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Originally published online as doi:10.1189/jlb.0704433 on January 24, 2005

Published online before print January 24, 2005
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(Journal of Leukocyte Biology. 2005;77:652-660.)
© 2005 by Society for Leukocyte Biology

V{gamma}9V{delta}2 T cells use a combination of mechanisms to limit the spread of the pathogenic bacteria Brucella

Jane Oliaro, Sherri Dudal, Janny Liautard, Jean-Baptiste Andrault, Jean-Pierre Liautard and Virginie Lafont1

Institut National de la Santé et de la Recherche Médicale Unité 431, Microbiologie et Pathologie Cellulaire Infectieuse, Université de Montpellier II, France

1 Correspondence: Institut National de la Santé et de la Recherche Médicale Unité 431, Microbiologie et Pathologie Cellulaire Infectieuse, Université de Montpellier II, Place Eugene Bataillon, CC 100, 34095 Montpellier Cedex 05, France. E-mail: vlafont{at}univ-montp2.fr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Human V{gamma}9V{delta}2 T cells play a crucial role in early immune response to intracellular pathogens. In brucellosis infection, this population of cells is drastically increased in the peripheral blood of patients during the acute phase of infection. In vitro, V{gamma}9V{delta}2 T cells exhibit strong cytolytic activity against Brucella-infected cells and are able to impair intracellular growth of Brucella suis in autologous macrophages. In this study, we have investigated the relative importance of contact-dependent mechanisms versus soluble factors in the intracellular growth and viability of B. suis. We show that V{gamma}9V{delta}2 T cells use contact-dependent mechanisms, such as the release of lytic granules and Fas-mediated signals, to decrease intracellular B. suis through lysis of infected macrophages, but these mechanisms have little impact on Brucella survival. Moreover, we demonstrate that soluble factors secreted by V{gamma}9V{delta}2 T cells can directly affect B. suis survival through their potent bactericidal effects. From these results, we conclude that V{gamma}9V{delta}2 T cells are able to use a combination of mechanisms that reduce the total numbers of B. suis and thus, may benefit the host by limiting the spread of this intracellular pathogen.

Key Words: human • T lymphocytes • bacterial infection • cytotoxicity • cytokines


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Control of infection by intracellular pathogens requires an orchestrated response by the immune system, involving a complex interaction of T cells with infected host cells [1 ]. Increasing evidence suggests that T cells of the {gamma}{delta} subtype may play an important role in the defense against intracellular pathogens [2 , 3 ]. V{gamma}9V{delta}2 T cells represent the major subtype of {gamma}{delta} T cells in human blood but make up only 1–5% of all circulating peripheral T cells [4 ]. However, the numbers of V{gamma}9V{delta}2 T cells found in the blood can dramatically increase in humans in response to infection by a number of intracellular pathogens of viral, bacterial, and parasitic origin [5 6 7 8 9 ].

Natural or synthetic, phosphorylated, nonpeptidic antigens, called phosphoantigens, activate these cells [10 11 12 ]. Natural phosphoantigens have been isolated from intracellular pathogens as metabolites involved in the isoprenoid pathway of biosynthesis [13 ]. Recognition of these phosphoantigens does not require antigen processing or presentation by major histocompatiblity complex molecules [14 , 15 ]. As a result of this property and their broad reactivity, V{gamma}9V{delta}2 T cells can respond extremely quickly and effectively at the onset of an immune response. As such, it is likely that they play a role in the first line of defense along with other cells of the innate immune response, before antigen-specific T cells have been recruited.

V{gamma}9V{delta}2 T cells are not found in nonprimate animals such as mice, but a contribution of these cells to the antimicrobial immune response in Rhesus monkeys has been reported [16 , 17 ]. Indeed, a correlation between the expansion of V{gamma}9V{delta}2 T cells and the clearance of Mycobacteria in Rhesus monkeys has been demonstrated recently [16 ]. Consistent with the responses seen in nonhuman primates, the antibacterial effect of human V{gamma}9V{delta}2 T cells has also been demonstrated in a chimeric human-severe combined immunodeficiency mouse model. In this model, human V{gamma}9V{delta}2 T cells have been shown to mediate resistance to acutely lethal infections with Staphylococcus aureus, Escherichia coli, and Morganella morganii [18 ]. Moreover, in vitro, human V{gamma}9V{delta}2 T cells reduce the number of intracellular Mycobacterium tuberculosis in infected macrophages [19 ] and inhibit the growth of the asexual blood stage of the Plasmodium falciparum parasite [20 ].

