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Immunology. Jan 2000; 99(1): 23–29.
PMCID: PMC2327132

Cellular interactions in bovine tuberculosis: release of active mycobacteria from infected macrophages by antigen‐stimulated T cells

Abstract

The outcome of Mycobacterium bovis infections depends on the interactions of infected macrophages with T lymphocytes. Several studies in humans and in mouse models have suggested an important role for cytotoxicity in the protective immune response to mycobacterial infections, and both CD4+ and CD8+ T cells have been shown to elicit appropriate cytolytic activity. The present study investigated in vitro interactions of T cells with M. bovis‐infected macrophages in bovine tuberculosis. The results showed that following interaction with antigen‐stimulated peripheral blood mononuclear cells (PBMC) from infected cattle, there was an increased presence of M. bovis in the extracellular compartment of infected macrophage cultures, as measured by incorporation of [3H]uracil into mycobacterial RNA. Furthermore, out of a panel of T‐cell clones from infected cattle, it was found that a higher proportion of CD8+ clones produced an increase in the number of metabolically active extracellular M. bovis organisms compared with CD4+ clones. Finally, a positive correlation between percentage of antigen‐dependent release of mycobacteria and total uracil uptake by M. bovis within culture systems was detected. This could be regarded as an indication of preferential intracellular control of mycobacteria by activated macrophages.

Introduction

Tuberculosis in animals continues to be an important disease that is present, not only in developing, but also in industrialized countries world‐wide. This zoonotic infection occurs within domestic and farmed animals (mainly cattle, goats and pets) and also in wildlife reservoirs (badgers, wild boars, deer, possums). The disease results in serious costs to farming economies, as well as a danger to human health. Mycobacterium bovis is the main causative agent of these infections, and belongs to the so‐called M. tuberculosis complex, which consists of a group of very closely related species of controversial classification. 1

Mycobacterium tuberculosis complex organisms are facultative intracellular pathogens that survive and multiply primarily within macrophages. The outcome of the infection largely depends on the interactions of infected cells with T lymphocytes. This results in the activation of mycobactericidal capabilities of proficient macrophages via secreted lymphokines or cell contact, or in destruction of the chronically infected macrophages by cytotoxic lymphocytes (CTL).

Different subpopulations of T cells have been shown to play vital roles in containing M. tuberculosis infection. The importance of CD4+ T cells is well defined in humans and murine models. 2,3 The function of these cells in response to mycobacteria is both to secrete cytokines capable of activating macrophages, 4 and to act as cytotoxic effector cells. 5,6 Recent evidence suggests that CD8+ T cells may also be important for protection against intracellular bacterial infections. 7 The functional role for the CD8+ subset has been identified in several studies in humans 810 and mice. 11,12 Functionally, this subset of T cells can exert both immunoregulatory functions and CTL activity, 1315 but there is still a lack of information on antigens and epitopes required for generation of CD8+ responses, as well as how these antigens are directed into the appropriate antigen‐processing pathways. While most reports have focused on the study of individual subpopulations, there is recent evidence in mice that either CD4+ or CD8+ T cells are required for protection, and when lacking both of these subpopulations the host fails to control mycobacterial infection. 16 The γδ T‐cell subset also seems to play a part in mycobacterial infections. It is believed that these cells participate in the early control of infection, and provide a link between immediate innate resistance and the subsequent specific response by αβ T lymphocytes. 17 Also, it has been shown recently that CD4 CD8 (double negative, DN) αβ T cells are able to respond to lipids or glycolipids from M. tuberculosis presented by CD1 molecules. 18

Several studies in the human and in mouse models suggest an important role for cytotoxicity in the protective immune response to mycobacterial infections. It has been suggested that specific lysis of chronically infected macrophages would enable uptake of the extracellular bacteria by infiltrating activated monocytes with better bactericidal potential. 19 All CD4+, CD8+ and γδ T cells have been proved to elicit cytolytic activity. 7,11,20

