<i>In vitro</i> T‐cell activation of monocyte‐derived macrophages by soluble messengers or cell‐to‐cell contact in bovine tuberculosis

Animal tuberculosis caused by Mycobacterium bovis is a disease with significant economic and public health implications. Organisms in the M. tuberculosis complex, such as M. bovis, are facultative intracellular pathogens which survive and multiply within the macrophages of the host. 1 It is believed that the interactions of these infected cells with T lymphocytes result either in the modulation of the different functions normally assigned to macrophages (mycobactericidal activity, antigen presentation capabilities), or in the destruction of the chronically infected macrophages by cytotoxic T lymphocytes (CTL). Furthermore, it is considered that interferon-γ (IFN-γ) plays an essential role in stimulation of macrophages, and is capable of up-regulating the pathways for several macrophage effector functions, although other factors may also be needed in combination.2,3 The macrophage plays a dual role in tuberculosis; promoting not only protection against the disease (effector role) but also survival of the pathogen. Mycobacterium tuberculosis complex organisms are efficient in entering the macrophage by specific receptor–ligand interactions, 4 and in surviving the hostile environment of phagolysosomes. However, there are some macrophage defences [such as release of tumour necrosis factor-α (TNF-α) and production of reactive nitrogen intermediates] which may restrict proliferation of these pathogens. The balance between mycobacterial virulence and these macrophage defences is fundamental to an understanding of the pathogenesis of tuberculosis. Correlations of in vitro growth rates of mycobacteria with in vivo virulence have been made previously for mice, 5 rabbits, 6 guinea-pigs, 7 humans 8–10 and cattle. 11 However, observations in one system may not necessarily translate to other species. 8 There is significant controversy on the feasibility of in vitro activation of mycobactericidal activity of macrophages by treatment with different cytokines. The ability of murine macrophages to inhibit the growth of mycobacteria following in vitro activation by soluble messengers has been demostrated. 12–16 In contrast, other studies have failed to show an increased ability to restrict mycobacterial growth by treatment with soluble factors in other species including humans. 12,17–19 The situation for cattle is still unclear and contradictory results have been reached.20,21 Some authors have suggested that the primary role of IFN-γ and other cytokines may be to regulate antigen processing and presentation of mycobacterial antigens to T cells rather than to stimulate macrophage activation for mycobacterial killing. 22 In the present study we have investigated the in vitro growth of M. bovis bacillus Calmette–Guérin (BCG) and virulent M. bovis in non-activated bovine monocyte-derived macrophages; also we assayed the use of T-cell supernatants (TCS) for in vitro activation of macrophages leading to changes in morphology, to modification of the antimycobacterial capabilities and to modulation of their potential as antigen-presenting cells (APC). We also evaluated the interaction of different T-cell populations with M. bovis-infected macrophages in terms of capacity to restrict intracellular mycobacterial growth. Field isolates of M. bovis (T/91/1378, Veterinary Sciences Division, Belfast, Northern Ireland) and M. bovis BCG (kindly provided by Dr P. Andersen, Statens Seruminstitut, Copenhagen, Denmark) were prepared for the experiments as previously described. 23 For production of M. bovis sonic extract (MBSE), mid-log phase cultures were treated as previously described. 23 Protein concentration was estimated by bicinchonicic acid (BCA) protein assay (Pierce, Rockford, IL). Friesian-cross, male calves, approximately 6 months old, 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 two different sets of animals, consisting of animals 01, 02 and 03, and animals 04 and 05, respectively. Animals 01–03 were experimentally infected as follows: the animals 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 04 and 05 were housed in normal farm boxes and were used as controls for the experiments. Blood samples utilized for the experiments were obtained at times varying between 18 and 36 weeks postinfection (p.i.). Monocytes were prepared as CD14+ cells