In addition to their role in these infections, V{gamma}9V{delta}2 T cells play a role in human brucellosis. Brucella are gram-negative bacteria that survive and replicate within phagocytic cells [21 ] and can cause chronic infection in humans, characterized by undulant fever, endocarditis, arthritis, and osteomyelitis. There is a significant increase in the number of V{gamma}9V{delta}2 T cells in the peripheral blood of patients in response to acute Brucella infection [6 ]. We have previously demonstrated that in vitro, Brucella suis release a nonpeptide antigen, which is able to stimulate V{gamma}9V{delta}2 T cells specifically and that soluble factors released by B. suis-infected macrophages are able to activate V{gamma}9V{delta}2 T cells [22 ]. Moreover, in an in vitro coculture infection model, V{gamma}9V{delta}2 T cells can impair intracellular growth of B. suis in autologous macrophages [23 ]. This result further suggested that V{gamma}9V{delta}2 T cells play a role in the immune response to B. suis, but the mechanisms involved were not identified. We were thus interested in determining whether the reduction in intramacrophagic bacterial numbers was a result of host cell lysis (and release of bacteria from their protective niche) or other mechanisms, which could affect the viability of bacteria found in the intracellular and extracellular compartments directly. We show that V{gamma}9V{delta}2 T cells decrease the total number of B. suis by a combination of mechanisms involving lysis of infected macrophages and secretion of antimicrobial factors. From these results, we conclude that V{gamma}9V{delta}2 T cells are able to use a combination of mechanisms that reduce the total numbers of B. suis and thus, may benefit the host by limiting the spread of this intracellular pathogen.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Antigens, antibodies, and reagents
Isopentenyl pyrophosphate (IPP) and strontium chloride were purchased from Sigma Chemical Co. (St. Louis, MO). Recombinant human tumor necrosis factor {alpha} (rhTNF-{alpha}), interferon-{gamma} (IFN-{gamma}), and perforin-phycoerythrin (PE)-conjugated antibodies were purchased from BD Biosciences (San Jose, CA). Recombinant human interleukin (rhIL-2) was purchased from Chiron (Emeryville, CA). Anti-IFN-{gamma} and anti-TNF-{alpha} antibodies were purchased from R&D Systems (Minneapolis, MN). Fas-activating antibody [immunoglobulin M (IgM)], Fas-blocking antibody (IgG1), anti-T cell receptor (anti-TCR) V{gamma}9 (IgG1), and isotypically matched control mice (conjugated or not) were all purchased from Beckman Coulter (Brea, CA). A monoclonal granulysin antibody, generously provided by Alan M. Krensky (Stanford University School of Medicine, CA), was used for fluorescein-activated cell sorter (FACS) analysis, and a polyclonal granulysin antibody purchased from Santa Cruz Biotechnology (CA) was used in the antimicrobial activity studies.

Cells
Peripheral blood mononuclear cells (PBMC) from healthy donors were prepared by density centrifugation on Ficoll-Paque (Eurobio, Les Ulis, France). Monocytes were purified from PBMC based on their adherence properties by incubating on gelatin (Sigma Chemical Co.)-coated culture flasks for 1 h and removed by incubating in detachment buffer [phosphate-buffered saline (PBS), 5 mM EDTA] for 15 min. This preparation yielded monocytes with >95% purity (CD3–CD19–CD14+), as assessed by FACS analysis. Autologous V{gamma}9V{delta}2 T cells were purified from nonadherent PBMC using an anti-{gamma}9 monoclonal antibody (mAb) and goat anti-mouse IgG-coated Dynal magnetic beads (Dynal, Compiégne, France), according to the manufacturer’s instructions. Following overnight incubation, the V{gamma}9V{delta}2 cells were separated spontaneously from the magnetic beads and then stimulated with IPP (20 µM) in a 24-well culture plate (Falcon, Becton Dickinson, Meylan, France) in the presence of autologous monocytes (1x106/ml) and rhIL-2 (20 ng/ml). Cells were maintained in complete medium (RPMI 1640/glutamax, Life Technologies, Paisley, UK), supplemented with 5% heat-inactivated fetal calf serum (FCS), 5% human AB serum, rhIL-2 (20 ng/ml), and gentamicin (30 µg/ml) at 37°C in a 5% CO2 humidified atmosphere. After a 3-week expansion in culture medium containing rhIL-2, the {gamma}{delta} cells were >98% CD3+V{gamma}9+V{delta}2+ as assessed by FACS analysis.

Infection of monocytes with Brucella
Purified monocytes were seeded into 24- or 48-well plates (Falcon) at a density of 0.7 x 106/ml in complete culture media (no gentamicin) supplemented with 10–7 M 1,25- dihydroxyvitamin D3 (VD; a generous gift of Hoffman La Roche, Basel, Switzerland) for 72 h. VD-differentiated monocytes display macrophagic cell properties and can be readily infected with Brucella [21 ]. They are hereafter referred to as macrophages. The culture media was then removed, and the adherent cells were infected with B. suis 1330 at a multiplicity of infection (MOI) of 30. After 1 h, the cells were washed twice with PBS and incubated with gentamicin for 1 h to kill nonphagocytosed bacteria in experiments that were carried out with gentamicin to measure intracellular bacteria only. In experiments that were performed in the absence of gentamicin, gentamicin treatment was carried out for 4 h, and the cells were washed to assess total bacterial counts. Following this, complete media, media containing V{gamma}9V{delta}2 T cells at a ratio of 1:1.5 (macrophage:V{gamma}9V{delta}2 T cell), or an appropriate reagent were added in the presence or absence of gentamicin as indicated. As all experimental and control groups were treated in the same way, no interference with residual gentamicin was observed, as shown with the macrophage controls, which do not have decreased bacterial numbers. Unless stated otherwise, the V{gamma}9V{delta}2 T cells were activated with 40 µM IPP, which is a well-described phosphoantigen that specifically stimulates V{gamma}9V{delta}2 T cells to produce cytokines [11 ]. Intracellular bacteria were estimated by lysing the infected monocytes with 0.2% Triton X-100, followed by removal of the supernatant, and by plating serial dilutions on tryptic soy broth (TS) agar plates. To quantitate the total number of viable bacteria, the supernatant was also assessed for colony-forming units (CFUs) by serial dilutions to include any bacteria that had been released as a result of lysis of the monocytes. CFUs were counted at 2 h (experiments with gentamicin) or 7 h (experiments without gentamicin), 24 h, and 48 h. In experiments performed with V{gamma}9V{delta}2 T cells in contact or separated, a two-chamber system using a 0.4-µM culture plate insert from Millipore (Bedford, MA) was used to separate the V{gamma}9V{delta}2 cells from the monocytes. For preparation of T cell supernatant, cells (1x106 cells/ml) were stimulated with 40 µM IPP for 6 h and centrifuged, and the supernatant was collected from the pellet before freezing for use at a later time.