There is a lack of in‐depth knowledge of the contribution of the different T‐cell subsets in the bovine response to M. bovis. Recent work in cattle has confirmed that all the major subsets are involved in response to the infection. 2123 The aim of the present study was to investigate the interactions of T cells and macrophages in bovine tuberculosis, and to compare such interactions for CD4+ and CD8+ subpopulations. The study was based around a method for the determination of mycobacterial metabolism. A [3H]uracil‐uptake assay 24 allowed relative measurement of the metabolic activity of intracellular, and released extracellular M. bovis following interaction with activated/non‐activated T cells. To our knowledge this is the first such a study in the course of M. bovis infections in cattle.

Materials and methods

Mycobacterial sonicate and cultures

A northern Irish field isolate of M. bovis (T/91/1378; Veterinary Sciences Division, Belfast, UK) and M. bovis bacillus Calmette–Guérin (BCG) (Statens Seruminstitut, Copenhagen, Denmark) were prepared for the experiments as described previously. 21

For production of M. bovis sonic extract (MBSE), mid‐log phase cultures were treated as described previously. 21 Protein concentration was estimated by bicinchonicic acid (BCA) protein assay (Pierce, Rockford, IL) as recommended.

Experimental animals

Friesian‐cross male calves of approximately 6 months of age were obtained from herds with no history of tuberculosis for at least 5 years. During the study all the animals were fed normal diets. The experiments were done in four different sets of animals, consisting of animals 01 and 02; animals 03, 04 and 05; animals 06 and 07; and animal 08, respectively. Animals 01–05 and 08 were infected experimentally as follows: they were housed in a high‐security isolation house under negative pressure with expelled air filtered through absolute filters, and were infected by intranasal instillation of 106 colony‐forming units (CFU) of M. bovis. Non‐infected animals 06 and 07 were housed in normal farm boxes and were used as controls for the experiments. Blood utilized for determination of T‐cell–macrophage interactions was obtained at times varying between 52 and 70 weeks post‐infection (p.i.) for animals 01 and 02, and around 20 weeks p.i. for animals 03, 04 and 05.

Generation of activated T cells

Peripheral blood mononuclear cells (PBMC) were separated from heparinized venous blood over Ficoll–histopaque gradients (Sigma Chemical Co., Poole, UK) and resuspended in culture medium [RPMI‐1640 (BioWhittaker UK Ltd, Wokingham, UK) containing 10 mm HEPES buffer (Gibco, Paisley, UK), 2 mm l‐glutamine (Gibco), 25 µg of gentamicin sulphate per ml, and 5% (v/v) fetal calf serum (FCS; Difco Laboratories Ltd, East Molsey, UK)]. Aliquots of PBMC at a concentration of 2 × 106 cells/ml were incubated for 5 days with or without antigen (MBSE; 4 µg/ml) at 37° in the presence of 6% CO2. Both antigen‐stimulated and control cultures were subsequently washed, counted, and resuspended in macrophage medium [RPMI‐1640 containing 10 mm HEPES buffer, 4 mm l‐glutamine, 1 mm sodium pyruvate (Gibco), 1 mm non‐essential amino acids (Sigma), 50 U/ml penicillin G (Sigma), and 10% (v/v) FCS (Difco)] at the appropriate dilutions to provide the required T‐cell–macrophage cell ratios.

Production and phenotyping of T‐cell clones

Antigen‐specific T‐cell lines were established at 15 weeks p.i. for animal 08, and 30 weeks p.i. for animal 02, using MBSE as stimulating antigen by methods described previously. 21 Proliferating clones were expanded for functional assays and phenotyping. The phenotype of each T‐cell clone was determined by flow cytometry using indirect staining with monoclonal antibody CC8, CC63 or CC15 25 in conjunction with the monoclonal CACT 116A. 26 Prior to functional experiments, the clones were rested for 24 hr in the absence of antigen or antigen‐presenting cells (APC).

