The recombinant TNF-α protein was expressed in a prokaryotic vector using the M15 Escherichia coli strain transformed with pQE-30/gpTNF-α[24]. The functional properties of rgpTNF-α, including bioactivity, were determined by measuring the cytotoxicity on L929 cells and cytokine mRNA expression by real time-reverse transcription–polymerase chain reaction (RT–PCR) and the anti-mycobacterial activity of macrophages by metabolic labelling of M. tuberculosis with [3H]-uracil, as reported previously [20,21,24,25].

Skin test response, necropsy and CFU assay

Six weeks after BCG vaccination, approximately 3 weeks following the last injection of rgpTNF-α, guinea pigs were injected with 0·1 ml purified protein derivative (PPD) (2 µg, kindly gifted by Dr Saburo Yamamoto, BCG Laboratories, Tokyo, Japan) on the ventral skin and the diameter of induration was measured 24 h later. The animals were then euthanized by the injection of 3 ml sodium pentobarbital (Sleepaway™, Fort Dodge Animal Health, Fort Dodge, IA, USA). Spleen, lymph node and peritoneal cells were collected for study. For assessing the effect of TNF-α injections on bacterial loads, lymph nodes and spleens were processed for CFU, as described previously [26]. Serial dilutions of tissue homogenates were plated on Middlebrook 7H9 agar and the colonies counted after 3 weeks. The CFU data were transformed into log10 per tissue from five to six guinea pigs per group.

Cell preparation

The lymph node and spleen cells were incubated in RPMI-1640 (Irvine Scientific, Santa Ana, CA, USA) medium supplemented with 2 µM glutamine (Irvine Scientific), 0·01 mM 2-mercaptoethanol [2-mercaptoethanol (ME); Sigma, St Louis, MO, USA], 100 U/ml of penicillin (Irvine Scientific), 100 µg/ml of streptomycin (Irvine Scientific) and 10% heat-inactivated fetal bovine serum (FBS) (Atlanta Biologicals, Norcross, GA, USA). Spleen cells were prepared by homogenizing the tissue in a glass homogenizer as described earlier [26]. Single cell suspensions obtained were centrifuged at 440 g for 10 min, the pellet resuspended in ammonium chloride (ACK) lysis buffer [0·14 M NH4Cl, 1·0 mM KHCO3, 0·1 mM Na2 ethylemediamine tetraacetic acid (EDTA) (pH 7·2 to 7·4)], washed three times in RPMI-1640 medium by centrifuging for 10 min at 320 g, and the viability determined by the trypan blue exclusion method.

The peritoneal cells were harvested as reported earlier [26,27]. After euthanizing the guinea pigs, the peritoneal cavity was flushed three to four times with 20 ml of cold RPMI-1640 containing 20 U of heparin (Sigma). The erythrocytes were lysed using the ACK lysing buffer, the cells were washed with complete RPMI-1640 medium and the viable cells were counted by the trypan blue exclusion method. The cells were suspended at 5 × 106 cells/ml in RPMI-1640 medium supplemented with glutamine, 2-ME, penicillin/streptomycin and 10% heat-inactivated FBS (Atlanta Biologicals). Peritoneal cells (2 × 106/ml) were incubated in 96-well microtitre plates (Becton Dickinson Labware, Franklin Lakes, NJ, USA) for 2–3 h, and non-adherent cells were removed. The adherent cells were comprised predominantly of macrophages (> 95%) determined by non-specific esterase staining, as reported previously [26,27]. The viability of spleen, lymph node and peritoneal cells was more than 95% as determined by the trypan blue staining method.

Lymphoproliferation assay

Spleen and lymph node cells cultured at 2 × 106/ml in 100 µl in 96-well microtitre plates (Becton Dickinson Labware, Franklin Lakes, NJ, USA) were stimulated with concanavalin a (ConA) (10 µg/ml) or PPD (25 µg/ml). The cells were incubated for 96 h at 37°C in 5% CO2 and labelled with [3H]-thymidine (1·0 µCi/well) for the final 6 h of incubation. Cells were harvested onto glass wool fibre filters using an automated cell harvester and the [3H]-thymidine uptake was measured in a liquid scintillation counter. The counts are expressed as a stimulation index (SI), which was calculated by dividing the counts per minute (cpm) of stimulated cells by the cpm of unstimulated cells.