Direct effect of V{gamma}9V{delta}2 T cells on Brucella
IPP-activated V{gamma}9V{delta}2 T cells (1x106/ml) were incubated directly with B. suis (MOI 30) for 24 h. Supernatants are prepared as described above, and the number of bacteria added per ml was identical to the number used for V{gamma}9V{delta}2 T cells. After 24 h, the total number of viable bacteria was quantitated by plating serial dilutions of cultures on TS agar plates. When indicated, polyclonal antigranulysin antibody was added at the same time as the bacteria at a concentration of 2 µg/ml.

Flow cytometry
To block nonspecific binding, 0.5 x 106 V{gamma}9V{delta}2 cells were incubated with 10% human AB serum for 30 min before labeling the cells with cell-surface markers. The cells were incubated with the relevant antibody [1 µg fluorescein isothiocyanate-labeled anti-TCR V{gamma}9 mAb] in PBS, supplemented with 10% FCS, 0.02% NaN3, on ice in a total volume of 50 µl. After 30 min, the cells were washed once, fixed in 1% paraformaldehyde, and analyzed on FACSCalibur (Becton Dickinson) using CellQuest software. For intracellular staining of perforin or granulysin, cell-surface staining was performed first, and then cells were perforated using the Cytofix/Cytoperm Plus kit (BD Biosciences) and incubated with PE-perforin mAb according to the manufacturer’s instructions or with granulysin mAb + PE-conjugated anti-mouse antibody.

Statistical analysis
The mean of triplicate samples is shown for each data point with their SD and is representative of a minimum of three experiments performed on separate human blood donors. P was calculated by using an unpaired Student’s t-test, where a significant difference was considered when P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
V{gamma}9V{delta}2 T cells decrease the survival of B. suis
Coculture of V{gamma}9V{delta}2 T cells with B. suis-infected human macrophages results in a significant reduction in intracellular numbers of bacteria [23 ]. However, as a result of the presence of gentamicin in the coculture medium (which kills any bacteria released from the intracellular compartment of infected macrophages), it was not clear whether a relationship between the decrease in intramacrophagic B. suis numbers and a direct effect on bacterial viability existed. To address this issue, we have now performed coculture experiments in the absence of gentamicin, so that subsequent colony assays for B. suis reflect the total number of organisms, intracellular and extracellular, which are present in the coculture. This method ensures that any observed decrease in intracellular bacteria is not simply attributable to a shift to the extracellular compartment following lysis of infected macrophages but truly reflects a difference in bacterial survival. In the absence of V{gamma}9V{delta}2 T cells, B. suis readily infected macrophages and their numbers increased exponentially over a 48-h period (data not shown). However, when the infection was performed in the presence of V{gamma}9V{delta}2 T cells, there was a tenfold reduction in the total number (intracellular and extracellular numbers combined) of B. suis after 48 h of culture (Fig. 1 ), and the largest effect occurred between 24 and 48 h (data not shown). When the T cells were activated by treatment with the synthetic phosphoantigen IPP, this effect was enhanced and resulted in a 100-fold reduction in total bacteria. Although B. suis lysate contains a nonpeptidic antigen(s), which can be used to activate V{gamma}9V{delta}2 T cells [22 ], we chose to use the purified antigen, IPP, to ensure consistency. These results confirmed that the ability of V{gamma}9V{delta}2 T cells to reduce bacterial numbers was not only attributable to lysis of infected macrophages but also to a decrease in B. suis survival. This inhibition could result from killing of the bacteria or an impairment of proliferation. We chose to carry out this study using IPP-activated V{gamma}9V{delta}2 T cells at 48 h postinfection for the following reasons: First, activation of V{gamma}9V{delta}2 T cells by infected macrophages leads to the same kind of activation and biological responses as IPP-activated V{gamma}9V{delta}2 T cells [23 ]. Second, we showed that nonactivated V{gamma}9V{delta}2 T cells produce the same effect on Brucella development as IPP-activated V{gamma}9V{delta}2 T cells but with a delay (72 h time-point of nonactivated V{gamma}9V{delta}2 T cells is comparable with 48 h time-point of IPP-activated V{gamma}9V{delta}2 T cells in the presence of gentamicin; data not shown). This delay represents the time necessary for infected macrophages to activate V{gamma}9V{delta}2 T cells, probably through the release of nonpeptidic antigen from killed bacteria. To avoid excessive levels of bacterial proliferation in the medium in the experiments without gentamicin, which would lead to nonanalyzable data, we chose to study Brucella development at 48 h postinfection using IPP-activated V{gamma}9V{delta}2 T cells.