Purification and culture of peripheral blood monocytes

CD14+ cells were selected positively from freshly isolated PBMC using the magnetic‐activated cell sorting system (MACS; Miltenyi Biotec, Bergisch Gladbach, Germany). Pelleted aliquots of 2 × 107 PBMC were resuspended in 100 µl of MACS flow [two parts of phosphate‐buffered saline (PBS) to one part of fluorescein‐activated cell sorter (FACS) flow to 1% bovine serum albumin (BSA; Sigma)] and incubated with anti‐human CD14 microbeads (Miltenyi Biotec) for 30 min at 4°. Thereafter, cells bound by MACS microbeads were separated using LS+ separation columns attached to Midi Magnets according to the manufacturer’s instructions (Miltenyi Biotec). The recovered cell population was resuspended in macrophage medium, and cell viability was checked by trypan blue exclusion. Assessment of cell purity was carried out by flow cytometry and was found to be greater than 98%. Purified monocytes were plated at 105 cells/well in flat‐bottomed 96‐well microtitre plates (Nunclon, Nunc, Denmark) and incubated at 37° in a 6% CO2 atmosphere for a further 7–10 days, with medium changes every 3 days before use.

Uracil uptake assay

In vitro intracellular and extracellular mycobacterial activity was determined in macrophage cultures infected with M. bovis and incubated with different T‐cell populations. At least quadruplicate wells containing approximately 105 macrophages per well were infected with 3 × 105 CFU of M. bovis/well (experiments for animals 01–07) or M. bovis BCG (experiment with clones from animal 08), to give a multiplicity of infection 3 : 1, and incubated for 4 hr at 37° in 5% CO2. Macrophages were then rinsed with warm PBS to remove extracellular bacteria (the possible presence of extracellular bacteria was excluded by acid‐fast staining of infected cells) and reincubated with autologous T cells (polyclonal or clonal) in macrophage media at a T‐cell–macrophage ratio of 10 : 1 for 16 hr. Quadruplicate wells of infected macrophages with no T cells were incubated in the same conditions, and used as controls for calculating the total mycobacterial load within the infected cells. After the incubation time, macrophages in the control wells were lysed by addition of 0·1% saponin (Sigma). For the experimental wells, supernatants (containing extracellular mycobacteria) were transferred to separate microtitre plates by careful pipetting to allow removal of the extracellular bacteria from the bottom of the wells, without detaching of the remaining adherent cells. Thereafter remaining adherent cells were lysed with saponin to give a suspension containing intracellular bacteria. Finally all samples were pulsed with 2 µCi/well of [3H]uracil (Amersham Life Science Ltd, Little Chalfont, UK) and incubated for 40 hr. Radioactivity incorporated into mycobacterial RNA was determined as counts per minute (c.p.m.) by liquid scintillation counting using a Wallac 1205 Betaplate counter (E. G. & G. Berthold and Wallac, Milton Keynes, UK). Percentages of uracil uptake (intracellular or extracellular) were calculated for each experiment and each animal in relation to the uracil uptake value of the total bacterial load in control infected macrophages (100%). This experimental model allowed estimation of the active mycobacteria in the intracellular compartment of the macrophages as well as total mycobacterial growth (intracellular + extracellular) after interaction with the T‐cell populations. We assessed release of active M. bovis by measuring uracil uptake in the supernatant of the cultures, and an antigen‐dependent release value was calculated for each experiment in each animal by the following formula:

[(mean uracil uptake (c.p.m.) in the extracellular compartment for the antigen stimulated T cells – mean uracil uptake (c.p.m.) in the extracellular compartment for the no antigen control T cells)/mean uracil uptake (c.p.m.) for the lysed control infected macrophages] ×100.

In experiments performed with T‐cell clones, the fact that pure cloned CD8+ or CD4+ cells were used ruled out the possibility of non‐specific activity by other cell populations, and in consequence the percentage of antigen‐dependent release of M. bovis was calculated as:

[ mean uracil uptake (c.p.m.) in the extracellular compartment/mean uracil uptake (c.p.m.) in the control infected macrophages] ×100.