positively selected from freshly isolated peripheral blood mononuclear cells (PBMC) using the magnetic-activated cell sorting system (MACS, Miltenyi Biotec, Bergisch Gladbach, Germany). Pelleted aliquots of 2 × 107 PBMC which had been separated from heparinized venous blood over Ficoll–Histopaque gradients (Amersham Pharmacia Biotech AB, Little Chalfont, UK) were resuspended in 100 µl of MACS flow [2 parts phosphate-buffered saline (PBS) : 1 part fluorescence-activated cell sorter flow (FACS flow; Becton Dickinson, Oxford, UK) : 1% bovine serum albumin (Sigma Chemical Co., Poole, UK)]. These were then 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 [RPMI-1640 (BioWhittaker UK Ltd, Wokingham, UK) containing 10 m m HEPES buffer (Gibco, Paisley, UK), 4 m m l-glutamine (Gibco), 1 m m sodium pyruvate (Gibco), 1 m m non-essential amino acids (Sigma), 50 U/ml penicillin G (Sigma) and 10% (v/v) fetal calf serum (Difco, East Molesley, Surrey, UK)] 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-bottom, 96-well polystyrene microtitre plates (Nunclon, Nunc, Denmark) and incubated at 37° in a 6% CO2 atmosphere for a further 5–7 days with medium changes every 3 days before they were used for the experiments. CD4+ and CD8+ T cells were positively selected by MACS from freshly isolated PBMC following previously described conditions. 23 The final cell population was resuspended in complete macrophage medium and cell viability was checked by trypan blue exclusion. Assessment of cell purity was carried out by flow cytometry after staining with the appropriate monoclonal antibodies and was always found to be greater than 98%. PBMC from animal 03 were resuspended in LTA culture medium [RPMI-1640 containing 10 m m HEPES buffer, 2 m m l-glutamine, 50 U/ml penicillin G, and 10% (v/v) fetal calf serum]. Then, 20-ml aliquots of PBMC at a concentration of 106 cells/ml were incubated in flasks for 2 days with concanavalin A (Con A; 4 µg/ml) or with live M. bovis (106 CFU/ml) at 37° in the presence of 6% CO2. Cultures were then pelleted by centrifugation, and supernatants were filtered through 0·22-µm pores and subsequently frozen at −20° in 0·5 ml aliquots. As a control of the cytokine production by the PBMC, the presence of IFN-γ was determined in the filtered supernatants by using a commercial enzyme-linked immunosorbent assay (ELISA; CSL, Parkville, Australia) and the optical density at 450 nm (OD450) values were always higher than 2·0. At least quadruplicate wells containing approximately 105 macrophages per well were infected with 105 CFU of M. bovis or M. bovis BCG/well to give a multiplicity of infection 1 : 1, and incubated for 4 hr at 37° in 5% CO2. Macrophages were then rinsed with warm PBS to remove possible extracellular bacteria (control wells were stained by Auramine-O acid-fast technique 24 to check that all bacilli were intracellular) and reincubated with fresh media for 24, 48, or 96 hr. After the incubation time, macrophages were lysed by addition of 0·1% saponin (Sigma). Thereafter, samples were pulsed with 2 µCi/well of 3H-labelled uracil (Amersham International, 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 (Wallac, Milton Keynes, UK). Metabolic labelling with 3H-labelled uracil makes it possible to measure intracellular mycobacterial growth, but it should not be considered as equivalent to CFU. The technique does not allow the differentiation of death and metabolic stasis of micro-organisms. At least quadruplicate wells, containing approximately 105 macrophages per well, were treated with TCS (25% v/v final concentration) produced with Con A or live M. bovis for 24 hr prior to the experiments. Thereafter, cells were infected with 3 × 105 CFU of M. bovis per well 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 any extracellular bacteria and reincubated with fresh medium containing 25% TCS for 48 hr at 37° in 5% CO2. Simultaneously, quadruplicate wells of control macrophages were left untreated, and infected and incubated in fresh medium in the same conditions. Also, quadruplicate wells of untreated macrophages were infected, and incubated under the same conditions with autologous T cells (PBMC or sorted cells) in macrophage medium at a T-cell : macrophage ratio of 20 : 1, 10 : 1, or 5 : 1 for PBMC, or 5 : 1 for sorted CD4+ or CD8+ T cells. After the incubation time, macrophages were lysed and viable mycobacteria were pulsed with uracil as