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  		Figure 1. Effect of V{gamma}9V{delta}2 T cells on total numbers of B. suis. Macrophages were infected with B. suis (MOI=30), treated for 4 h with gentamicin (30 µg/ml) to kill extracellular bacteria, and then incubated alone (M) with {gamma}{delta} T cells or with {gamma}{delta} T cells + 40 µM IPP in the absence of gentamicin. Intracellular and extracellular bacteria present at 7, 24, and 48 h postinfection were pooled and counted by plating serial dilutions on TS agar under each condition. Total numbers of B. suis present at the 48-h time-point are shown in the graph. Data are shown as the means of triplicate wells ± SD. *, Significant differences between the condition studied and macrophages alone are indicated (*, P<0.05; **, P<0.01). These data are representative of three separate experiments performed with different human donors.

 
Role of contact-dependent mechanisms versus independent mechanisms
We have previously established that when V{gamma}9V{delta}2 T cells are allowed to interact with macrophages, infected cells are lysed [23 ], but the impact on Brucella survival remains to be determined. To better understand the mechanisms used by V{gamma}9V{delta}2 T cells in impairing Brucella development, we determined the relative involvement of contact-dependent mechanisms versus soluble factors and their role in infected cell lysis and bacterial development. To differentiate between contact-dependent mechanisms and soluble factors, a two-chamber system was used, where a permeable membrane separates the infected macrophages from the V{gamma}9V{delta}2 T cells and allows the interchange of soluble factors. In the presence of gentamicin, we found that V{gamma}9V{delta}2 T cells were more effective at reducing intracellular B. suis when they were in contact with the macrophage (approximately 100-fold decrease) than when they were separated (approximately fivefold reduction; Fig. 2 , striped columns). Based on these data and our previous work, we can surmise that although soluble factors are capable of reducing intracellular bacterial numbers, cytolytic activity through contact-dependent mechanisms plays a more important role in reducing the numbers of intracellular B. suis.



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  		Figure 2. Effect of contact versus noncontact between V{gamma}9V{delta}2 T cells and infected macrophages on intracellular B. suis and total bacteria. Macrophages were infected with B. suis (MOI=30) and then incubated alone (M) with {gamma}{delta} T cells + 40 µM IPP (M+{gamma}{delta} cells contact) or {gamma}{delta} T cells + 40 µM IPP, separated by a two-chamber system (M+{gamma}{delta} cells separated) in the presence or absence of gentamicin. When the experiment was performed in the presence of gentamicin (striped columns), intracellular bacteria were counted by lysing infected macrophages at 48 h postinfection and plating serial dilutions on TS agar. For the experiment performed in the absence of gentamicin (solid columns), macrophages were infected with B. suis, as described above, with the exception that all wells contained filters as a control to account for loss of extracellular bacteria as a result of adherence to the filter. Following the relevant time-point, filters were removed, and intracellular and extracellular bacteria were counted at 48 h postinfection as described previously. The CFUs of intracellular and extracellular were pooled to give the amount of total bacteria. All data are shown as the mean of triplicate wells plus SD. *, Significant differences between the condition studied and macrophages alone at 48 h postinfection are indicated by P < 0.01. These data are representative of three separate experiments performed with different human donors.

 
When we performed the assay in the absence of gentamicin to assess the effects of V{gamma}9V{delta}2 T cells on bacterial viability, we found that the total number of bacteria in the media control was ~270-fold higher (Fig. 2 , solid column) than the control with gentamicin (Fig. 2 , striped column). This was expected, as during the time-course of infection, live bacteria can be released from infected macrophages (death of infected macrophages). Conversely, in the absence of gentamicin, released bacteria are contained in close contact with noninfected macrophages, which can readily phagocytose them, leading to an increase of intracellular bacterial numbers, and this can be reproduced throughout the experiment. Conversely, released bacteria are in a media conducive to their growth and rapidly proliferate, leading to an exponential increase in extracellular bacterial numbers. Indeed, microscopy analysis of macrophages infected with a B. suis-green fluorescent protein strain confirmed that previously uninfected macrophages phagocytosed bacteria, which had been released into the media within the first 24 h of the infection by lysed, infected macrophages (data not shown). In the absence of gentamicin, V{gamma}9V{delta}2 T cells in contact with infected macrophages were still able to reduce the total numbers of bacteria by approximately 30-fold compared with the media control (Fig. 2 , solid column). However, when V{gamma}9V{delta}2 T cells were separated from infected macrophages, and only soluble factors could act on B. suis development, they were more effective at reducing the total numbers of bacteria present in the coculture (approximately 500-fold) than the V{gamma}9V{delta}2 T cells that were in contact with the infected macrophages. Based on this data, we suggest that infected cells are lysed through contact-dependent mechanisms, leading to the release of bacteria into the media. In a gentamicin-free environment, bacteria survive, proliferate, and infect new macrophages, leading to an increase in the total number of Brucella. On the contrary, when V{gamma}9V{delta}2 T cells are separated from infected macrophages, host cell lysis cannot occur, and only soluble factors can act on Brucella development through indirect or direct mechanisms—by activating macrophages, which then eliminate Brucella, or by killing intracellular or extracellular Brucella directly.