Results

Release of M. bovis from infected macrophages after interaction with antigen‐activated PBMC

Figure 1 shows the results of a series of experiments with cells from five infected and two non‐infected control animals. PBMC were either activated in vitro with MBSE, or remained unstimulated and used as control cells to study interactions with infected macrophages in bovine tuberculosis. For the infected animals, activated T cells resulted in release of mycobacteria into the extracellular compartment, which gave 38–80% of the uracil uptake seen due to total saponin‐lysis of infected macrophages in the absence of T cells. When non‐antigen activated T cells were used, this level of uracil uptake was reduced to 3–20%. Importantly, when these experiments were repeated for non‐infected control animals there was little evidence of antigen‐dependent release of mycobacteria, with extracellular uracil uptake values under 11% of maximal lysed values. When percentages of antigen‐dependent release of bacteria were calculated for all animals, values between 19·1% and 76·3% were obtained for the infected cattle; for the non‐infected animals, antigen‐dependent release values between 3·6% and 7·7% were obtained (Table 1).

Figure 1
[3H]Uracil uptake by Mycobacterium bovis in extracellular and intracellular compartments of infected macrophages after incubation with MBSE‐stimulated and control (no antigen) PBMC. Animals 01–05 were experimentally infected cattle, and ...
Table 1
[3H]Uracil uptake values (c.p.m.) by M. bovis in extracellular and intracellular compartments of infected macrophage cultures after interaction with T‐cell populations [MBSE‐stimulated PBMC or control (no antigen) PBMC] from M. bovis‐infected ...

Effects of CD4+ and CD8+ T‐cell clones on infected macrophages

A total of 18 T‐cell clones (four CD4+ and 14 CD8+ clones) was obtained from animal 02 at 30 weeks p.i. Similarly, 16 T‐cell clones (15 CD4+ and one CD8+ clone) were derived from animal 08 at 15 weeks p.i.. Maintenance of the clones was dependent on the presence of MBSE and APC. In order to investigate the nature of the interactions with macrophages within these populations, representative clones were selected from both animal 02 (two CD4+ and seven CD8+) and animal 08 (three CD4+ and one CD8+) for assessment of their activity against M. bovis‐infected autologous macrophages. The results from the experiments are shown in Fig. 2. These data demonstrate that, for both the animals, the CD8+ clones were able to release more M. bovis from the infected cells than the CD4+ clones. The percentage of antigen‐dependent release for the CD8+ clones ranged between 17·7% and 43%, and for the CD4+ clones between 5·1% and 15·2%.

Figure 2
[3H]Uracil uptake by Mycobacterium bovis BCG (figure on the left) or M. bovis (figure on the right) in extracellular and intracellular compartments of infected macrophages after incubation with CD4+ or CD8+ T‐cell clones. The figure on the left ...

Growth inhibition of M. bovis by bovine macrophages after interaction with different T‐cell populations

The technique that was used in this study permitted estimation of the replicating mycobacteria within the target cells, and of the metabolic activity present within total mycobacteria (intracellular + extracellular) following interaction with the T cells. In general terms, in macrophages incubated with control PBMC T cells (no antigen), the metabolic activity of intracellular M. bovis organisms was greater than in those wells incubated with MBSE‐stimulated T cells (Fig. 1). This result was expected as a lower number of extracellular active M. bovis organisms was observed after interaction with these control T cells. Data in Fig. 3 may suggest that CD4+ clones are better in activating anti‐mycobacterial capabilities in macrophages than CD8+ clones, although we only tested five CD4 clones and one of them did not behave following this rule. Further work is intended to clarify this matter.

Figure 3
Relationship between percentage of antigen‐dependent release of mycobacteria and total [3H]uracil uptake by viable Mycobacterium bovis remaining after interaction of infected macrophages with in vitro‐stimulated T cells. (a) PBMC; (b) ...