Flow cytometry

The phenotypic changes in the lymph node or spleen cells after TNF-α injection were assessed by staining the cells immediately after isolation with monoclonal antibodies (mAbs) against guinea pig major histocompatibility complex (MHC) class II, pan T (CT5), CD4 (CT7) and CD8- T cell (CT6) phenotypic markers (Serotec, Oxford, UK) using our previously published procedures [26,28]. For each mAb or control, 5–10 × 105 cells were incubated with mouse serum (Sigma) for 10 min to block FcR binding. This was followed by the addition of 50 µl of the appropriate antibodies followed by secondary staining with the fluorescein isothiocyanate (FITC)-conjugated AffiniPure goat anti-mouse immunoglobulin G (IgG) (H + L) (Jackson ImmunoResearch Laboratories, Inc., West Grove, CA, USA). The proportions of positive cells were determined with a fluorescence activated cell sorter (FACS)Calibur flow cytometer and CellQuest software (Becton Dickinson Immunocytometry Systems, San Jose, CA, USA).

Total RNA isolation and real-time RT–PCR

Spleen and lymph node cells were seeded into 24-well tissue culture plates (1 × 106 cells/well) and were stimulated with PPD (25 µg/ml) at 37°C in 5% CO2 for 24 h. Similarly, the peritoneal macrophages were cultured in the presence of PPD (25 µg/ml) or live M. tuberculosis[multiplicity of infection (MOI): 0·1] for 24 h. At the end of the incubation period, supernatants were removed and the cells were washed with phosphate-buffered saline (PBS), lysed with RLT buffer (Qiagen), and the lysates frozen at −80°C until RNA extraction. The total RNA from the spleen, lymph node and peritoneal macrophages were isolated using the RNeasy kit (Qiagen, Valencia, CA, USA), as published earlier [29]. Taqman reverse transcription reagents (Applied Biosystems, Foster City, CA, USA) were used for reverse transcription and real-time RT–PCR was carried out using SYBR Green I double-stranded DNA binding dye (Applied Biosystems) and the ABI Prism 7700 sequence detector, as reported previously [26,29,30]. Real-time primers for guinea pig TNF-α, IFN-γ, IL-12p40, IL-10 and hypoxanthine–guanine phosphoribosyltransferase (HPRT) were designed using Primer Express software (Applied Biosystems), as reported previously [24,25,29]. Fold induction levels of mRNA were determined from the cycle threshold (Ct) levels normalized for HPRT expression and then to the Ct levels from unstimulated cells cultured for 24 h.

Histopathology

Portions of lung, lymph node and spleen tissues from both TNF- and BSA-treated guinea pigs were fixed in 10% buffered formalin, embedded in paraffin, sectioned at 5 µm and stained by the haematoxylin and eosin (H&E) method. The histological changes were evaluated in a blinded fashion by Dr Bradley Weeks (Department of Veterinary Pathology, Texas A&M University, College Station, TX, USA).

Statistical analysis

Results are displayed as the mean ± standard error of the mean (s.e.m.) of five to six animals per group. These experiments were repeated three times. Differences between groups for skin test, CFUs and flow cytometric results were compared by Student's two-tailed t-test. The real-time RT–PCR data were analysed by the GraphPad Prism (version 4·03, 2005; GraphPad, Inc., San Diego, CA, USA) software package for the Mann–Whitney non-parametric test to compare BSA-treated and TNF-α treated guinea pigs. P-values of < 0·05 were considered statistically significant.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