Taken together, these results show that contact-dependent mechanisms play a more important role in direct cytotoxic activity against infected macrophages and thus, host cell lysis, and soluble factors produced by V{gamma}9V{delta}2 T cells may affect the bacteria themselves. To determine the contact-dependent mechanisms involved in cytotoxicity, we studied the granule exocytosis pathway and Fas-Fas ligand (FasL) interactions.

Role of granule exocytosis pathway
The release of lytic granules is one of the contact-dependent mechanisms used by cytotoxic cells to kill their target. Lytic granules contain a family of proteins, including perforin and granzymes [24 ], and more recently, granulysin has also been found in the cytotoxic granules of T cells and natural killer (NK) cells [25 ]. Perforin forms a pore in the membrane of the target cell through which proteolytic enzymes, such as granzymes and granulysin, can pass and trigger cell apoptosis. Several studies and our own unpublished data have shown that V{gamma}9V{delta}2 T cells express perforin, granzymes, and granulysin [26 ] and that release of lytic granules is one of the major cytotoxic mechanisms used by V{gamma}9V{delta}2 T cells to lyse infected cells.

To investigate the exact role of granule exocytosis in the inhibition of Brucella development, we treated V{gamma}9V{delta}2 T cells with strontium chloride, which induces degranulation and depletion of intracellular granule stores [27 ]. We tested several concentrations of strontium chloride and followed the depletion of intracellular granule stores by analyzing V{gamma}9V{delta}2 T cells for levels of one of the granule proteins, perforin. Treatment of V{gamma}9V{delta}2 T cells with 50 mM and 25 mM strontium chloride for 18 h markedly reduced the levels of perforin, indicating that degranulation had occurred (Table 1 and Fig. 3A ). The ability of degranulated V{gamma}9V{delta}2 T cells to control intracellular bacteria was then assessed. Incubation of untreated V{gamma}9V{delta}2 T cells with infected macrophages resulted in 80% inhibition of intracellular bacterial growth compared with macrophages alone. This inhibition is decreased when V{gamma}9V{delta}2 T cells are treated with strontium chloride in a dose-dependent manner (Fig. 3B) . From these results, we can conclude that granule exocytosis is a mechanism used by V{gamma}9V{delta}2 T cells to reduce the numbers of intracellular B. suis by lysing infected macrophages.


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  		Table 1. Effect of Strontium Chloride Treatment on Perforin Level in V{gamma}9V{delta}2 T Cells

 


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  		Figure 3. (A) Analysis of intracellular perforin stores in V{gamma}9V{delta}2 T cells after activation or strontium treatment. V{gamma}9V{delta}2 T cells were degranulated with 50 mM strontium chloride solution for 18 h. Following incubation, treated and untreated cells were collected and analyzed for surface expression of TCR V{gamma}9 and intracellular expression of perforin. (B) Effect of strontium-treated V{gamma}9V{delta}2 T cells on intracellular B. suis development. V{gamma}9V{delta}2 T cells were treated or not with different concentrations of strontium chloride (SR++) for 18 h and then added in coculture with autologous-infected macrophages for 48 h in the presence of gentamicin. Intracellular bacteria were counted by lysing infected macrophages at 48 h postinfection and plating serial dilutions on TS agar. *, Significant differences between the condition studied and {gamma}{delta} T cells at 48 h postinfection are indicated by P < 0.01. Data are represented as the percent inhibition of intracellular B. suis numbers, where 100% is the total number of intracellular bacteria in the media-alone control. Significant differences between {gamma}{delta} T cells and strontium-treated {gamma}{delta} T cells are indicated directly on the graph. These data are representative of two experiments performed with different human donors.

 
Fas-FasL interactions contribute to macrophage death and reduction of intracellular Brucella without affecting bacterial viability
Fas-FasL interactions are an alternative pathway used by T cells to trigger death of target cells [28 ]. FasL expressed on the cell surface of activated V{gamma}9V{delta}2 T cells is able to trigger contact-dependent apoptosis in host cells expressing Fas [29 ]. In light of this information, we first analyzed Fas expression on macrophages before infection and 48 h postinfection with B. suis. The macrophages used in our experiments express low levels of Fas on their surface, and there was no apparent modulation of Fas expression in B. suis-infected cells (data not shown). Following this, we determined the effect of the Fas-FasL pathway on B. suis-infected macrophages. Infected macrophages were activated to undergo apoptosis by incubating them with activating anti-Fas antibody. Figure 4 (left panel) demonstrates that activation of the Fas-FasL pathway in infected macrophages in the presence of gentamicin reduced the numbers of intracellular B. suis fivefold. This effect is reversed when macrophages are pretreated with a blocking Fas antibody, suggesting that a pretreatment with a blocking antibody can abrogate the triggering of apoptosis induced by the recruitment of the Fas pathway. In the presence of the blocking Fas antibody, the effect of V{gamma}9V{delta}2 T cells is decreased. Overall, these data indicate that this pathway plays a role in the death of infected macrophages. Following this, we demonstrated the role of Fas pathway on bacteria viability by performing the same experiments in the absence of gentamicin. The recruitment of the Fas pathway with an activating Fas antibody does not influence the total number of bacteria, suggesting that Fas-mediated apoptosis of the infected macrophages had no effect on B. suis viability (Fig. 4 , right panel). The pretreatment with a blocking Fas antibody does not modify the effect of V{gamma}9V{delta}2 T cells. Taken together, these data show that Fas-FasL interactions contribute to the death of infected macrophages without any direct effect on B. suis viability.