In the present in vitro model it was observed consistently that, after incubation with the PBMC populations, the higher the percentage of antigen‐dependent release of mycobacteria, the greater the total (intracellular + extracellular) growth of M. bovis. When these values were compared, a high level of correlation was revealed (Fig. 3a). A similar situation was observed with the T‐cell clones that were tested with M. bovis‐infected macrophages (Fig. 3b); after interaction of these clones with the infected macrophages at the same cell ratio used for the PBMC, we observed that the total percentage of uracil uptake (indicative of the metabolic labelling of active M. bovis) was lower when clones were used compared with PBMC.

Discussion

The results of this study suggest a role for T cells in the release of mycobacteria from infected macrophages in bovine tuberculosis. Furthermore, the use of soluble antigen in the in vitro re‐stimulation of PBMC from infected cattle resulted in increased release of bacilli from M. bovis‐infected cells, with possible implications for vaccine design. Antigenic stimulation of the PBMC effectors was essential to achieve a noticeable release of intracellular bacteria, and individual variation in this activity was detected for each of the animals. Importantly, there was no clear evidence of induction of a T‐cell mediated release of mycobacteria using cells from uninfected cattle, indicating that the response was antigen specific.

In order to ascertain the interactions of CD4+ and CD8+ T‐cell subsets with M. bovis‐infected macrophages, we performed T‐cell cloning experiments in two infected animals. A higher number of CD8+ T‐cell clones was obtained from animal 02 at 30 weeks p.i., than from animal 08 at 15 weeks p.i.. Although individual variation in the immune response is likely to be part of the explanation, this result is in agreement with previous studies in bovine tuberculosis that suggested increased importance of CD8+ responses with the progression of infection. 23 In the present study, CD8+ T‐cell clones were clearly superior in their ability to cause release of active mycobacteria from macrophages compared with CD4+ clones. Previous reports in humans and mice have shown that CD4+ cells may act as CTL effectors in M. tuberculosis infections, but the present study provides no evidence that this is an important function in cattle. However, we cannot conclude that the bovine host is different in terms of CD4+ responses, as our results reflect only a particular condition of the selected CD4+ clones.

The presentation of mycobacterial antigens remains a complex issue. 13 There is evidence for major histocompatibility complex (MHC) class II‐restricted CD4+, 5,27 MHC class I‐restricted CD8+, 8,13,21 CD1‐restricted CD8+ 28 and CD1‐restricted CD4 CD8 T cells. 29 We recently demonstrated presentation of soluble antigen to CD8+ T cells by an endogenous pathway in bovine tuberculosis. 21 There is not yet a clear picture on how such antigens gain access to the MHC class I pathway. Whether mycobacteria or their products escape from the phagosome to the cytoplasm remains a question open to debate. 13,3032 In addition to the reasons discussed in our previous work, 21 the generation of CD8+ clones to MBSE could have been facilitated by the use of interleukin‐2 (IL‐2) in the cloning medium. Similarly, other authors using IL‐2 in conjunction with soluble antigens were able to activate class I MHC‐restricted CD8+ CTL populations. 7

For this study we adopted a method based on labelling of active mycobacteria released from macrophages by means of a [3H]uracil isotope that is incorporated into the bacterial RNA. This technique has proved useful previously for assessment of mycobacterial growth within murine, human and bovine macrophages. 24,33,34 The measurement of uracil incorporation by metabolically active bacteria has been shown to provide a reliable indication of the metabolic status of growing organisms. 3537 To our knowledge this is the first time the technique has been applied to determine release of bacteria from infected cells. The technique has the added advantage of allowing estimation of the proportion of replicating bacteria (synthesizing RNA) in the intracellular compartment of the target cells, as well as an estimation of the total number of growing mycobacteria remaining after interaction with the T cells.

Of great importance to understanding of bovine anti‐mycobacterial responses, the results of this study are indicative of the existence of cytotoxicity responses during bovine tuberculosis. Further work is planned to investigate such responses using conventional CTL techniques.