Effect of TNF-α on skin test response and bacillary loads

As shown in Fig. 1a, 6 weeks after vaccination BCG-vaccinated guinea pigs exhibited a strong skin test response 24 h after injection with 2 µg of PPD, while TNF-α-treated animals showed a significantly (P < 0·03) enhanced dermal response when compared to the BSA-injected group. Lymph nodes draining the site of vaccination were homogenized and plated for viable BCG. As shown in Fig. 1b, the CFUs were reduced significantly (P < 0·006) in the lymph nodes of TNF-α-treated guinea pigs when compared with the BSA-injected animals. No significant differences in the CFUs were seen in the spleen after TNF-α injection (Fig. 1b).

image

Figure 1. Effect of tumour necrosis factor (TNF)-α on purified protein derivative (PPD) skin test response and lymph node bacillary loads. Guinea pigs were vaccinated with Mycobacterium bovis bacille Calmette–Guérin (BCG) and injected with either recombinant guinea pig (rgp)TNF-α (25 µg/animal) or 1% bovine serum albumin (BSA) for a total of 12 injections given every other day. Six weeks after vaccination, the guinea pigs were injected with 0·1 ml PPD (2 µg) and the diameter of the induration was measured 24 h later (a). Lymph node and spleen homogenates were inoculated onto 7H10 agar plates, the colonies were counted 21 days later and converted into log10 colony-forming units (CFU) per tissue (b). The results are expressed as mean ± standard error of mean (s.e.m.) from five to six guinea pigs each. Differences between TNF-α- and BSA-injected guinea pigs were tested by Student's t-test. P < 0·03 (a) and *P < 0·006 (b).

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Effect of TNF-α on T cell proliferation

The T cell proliferative ability of lymph node and spleen cells from TNF-α- and BSA-injected guinea pigs vaccinated with BCG was determined by the [3H]-thymidine uptake assay after culturing the cells for 4 days in the presence of ConA or PPD. As depicted in Fig. 2, both lymph node and spleen cells proliferated well to ConA (Fig. 2a), although the response was much higher in the lymph node cells. There was no significant difference in the T cell response between TNF-α- and BSA-injected guinea pigs. Similarly, lymph node and spleen cells proliferated well after PPD stimulation (Fig. 2b), and the response was similar in both cell types. However, T cell proliferation was enhanced significantly (P < 0·04) in the lymph node cells of TNF-α-injected guinea pigs compared to the BSA controls (Fig. 2b).

image

Figure 2. Effect of tumour necrosis factor (TNF)-α treatment on proliferation in lymph node and spleen cells. Lymph node and spleen cells (2 × 106/ml) were stimulated with concanavalin A (ConA) (10 µg/ml, a) or purified protein derivative (PPD) (25 µg/ml, b) for 4 days. Cells were harvested after the addition of [3H]-thymidine 6 h earlier. The results are expressed as a stimulation index (stimulated over unstimulated) and represent the mean ± standard error of mean (s.e.m.) from six guinea pigs each. P < 0·04 denotes the differences in the proliferation between the TNF-α and bovine serum albumin (BSA) groups as determined by Student's t-test.

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Lymphoid cellularity after TNF-α injection

The effect of TNF-α injection on the proportions of immune cells in the lymph nodes and spleen was carried out by flow cytometry after staining the cells with the mAbs against guinea pig MHC class II, pan (CD3+) T, CD4 and CD8 T cell phenotypic markers. TNF-α injection resulted in a significant increase in the proportion of CD3+ T cells (P < 0·03) in the lymph nodes (Fig. 3a). There was no significant treatment effect on the proportions of MHC class II, CD4 or CD8+ cells in the lymph nodes (Fig. 3a) or spleen (Fig. 3b) of guinea pigs.

image

Figure 3. Cellularity changes after tumour necrosis factor (TNF)-α treatment in lymph nodes and spleen. Lymph node (a) and spleen (b) cells (5 × 105) were assessed for the proportions of immune cells by staining with monoclonal antibodies (mAbs) against guinea pig major histocompatibility complex (MHC) class II, pan T (CD3, CT5), CD4 (CT7) and CD8- T cell (CT6) phenotypic markers followed by the secondary antibody staining with the fluorescein isothiocyanate (FITC)-conjugated AffiniPure goat anti-mouse immunoglobulin G (IgG). The proportions of positive cells were determined with a fluorescence activated cell sorter (FACS)Calibur flow cytometer. Results are expressed as mean ± standard error of mean (s.e.m.) from six guinea pigs each. The differences in the proportions of cells between TNF-α- and bovine serum albumin (BSA)-injected animals were determined by the Student's t-test. P < 0·03.