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  		Figure 4. Role of Fas-FasL interactions on Brucella survival. Macrophages were infected with B. suis (MOI=30), as described previously, and incubated alone (Macrophages) or with 2 µg/ml activating Fas antibody (Act Fas Ab), 5 µg/ml blocking Fas antibody (Block Fas Ab), Act Fas antibody and block Fas antibody, or with IPP-activated V{gamma}9V{delta}2 T cells ({gamma}{delta} T cells) in the presence [intracellular (i)] or absence [total bacteria (ii)] of gentamicin. Numbers of intracellular bacteria were assessed by lysing infected macrophages at 48 h postinfection and plating for CFUs. Data shown are the means of triplicate plates plus SD. *, A significant difference between the condition studied and macrophages alone is indicated by P < 0.04 (*), P < 0.01 (**), and P < 0.015 (***). Significant differences between Fas antibodies and {gamma}{delta} T cells are indicated directly on the graphs. These results are representative of three experiments performed with different human donors.

 
IFN-{gamma} and TNF-{alpha}, two major cytokines produced by V{gamma}9V{delta}2 T cells, do not have a significant impact on bacterial viability
Activation of V{gamma}9V{delta}2 T cells by a nonpeptidic antigen, killed bacteria, or infected macrophages leads to the release of proinflammatory cytokines such as IFN-{gamma} and TNF-{alpha} in the supernatants as well as other undetermined factors [23 ]. We showed in Figure 5 that supernatants from IPP-activated V{gamma}9V{delta}2 T cells decrease intracellular (Fig. 5A , left panel) and total (Fig. 5B) numbers of bacteria, suggesting that secreted molecules are involved in Brucella development. To define the part played by IFN-{gamma} and TNF-{alpha} in the effects of supernatants from IPP-activated V{gamma}9V{delta}2 T cells, Brucella-infected macrophages were treated with IFN-{gamma}, TNF-{alpha}, or a combination of both cytokines in the presence of gentamicin (Fig. 5A) or without gentamicin (Fig. 5B) . The results show that the addition of IFN-{gamma} has a small but significant (P<0.05) effect on the survival of intracellular bacteria, whereas TNF-{alpha} had a negligible effect on bacterial numbers (Fig. 5A , left panel). Nevertheless, the effect of IFN-{gamma} is lower than the effect obtained with supernatants from activated V{gamma}9V{delta}2 T cells. To confirm that both cytokines were effective in activating macrophages at the concentrations used in our experiments, phosphorylation of signal transducer and activator of transcription (STAT)1 and p38 mitogen-activated protein kinase proteins was confirmed by Western blot of macrophage lysate proteins for IFN-{gamma} and TNF-{alpha}, respectively (data not shown). Moreover, an anti-IFN-{gamma} mAb reduces the effect of V{gamma}9V{delta}2 T cells (Fig. 5A , right panel) or the effect of supernatants from activated V{gamma}9V{delta}2 T cells (data not shown), and an anti-TNF-{alpha} mAb has no effect, confirming the data obtained with recombinant cytokines. However, when experiments were performed in the absence of gentamicin, no significant reduction in total numbers of Brucella was observed in the presence of both cytokines (Fig. 5B) . This is in contrast to supernatants from activated V{gamma}9V{delta}2 T cells, which reduced bacterial numbers by tenfold. These results suggest that IFN-{gamma} induces macrophage cell lysis and thus, the release of intracellular bacteria, without significantly affecting bacterial viability, and V{gamma}9V{delta}2 T cells produce other soluble factors with antimicrobial activities.



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  		Figure 5. Effects of TNF-{alpha}, IFN-{gamma}, and V{gamma}9V{delta}2 T cells on Brucella viability. Macrophages were infected with B. suis (MOI=30) and incubated alone (M) or with 50 U/ml IFN-{gamma}, 500 pg/ml TNF-{alpha}, IFN-{gamma} and TNF-{alpha}, supernatant from IPP-activated V{gamma}9V{delta}2 T cells ({gamma}{delta} SN), or IPP-activated V{gamma}9V{delta}2 T cells ({gamma}{delta} cells) in the presence or not of 2 µg/ml anti-IFN-{gamma} antibody or 3.2 µg/ml anti-TNF-{alpha} antibody. Infection was performed in the presence of gentamicin, and intracellular CFUs were assessed (A) by plating on TS agar at 48 h or in the absence of gentamicin, in which total bacteria (intracellular+extracellular) were assessed (B). Data are shown as the mean of triplicate wells plus SD and are representative of three similar experiments performed with different human donors. Significant differences between the condition studied and macrophages alone are indicated (*, P<0.05; **, P<0.01; ***, P<0.005).