In the present study, a positive correlation between the percentage of antigen‐dependent release of mycobacteria and uracil uptake for the total (intracellular + extracellular) M. bovis population was identified. The cloned effectors caused more inhibition of the uracil uptake in M. bovis than the PBMC. A possible explanation for this finding could be that the cloned cells are responding to mycobacterial antigen at a higher rate, thus releasing into the media a greater concentration of products to activate macrophages and/or directly damage or kill M. bovis. Taken together these results could indicate that intact macrophages, at least during the early stages (16 hr) of the interaction with the T cells, are able to induce stasis in the mycobacterial population. The uracil uptake technique does not allow differentiation of stasis and killing of M. bovis. Therefore we cannot conclude if at later stages M. bovis infection would be able to progress and overwhelm the mycobacteriostatic capabilities of the macrophage.

The present observation that T‐cell interactions with infected macrophages result in release of active M. bovis from the cells may appear contrary to new findings that CD8+‐mediated CTL is an effective mechanism for control of M. tuberculosis. 29 It seems probable, however, that in the in vitro model for the present study, molecules such as perforin and granulysin were not released from cytotoxic granules to give effective concentrations. In vivo, additional factors such as rephagocytosis and concomitant killing by freshly activated macrophages may play a part in clearing the bacteria released as a result of CTL responses. In our model, as a result of the T‐cell activity (during the 16‐hr contact), viable mycobacteria were released in the extracellular media, where they were unaffected by the inhibitory effect of the macrophages.

In the present experiments cell contact of macrophages with mycobacteria‐reactive T‐cell lines, or release of factors such as interferon‐γ (IFN‐γ), or probably both mechanisms, may have enabled intact macrophages to restrict replication of intracellular bacilli more efficiently. 8

When non‐in vitro stimulated PBMC from the infected animals were used in the experiments a noticeable decrease in the metabolic activity of M. bovis was observed after 16 hr of contact, compared with control macrophages (no T cells). It is notable that these cells came from infected animals and therefore a proportion of clones within the PBMC were already primed to recognize mycobacterial antigen presented by the infected macrophages, leading to activation of macrophages and anti‐mycobacterial activity.

We did not find antigen‐specific release of M. bovis after interaction with PBMC from the two non‐infected animals. Our data from these animals indicated, however, that even the non‐stimulated PBMC interact in vitro in some way with the M. bovis‐infected macrophages, inducing stasis of over 50% of the mycobacterial load in comparison with the control infected macrophages. At present we do not have an explanation for this observation; a possible theory is that there is a degree of innate resistance to the mycobacterial infection in these cellular interactions. Other authors have found similar results in M. bovis BCG‐infected macrophages. 38

In conclusion, we have provided evidence for release of viable M. bovis from macrophages by interaction with activated T cells, pointing to the presence of CTL responses in M. bovis‐infected cattle, which could have potential implications in the protective immunity against this pathogen. It is believed that, in humans and mice, cytolysis may contribute to host protection as well as to tissue destruction and consequent dissemination of tuberculosis. 5,39,40 The present study is the first to demonstrate that MBSE‐stimulated T cells are able to induce release of M. bovis from autologous infected macrophages in the course of bovine tuberculosis. Further knowledge of these systems is essential to the understanding of protective immunity and vaccine development.

Acknowledgments

This work was performed completely in the Department of Veterinary Science (Queen’s University of Belfast) and was supported by the European Contracts ERBFMBICT972853 and FAIRBM971136, the British Council, and funds from DANI.

Abbreviations

APC
antigen‐presenting cell
BCA
bicinchonicic acid
CFU
colony‐forming units
c.p.m.
counts per minute
CTL
cytotoxic lymphocytes
MACS
magnetic‐activated cell sorting system
MBSE
Mycobacterium bovis sonic extract
MHC
major histocompatibility complex
OADC
oleic acid albumin dextrose catalase
PBMC
peripheral blood mononuclear cells
PPD
purified protein derivative of Mycobacterium bovis

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