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Effect of TNF-α on cytokine mRNA expression

Lymph node and spleen cells were cultured with PPD and peritoneal cells were stimulated with PPD or live M. tuberculosis for 24 h. The level of TNF-α mRNA expression in both lymph nodes and spleen after PPD stimulation was similar in TNF-α- and BSA-injected guinea pigs, although the response was higher in spleen cells (Fig. 4a). IL-12p40 mRNA levels (Fig. 4b) were increased significantly in both lymph nodes (P < 0·005) and spleen (P < 0·01) after TNF-α injection. In contrast, the levels of IFN-γ (Fig. 4c) and IL-10 (Fig. 4d) mRNA expression remained unchanged after TNF-α injection compared to the BSA-injected group. The magnitude of the IFN-γ response was much higher compared to the low levels of IL-10 mRNA in both lymph nodes and spleen, indicating that Th1 cytokines predominate in guinea pigs 6 weeks after BCG vaccination.

image

Figure 4. Effect of recombinant guinea pig tumour necrosis factor recombinant guinea pig tumour necrosis factor (rgpTNF)-α (rgpTNF-α) on mRNA expression in the lymph nodes and spleen. Lymph node and spleen cells were stimulated with purified protein derivative (PPD) (25 µg/ml) for 24 h and the RNA was subjected to real-time reverse transcription polymerase chain reaction (RT–PCR). The fold induction of mRNA expression [a, TNF-α; b, interleukin (IL)-12p40; c, interferon (IFN)-γ; and d, IL-10] was calculated from the threshold cycle (Ct) values normalized first to hypoxanthine–guanine phosphoribosyltransferase (HPRT) Ct values and then to the unstimulated cells cultured for 24 h. The results are expressed as mean ± standard error of mean (s.e.m.) from six guinea pigs. P < 0·01 and *P < 0·005 show the differences between the TNF-α- and bovine serum albumin (BSA)-treated groups as determined using the non-parametric Mann–Whitney U-test.

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Peritoneal cells were stimulated with PPD or live M. tuberculosis for assessing the effect of TNF-α injection on mRNA expression. In the TNF-α-injected guinea pigs, stimulation of peritoneal cells in vitro with live M. tuberculosis caused a significant increase (P < 0·01) in the mRNA response at 12 h (Fig. 5a), and a further increase at 24 h (Fig. 5b) compared to the BSA-treated guinea pigs. Similarly, PPD caused a significant increase (P < 0·01) in the TNF-α mRNA at 12 h (Fig. 5a) but a decrease (P < 0·05) at 24 h (Fig. 5b). Both M. tuberculosis and PPD stimulation induced similar levels of TNF-α mRNA in the peritoneal cells from BSA-injected guinea pigs (Fig. 5a,b). Peritoneal cells showed a high level of IL-12p40 mRNA expression after stimulation with M. tuberculosis (P < 0·005) compared to PPD in both TNF-α- and BSA-injected guinea pigs (Fig. 5c) but there was no difference in the response between the two groups. Although PPD induced a lower level of IL-12p40 mRNA expression in the peritoneal cells of both TNF-α- and BSA-injected guinea pigs compared to M. tuberculosis stimulation, the response was significantly lower (P < 0·05) in the TNF-α-injected guinea pigs (Fig. 5c). The IL-10 mRNA expression was significantly lower (P < 0·05) when peritoneal cells from TNF-α-injected guinea pigs were stimulated with either M. tuberculosis or PPD (Fig. 5d) compared to the BSA-injected group. In the BSA-injected guinea pigs, peritoneal cells stimulated with PPD had four times higher levels of IL-10 mRNA than the M. tuberculosis-stimulated cells.