 
Soluble factors produced by V{gamma}9V{delta}2 T cells play an important role in limiting the growth of Brucella
As we observed a strong reduction in bacterial numbers from infected macrophages from soluble factors produced by activated V{gamma}9V{delta}2 T cells, we next determined if supernatants derived from activated V{gamma}9V{delta}2 T cells could have a direct effect on bacterial growth and survival outside of the macrophage. In these experiments, V{gamma}9V{delta}2 T cells or supernatants from activated V{gamma}9V{delta}2 T cells were incubated directly with B. suis without the presence of macrophages, as described in Materials and Methods. In the presence of V{gamma}9V{delta}2 T cells, a reduction in total viable bacteria was observed (Fig. 6 ). It is most interesting that supernatant from activated V{gamma}9V{delta}2 T cells alone was able to affect bacterial viability directly, although to a lesser extent than V{gamma}9V{delta}2 T cells. Thus, the supernatant from activated V{gamma}9V{delta}2 T cells contained factor(s) that were able to affect bacterial growth and survival in the absence of host cells, indicating a potent, antimicrobial effector function of V{gamma}9V{delta}2 T cells. As V{gamma}9V{delta}2 T cells express granulysin in their lytic granules, and granulysin has antimicrobial properties, granulysin could play a role in the antimicrobial effects of V{gamma}9V{delta}2 T cells. We repeated the same experiments in the presence of antigranulysin antibody. No difference was observed in the presence of antigranulysin antibody, suggesting that granulysin is not involved in the antimicrobial effect of V{gamma}9V{delta}2 T cells against Brucella bacteria.



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  		Figure 6. Direct effect of V{gamma}9V{delta}2 T cells and {gamma}{delta} T cell supernatants on Brucella viability. A B. suis culture was incubated with media alone, {gamma}{delta} T cells + 40 µM IPP ({gamma}{delta} T cells), or supernatant prepared from {gamma}{delta} T cells + 40 µM IPP ({gamma}{delta} SN) in the presence or not of antigranulysin antibody as indicated. After 24 h, bacteria were recovered from the wells, and serial plating on TS media assessed the number of CFUs. Data are shown as the mean of triplicate wells plus SD; *, significant differences between the conditions studied and media are indicated by P < 0.001. These results are representative of three experiments performed with {gamma}{delta} T cells from different human donors.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
There is mounting evidence to suggest that {gamma}{delta} T cells play a role in the early defense against intracellular pathogens. Our own studies with Brucella have shown that primary V{gamma}9V{delta}2 T cells can reduce the numbers of intracellular bacteria through soluble and contact-dependent mechanisms [23 ]. In this study, we have investigated the mechanisms used by V{gamma}9V{delta}2 T cells against infected host cells and explored their effect on the relationship between lysis of B. suis-infected macrophages and bacterial survival and growth in an attempt to clarify their role in host immunity against intracellular bacterial infections. Using an in vitro infection system and several experimental protocols, we could monitor the effect of V{gamma}9V{delta}2 T cells on B. suis infection of autologous macrophages. When the experiments are carried out in the presence of gentamicin, the observed effects represent the combination of killing of bacteria by macrophages (bactericidal activity of macrophages) and death of bacteria released in the gentamicin-containing medium after lysing infected macrophages by V{gamma}9V{delta}2 T cells; both of these mechanisms can occur simultaneously. Conversely, in the absence of gentamicin, the observed effects represent killing of bacteria by macrophages and by soluble antimicrobial factors produced by V{gamma}9V{delta}2 T cells, which can be observed in gentamicin-free conditions. Thus, we demonstrated that coculture of V{gamma}9V{delta}2 T cells with infected macrophages resulted in an overall decrease in B. suis numbers, indicating a loss in viability or inhibition of bacterial growth.

The use of a two-chamber system demonstrated that contact-dependent mechanisms and soluble factors contributed to the reduction in intramacrophagic B. suis. It is interesting that in the absence of gentamicin, contact-dependent mechanisms of V{gamma}9V{delta}2 T cells (mainly infected cell lysis) had less effect on total bacteria compared with separated V{gamma}9V{delta}2 T cells, suggesting an important role for soluble factors in antimicrobial activity. This result may have important implications during infection in vivo, as it suggests that lysis of the host cell can result in exacerbation of the infection through reinfection of surrounding macrophages as a result of bacterial release into the media. However, our model may not reflect the true environment that the bacteria encounter on their release from lysed cells. In our system, the bacteria remain in a media conducive to their growth and are in close contact with other macrophages that can readily phagocytose released bacteria. Moreover, as B. suis organisms can only survive intracellularly in the host through evasion of the immune system, other immune cells may play a part in eliminating extracellular bacteria in vivo.