above. Microtitre macrophage cultures were treated for 24 hr with Con-A-generated TCS (25%). Subsequently, medium was removed and adherent cells were methanol-fixed to the bottom of the wells, stained with methylene blue and eosine (Speedy-Diff kit, Clin-Tech, Clacton-on-Sea, Essex, UK) and examined microscopically (Nikon TMS, Tokyo, Japan). Untreated control wells, and Con-A-treated (1 µg/ml) wells were also stained for comparison. To determine the effects of TCS on the capacity of macrophages to present antigen, proliferation assays were set up for CD4+ sorted T cells. Microtitre cultures were established with 2·5 × 105 CD4+ T cells, and 105 autologous macrophages in 200 µl of medium. For the comparative experiments, macrophages were pretreated for 24 hr with 25% Con A–TCS, and control macrophages were left untreated. The macrophage cultures were gently washed before the addition of the lymphocytes to prevent any direct effect of the TCS on the proliferation results. For both types of APC (controls and TCS-treated cells) triplicate wells were set up for the antigen (MBSE) as well as negative controls (PBS). Cultures were incubated for 5 days in the same conditions described previously. 23 Aliquots of 5 × 106 CD14+ sorted cells in 10 ml of medium were incubated in 50 ml Teflon Erlenmeyer flasks (Nalge Company, Rochester, NY) at 37° in the presence of 5% CO2 for 6 days; the flasks were then incubated for a further 24 hr with or without Con A–TCS (25%). Control cultures were also treated with Con A (1 µg/ml) to rule out any direct effect of residual mitogen present in the TCS. After the period of culture, the flasks were incubated on ice for 15 min and cells were harvested and incubated with optimal concentrations (in RPMI : 10% normal rabit serum) of anti-ovine CD14+ monoclonal antibody [mouse immunoglobulin G1 (IgG1); Serotec, Oxford, UK] and anti-bovine MHC class II monoclonal antibody (ILA-21, mouse IgG2a) (ECACC, Salisbury, UK) for 30 min at 4°. After washing twice with PBS : 0·1% NaN3, positive cells were identified using a mix of fluorescein isothiocyanate (FITC) -conjugated goat anti-mouse IgG2a, and phycoerythrin (PE) -conjugated goat anti-mouse IgG1 (Southern Biotechnologies Associates Inc., Birmingham, AL). After a further 30 min incubation at 4°, the cells were washed and fixed in 1% paraformaldehyde in PBS before analysis. Flow cytometry analysis was performed using a FACS Vantage (Becton Dickinson) with an the cell the population was identified on the of and and of the cells with antibody of a macrophage population after the in vitro was for the population and cells were for were using to determine in of MHC class II between control and TCS-treated 1 the results of experiments with cells from animals 02 and of mycobacteria and well in macrophage the in the uracil assay for the were and for M. bovis and M. bovis the in the uracil assay for the final were and for M. bovis and M. bovis respectively. in the presence of bovine monocyte-derived macrophages both of uracil to the cultures in medium The of uracil by bacteria in the presence of macrophages on for both in both animals of the values for 24 hr growth in medium that macrophages were in the survival and growth of these In both animals, by 24 hr of uracil by both of M. bovis was However, at by virulent M. bovis was greater than by M. bovis that macrophages were only to the survival and growth of virulent bovis. an period of 96 M. bovis not M. bovis growth was increased within the macrophage it was that bovine monocytes in vitro were to metabolic stasis of M. bovis BCG for up to 96 hr they not control by virulent M. bovis. of M. bovis-infected cultures at hr that cell were with no of extracellular and of some macrophages was microscopically in cultures at 96 hr in the presence of extracellular bacteria to and from the intracellular the in M. bovis uracil at 96 hr is to both and extracellular growth. growth of M. bovis and M. bovis BCG in bovine monocyte-derived were infected with mycobacteria at a ratio of 1 : 1 and were for 24, and 96 hr. At the of macrophages were and mycobacterial growth was determined by of 3H-labelled are values of experiments with cells from two infected For the values for bacteria in medium for the and for M. bovis and M. bovis for the and for M. bovis and M. bovis respectively. 