image

Figure 5. Effect of recombinant guinea pig tumour necrosis factor (rgpTNF)-α on mRNA expression in peritoneal macrophages. Peritoneal macrophages were stimulated with live Mycobacterium tuberculosis[multiplicity of infection (MOI): 0·1] or purified protein derivative (PPD) (25 µg/ml) for 12–24 h. TNF-α (a, 12 h; b, 24 h), interleukin (IL)-12-p40 (c, 24 h) and IL-10 (d, 24 h) mRNA expression was quantified using real-time reverse transcription polymerase chain reaction (RT–PCR). Other details are as in the legend for Fig. 4. Results are displayed as the mean ± standard error of mean (s.e.m.) of six animals per group. Differences between the TNF-α-treated or bovine serum albumin (BSA)-treated guinea pigs were compared using the non-parametric Mann–Whitney U-test. P < 0·05.

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Histological changes after TNF-α injection

Lymph node, spleen and lung tissues from TNF-α- and BSA-injected animals were processed for histological studies to determine whether TNF-α altered the cellular response to BCG vaccination. The H&E staining of the lymph nodes indicated that there was an increase in the infiltration of mononuclear cells in the lymph nodes of TNF-α injected animals (Fig. 6). As clear from the figure, this was seen throughout the lymph nodes in the TNF-α-injected guinea pigs, while in the BSA-injected animals they were mainly in the cortical areas (indicated by arrows). There were no significant histological changes in the lung or spleen tissues between the TNF-α- or BSA-injected guinea pigs.

image

Figure 6. Effect of tumour necrosis factor (TNF)-α on histological changes in the lymph nodes. Lymph modes from TNF-α- or bovine serum albumin (BSA)-treated guinea pigs were processed for histological studies by haematoxylin and eosin (H&E) staining. Note mononuclear cell infiltration throughout the lymph node in the TNF-α-treated guinea pigs and only in the cortical areas of BSA-injected group (arrows). (a,b) Magnification ×100; (c,d) magnification ×400.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

Previously, we have reported that treatment with rgpTNF-αin vitro enhanced T cell and macrophage functions and injection of anti-TNF antibody into non-vaccinated or BCG-vaccinated M. tuberculosis-infected guinea pigs or animals with experimental tuberculous pleuritis enhanced splenic granuloma organization and inflammatory processes [20–25]. This is the first study that demonstrates that rgpTNF-α exerts immunomodulatory effects when injected after BCG vaccination in guinea pigs. The dose of TNF-α was selected on the basis of previous studies in mice [13,16,31]. TNF treatment was not associated with overt toxicity, as the guinea pigs did not display weight loss, morbidity or mortality.

TNF-α is known to mediate a number of immunological functions after M. tuberculosis infection including cell recruitment, induction of chemokine and cytokine secretion, macrophage activation and apoptosis, in addition to synergizing with IFN-γ in the formation and maintenance of granuloma [19,32–34]. Injection of guinea pigs with rgpTNF-α induced an increase in the PPD skin test response (Fig. 1a), suggesting that it may enhance leucocyte recruitment and/or other aspects of the dermal inflammatory responses at the site of antigen challenge in the M. bovis BCG-vaccinated animals. TNF-α treatment also resulted in an increase in the infiltration of mononuclear cells in the lymph nodes draining the vaccination site (Fig. 6), as well as an increase in the proportions of CD3+ T cells (Fig. 3a). An increase in CD3+ T cells after TNF-α treatment was not accompanied by an increase in the number of CD4 or CD8+ T cell subsets. One explanation for this result could be that while all α and β T cell receptor-positive T cells express CD3 antigen on their surface, cells other than CD3+ T cells, such as macrophages or dendritic cells, are also known to express CD4 or CD8 markers [35]. Thus, a concomitant change in the CD4 or CD8+ T cells may not be evident in these experiments, and in future this can be addressed by the double staining of cells against CD3 and CD4 or CD8 T cell phenotypic markers. In addition, antigen-specific T cell proliferation to PPD was enhanced in the lymph nodes of guinea pigs treated with rgpTNF-α, while Con-A-induced proliferation of T cells remained unaltered in these animals (Fig. 2c). The results from these in vivo studies are consistent with the in vitro observations reported earlier from our laboratory, that treatment with rgpTNF-α of spleen cells from BCG-vaccinated guinea pigs enhanced the T cell proliferation to PPD and not ConA [21]. The differential effect of TNF-α on PPD or ConA-induced T cell proliferation may be attributed to the differential contributions of co-stimulation by antigen-presenting cells (APC), as reported by others [36,37]. From our study, as well as from others, it is clear that TNF-α causes further proliferation of T cells but TNF blockade enhances both Th1 (IFN-γ and IL-12p40) and Th2 (IL-10) cytokine responses in mice with chronic tuberculosis infection [13,21]. It is also likely that TNF-α activation causes a transition of T cells to a memory phenotype in the animals [15,38].

An enhanced skin test response to PPD after TNF-α treatment was associated with a reduction in the BCG bacillary loads in the lymph nodes when compared to the BSA-injected guinea pigs (Fig. 1b). In the present study, no viable M. bovis BCG were detected in the spleen of either TNF-α- and BSA-injected guinea pigs 6 weeks after M. bovis BCG infection. This can be explained on the basis of studies by others that a maximum level of viable BCG organisms in spleen was seen 20 days post-vaccination, after which there was a significant decrease in the bacilli in spleen [39]. It is known that in vivo injection of TNF-α increases the resistance of mice to virulent M. tuberculosis or M. avium complex, as it resulted in decreased bacteria in the tissues [16,31]. Conversely, treatment with anti-TNF-α antibody enhanced the susceptibility of mice to tuberculosis [2,13]. In M. marinum-infected zebra fish, loss of TNF-α signalling accelerated bacterial growth and caused increased mortality, although TNF-α was not required for tuberculous granuloma formation [40]. In vitro studies from our laboratory also support our findings, as rgpTNF-α and rgpIFN-γ, alone or in combination, inhibited the intracellular growth of M. tuberculosis in guinea pig macrophages in vitro[25]. Conversely, alveolar and peritoneal macrophages from BCG-vaccinated guinea pigs treated with anti-gpTNF-α antibody in vitro showed increased mycobacterial growth [20]. Furthermore, we reported that injection of anti-TNF antibody into BCG-vaccinated and non-vaccinated guinea pigs following aerosol challenge with virulent M. tuberculosis resulted in splenomegaly and presence of plasma cells in the granulomas in the BCG-vaccinated guinea pigs, while splenic granulomas were more organized in the non-vaccinated guinea pigs [24]. Thus, anti-TNF-α seems to have a differential effect after M. tuberculosis infection, as large amounts of TNF-α and greater number of bacillary loads occur in non-vaccinated guinea pigs versus lower levels of TNF-α and reduced numbers of bacilli in the vaccinated animals [26,41,42]. In the tuberculous pleurisy model, no necrosis was evident after the anti-TNF-α treatment, while the treatment altered the cellular composition of the pleural effusion, as well as increasing the cell-associated mycobacterial loads in the granulomas [23].