2 a of in of cultures after a incubation period in the presence of 25% TCS. the controls and Con-A-treated cells, TCS-treated cells were of increased and increased to These monocytes and were found in of several cells, with cells. These changes in were of in vitro activation of the of control and Con bovine 105 cells per well were for 6 days in 96-well polystyrene plates before stimulation with TCS for a further 24 hr. cells with of and of untreated cells the × We to determine treatment with factors the growth of M. bovis within bovine 3 the results of a of experiments with cells from infected and two The of 3H-labelled uracil incorporated by bacteria within the treated or T cells were as a of incorporated by bacilli in untreated In the use of soluble TCS for the in vitro activation not the antimycobacterial activity of bovine macrophages to the control untreated cells Furthermore, on several the treated cells were to M. bovis growth, as by a uracil in the cultures greater than were found between TCS with Con A or with live M. bovis infection of the a of of TCS production cell incubation prior to or per v/v of supernatants was in terms of the antimycobacterial effect within the macrophage cultures not growth of M. bovis in bovine monocyte-derived macrophages from infected and and two and of the effects of TCS and T-cell populations on Macrophages were pretreated with M. or Con A–TCS for 24 hr and then infected at a multiplicity of infection of 3 : macrophages were infected and subsequently autologous T cells were to the wells at the concentrations in the Cultures were incubated for 2 then and incubated for 40 hr in the presence of 3H-labelled are as of incorporated by bacteria within the treated or macrophages, in to the incorporated by bacilli in untreated The 3H-labelled uracil control values (c.p.m.) were as follows: 50 and The result are as for experiments In to these in the present in vitro incubation of infected monocytes with T-cell populations in increased of M. bovis uracil the for for activation of antimycobacterial capabilities in bovine T-cell populations were in their ability to M. bovis a PBMC population was a M. bovis stasis was with values of uracil between and of the values for the control infected cells. In a between was an of the of PBMC per macrophage in a uracil were found between the of infected and animals, that of be to to the In an to the potential of CD4+ and CD8+ cells to such activity, populations were used in the experiments. The M. bovis growth was than with the with of for the CD4+ cells and for the CD8+ cells. TCS not the ability of macrophages to restrict M. bovis growth, we to TCS treatment antigen presentation of these cells to CD4+ T 4 the in antigen (MBSE) of sorted CD4+ cells in the presence of TCS The were as proliferation of CD4+ cells. A in proliferation was found after treatment of the APC with Con the effect was only found in the presence of antigen and any effect of soluble factors or Con A treatment was out by the of the PBS proliferation of CD4+ sorted cells to soluble mycobacterial antigen For the comparative experiments, macrophages were pretreated for 24 hr with 25% Con A–TCS, and control macrophages were left untreated. The macrophage cultures were gently washed before the addition of the lymphocytes to prevent any direct effect of the TCS on the proliferation results. are the of one for animal 02 and experiments for animal To determine an in the MHC class II was a possible of a antigen-presenting activity, cultures of monocytes from animals 03 and 04 were established in and treated with TCS. Subsequently, MHC class II was by The experiments were twice in both animals with results. were by 5 a that there is an of class II in the treated cultures to untreated cells. Flow analysis of MHC class II CD14+ monocytes from animal 03 in were treated for 24 hr with Con A–TCS or left untreated as Subsequently, MHC class II was by cells were selected on was for the and in for control and TCS-treated cultures were using cell and The uracil technique has previously for of intracellular mycobacterial growth. The of uracil a of the metabolic of The present study is the to the growth of a virulent M. bovis and M. bovis BCG in bovine monocyte-derived staining of cultures that the bacilli were intracellular following infection of and the uracil counts intracellular growth of The in M. bovis uracil at 96 hr is to both and extracellular growth to destruction of study that bovine macrophages are capable of M. bovis BCG growth but