In order to determine whether TNF-α treatment also altered the cytokine mRNA expression after BCG vaccination, lymph node and spleen cells were stimulated in vitro with PPD. TNF-α treatment enhanced the IL-12p40 mRNA expression in both lymph node and spleen cells upon antigen restimulation (Fig. 4a). These results are in agreement with previous reports as well as our in vitro experiments in which rgpTNF-α enhanced both IL-12p40 and IFN-γ mRNA expression [20,21]. In the present study, in vivo injection of TNF-α did not enhance the IFN-γ mRNA expression in the lymph nodes or spleen; in fact, the levels of expression were higher than the IL-10 mRNA in both groups of guinea pigs, confirming that Th1 cytokines are predominant after BCG vaccination. There was an increase in the TNF-α mRNA in the peritoneal cells stimulated with live M. tuberculosis or PPD. In fact, with the live M. tuberculosis stimulation the mRNA expression was sustained beyond 12 h with a further increase at 24 h compared to PPD. Previous reports from our laboratory have shown clearly that after aerosol challenge with virulent M. tuberculosis (H37Rv), high levels of TNF-α mRNA expression were evident in the laser capture micro-dissected discrete granulomatous lesions in non-vaccinated, but not in BCG-vaccinated guinea pigs [41,43]. This was also evident when peritoneal, bronchoalveolar lavage cells, spleen or lung digest cells from M. tuberculosis-infected guinea pigs were restimulated in vitro with PPD [26,42]. However, recent reports have indicated that secretion of TNF-α was dependent on the virulence of M. tuberculosis, as cytokine (TNF-α, IL-6, IL-10) or chemokine [growth-regulated oncogene (GRO)-α] secretion was found to be reduced significantly when human macrophages or dendritic cells were infected with the Beijing strains of M. tuberculosis compared to the H37Rv strain [44]. Patients infected with Beijing strains were more prone to disease progression, had higher risk of extrapulmonary tuberculosis or were less likely to respond to treatment [45,46]. Previous studies from our laboratory have indicated that in vitro treatment of peritoneal or alveolar macrophages with rgpTNF-α enhanced the TNF-α and IL-12p40 mRNA expression [24,25]. Again, other studies as well as ours have demonstrated that TNF-α alone or in combination with rgpIFN-γin vitro-induced expression of MHC class II molecules on macrophages and T cell IL-2 receptors [25,47,48], although TNF-α injection had no effect on MHC class II expression. It is quite possible that TNF-α had an immediate effect on MHC class II expression, but the effect was not long-lasting until 6 weeks of vaccination. In vitro studies have also shown that TNF-α alone or together with IFN-γ induced an enhanced expression of IL-10 mRNA in peritoneal macrophages from BCG-vaccinated guinea pigs [25]. Injection of TNF-α may be causing intrinsic changes in macrophages in the BCG-vaccinated guinea pigs, as it is known that TNF-α is essential for the differentiation of macrophages into epithelioid cells and in the aggregation of leucocytes into functional granulomas for controlling virulent mycobacterial infection [34]. Clearly, TNF-α injection caused a better clearance of M. bovis BCG in the lymph nodes of these guinea pigs.

These results indicate that in vivo administration of rgpTNF-α decreased M. bovis BCG CFUs, increased the PPD skin test response and the proliferative ability of T cells and altered cytokine mRNA expression, thus modulating the function of both T cells and macrophages in guinea pigs after M. bovis BCG vaccination. Additional experiments investigating the effect of TNF-α on chemokine expression need to be carried out in order to elucidate further the cellular mechanisms that occur after TNF-α injection. As rgpTNF-α acted synergistically with rgpIFN-γ in our in vitro studies, it will be interesting to investigate the effect of a combination of these two cytokines on virulent M. tuberculosis infection in the guinea pig model. It is becoming clearer that immune response to M. tuberculosis is mediated by multi-functional T cells [49], and a vaccine superior to BCG is yet to be identified to combat tuberculosis. Efforts leading to enhancing the immunomodulatory properties of BCG vaccine with recombinant BCG vaccine strains expressing multiple functional cytokines [50] in a relevant animal model of pulmonary tuberculosis would certainly boost our knowledge of the mechanisms of anti-bacterial immunity.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

This study was supported in part by USPHS, NIH grant R01-15495 and a subcontract awarded to D. N. M. from Colorado State University under the NIH contract HHSN 266200400091c. The authors are thankful to Dr. Robert Alaniz and Jane Miller for their valuable assistance with the flow cytometry experiments. The authors greatly appreciate the help from Dr. Bradley Weeks for evaluating the histological changes in the tissues.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References
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