they allow intracellular growth of virulent M. bovis. We only an ability to the growth of bovis 24 with effect on surviving intracellular bacilli which were to over an period of study A in monocyte-derived macrophages the same studies in cattle have also found that bovine macrophages are capable of metabolic stasis of BCG for up to 96 hr M. bovis is to in the In with these found that M. bovis BCG was to monocyte-derived 20 The between studies be to the higher multiplicity of infection : 1 as to 1 : 1 in the present used for experiments. have been to virulent M. bovis is to the activity of has suggested that of may and that soluble factors as may differentiation and of these cells into cells. study that treatment of monocytes with TCS changes and cell which an activation of the cells. A study with monocytes treated with TCS rates between 10 and The role of cell in an in vivo be to mycobacteria cells which are to the pathogens and to prevent from the of In to out the of the changes by TCS treatment we performed experiments to determine antimycobacterial activity, and capacity to present antigen to CD4+ T cells. We found that the use of soluble TCS for the in vitro activation not the antimycobacterial activity of bovine macrophages to the control untreated cells. Furthermore, on several the treated cells were to intracellular mycobacterial growth. studies have suggested the for several cytokines in the macrophage In to a of cytokines and other factors an in vivo situation we to the use of a complex TCS with Con A or by infection of PBMC with live M. bovis. It be that stimulation with mitogen not of the soluble that is was also by supernatants with live M. bovis from cells from infected cattle. of these were to antimycobacterial experiments in the system also that or treatment of M. macrophages with different cytokines not have an effect on the growth of that A situation was for M. bovis in macrophages, macrophages had but failed to control the intracellular and for M. bovis BCG in cattle 20 However, other studies with bovine macrophages found that with IFN-γ or with IFN-γ and in a significant of M. bovis and M. bovis BCG A to be for the murine These results may in the of macrophage of of mycobacteria and conditions of Macrophages from different may also be to different The in macrophage activation found in study was not to of the TCS experiments with changes as well as in the antigen-presenting capacity of the cells. To rule out the that the were at we performed experiments with of TCS but not an on the of in a of the macrophage to mycobacterial growth was macrophages for growth of mycobacteria have been described the that cell may be as for activation of macrophages as the presence of soluble In the present experiments, cell of macrophages with autologous T cells (PBMC or sorted in with the release of factors such as macrophages to restrict of intracellular bacilli In the of the PBMC, the of M. bovis growth to a has been for M. bovis 20 In the present study and for the same T-cell : macrophage ratio : CD4+ or CD8+ sorted cells were to inhibit M. bovis growth to that both are needed for antimycobacterial from both infected and animals that PBMC in vitro in some with the M. bovis-infected macrophages, significant stasis of the mycobacterial in with the control infected At present we not have an for a possible that there is a of to the mycobacterial infection in these In with autologous PBMC from both and have been found to restrict the growth of BCG monocyte-derived 20 Macrophages are primary effector cells but also are essential for processing and presentation of antigens to T cells. We have that in vitro treatment of macrophages with Con A–TCS the antigen presentation capabilities of these cells. In experiments, presentation of a soluble mycobacterial antigen (MBSE) to CD4+ T cells was as by an increased proliferation of the sorted cells. Furthermore, we the ability of TCS to MHC class II on the bovine authors in the have found results in In although treatment of bovine monocytes with TCS caused increased MHC class II and increased proliferation of CD4+ sorted cells against mycobacterial we were to increased in vitro antimycobacterial The observations described may be of in the study of and in the of for bovine tuberculosis. was performed in the of Veterinary of and was by the and and from the of for Northern

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