Granule exocytosis is one of major cell-cell contact mechanisms of {gamma}{delta} T cells involved in cell cytotoxicity [30 ]. We observed a decrease in the effect of degranulated V{gamma}9V{delta}2 T cells in comparison with nontreated cells, demonstrating that granule exocytosis is one of the mechanisms used by V{gamma}9V{delta}2 T cells to lyse B. suis-infected macrophages and decrease the number of intracellular bacteria present in cells. These results are in agreement with several results obtained for other pathogens [19 ]. Studies have demonstrated that the granule exocytosis pathway is the major pathway used by CD8+ T cells to reduce intracellular M. tuberculosis [31 , 32 ] and that this granule-mediated pathway results in a significant loss of viable M. tuberculosis during lysis of infected macrophages. However, other studies state that the granule exocytosis pathway is not directly responsible for the killing of intracellular M. tuberculosis [33 , 34 ]. Although data concerning the role of V{gamma}9V{delta}2 T cells in the killing of intracellular bacteria in vitro are scarce, recent studies have shown that V{gamma}9V{delta}2 T cells play an active role in the reduction of M. tuberculosis in macrophages and also have the capacity to kill bacteria directly [19 , 35 ]. It has been suggested that granulysin, one of proteins contained in lytic granules, which possesses antimicrobial activity, could be responsible for the killing of intracellular bacteria. This is in contrast to a similar study that found cytotoxic activity against M. tuberculosis-infected macrophages by {gamma}{delta} T cells had no effect on bacterial viability [33 ]. In regard to Brucella, our data show that the granule exocytosis pathway is involved in the lysis of infected cells rather than bacterial viability. We cannot totally exclude that the granule exocytosis pathway (through granulysin) has no effect on the viability of intracellular Brucella, as any weak effects would be masked in our system by cell lysis and release of bacteria into the media.

Fas-FasL interactions represent another mechanism by which V{gamma}9V{delta}2 T cells can mediate host cell death. FasL is expressed on the membranes of activated V{gamma}9V{delta}2 T cells but can also be released as a soluble form [36 ]. Thus, the recruitment of the Fas pathway on infected macrophages could occur through contact-dependent or -independent mechanisms. We therefore investigated what effects the direct activation of Fas-induced apoptosis (using an activating Fas mAb) and the blockage of Fas-induced apoptosis by V{gamma}9V{delta}2 T cells (using a blocking Fas mAb) would have on Brucella-infected macrophages. We found that although this pathway can contribute to a reduction in intracellular bacterial numbers, it does not affect the viability of the bacteria significantly. Moreover, we found no modulation of Fas expression on the surface of the macrophage in response to Brucella infection (data not shown). The constitutively low expression of Fas may explain why there was only a small contribution of the Fas-FasL pathway to lysis of Brucella-infected macrophages and a small reduction in intracellular bacterial numbers. Alternatively, it is possible that Brucella species may protect themselves and maintain their ecological niche by interfering with apoptotic signals in infected macrophages, as previously suggested by our laboratory [37 ]. This phenomenon is not exclusive to Brucella, as inhibition of host cell apoptosis is a strategy used by other intracellular microorganisms for survival in the host [38 39 40 ]. These data support the hypothesis that Fas-FasL-induced apoptosis does not contribute to killing of intracellular B. suis and that FasL is not one of the soluble factors that can have an effect on Brucella viability.

To further investigate the effects of soluble factors on infected macrophages, we analyzed two cytokines that are produced by activated V{gamma}9V{delta}2 T cells, IFN-{gamma} and TNF-{alpha} [23 ]. Cytokine treatment during infection showed that IFN-{gamma} but not TNF-{alpha} could induce some of the cytotoxicity exhibited by V{gamma}9V{delta}2 T cells, but neither cytokine appeared to have a significant effect on the total number of bacteria. This is confirmed by the data obtained with blocking IFN-{gamma} and TNF-{alpha} antibodies. These results support and extend our previous observation that treatment of human macrophages with IFN-{gamma} following infection does not suppress Brucella development in THP-1 cells [37 ]. Hence, Brucella may be able to evade IFN-{gamma}-induced microbicidal activity by an as-yet unidentified mechanism currently under investigation in our laboratory. Evasion of cytokine-mediated effects by intracellular pathogens is not unprecedented [41 , 42 ]. For instance, IFN-{gamma} does not activate human macrophages to kill intracellular M. tuberculosis, and macrophages infected with M. tuberculosis become resistant to the IFN-{gamma} signaling pathway by inhibiting the interaction of STAT1 with other proteins to form an active transcriptional complex [43 ].

In this study, we have demonstrated for the first time that V{gamma}9V{delta}2 T cells can affect the growth and survival of bacteria directly, independent of host cell environment. As granulysin is expressed by V{gamma}9V{delta}2 T cells and presents antimicrobial properties against several strains of bacteria, we have therefore investigated the role of granulysin in the direct antimicrobial effect against Brucella. We showed that the antimicrobial activity of V{gamma}9V{delta}2 T cells is not modified in the presence of antigranulysin antibody, suggesting that granulysin is not involved in antimicrobial properties of V{gamma}9V{delta}2 T cells against Brucella bacteria. Moreover, we showed that V{gamma}9V{delta}2 T cells release soluble factors responsible for direct effects on B. suis organisms. Also, soluble factors released from activated V{gamma}9V{delta}2 T cells are able to activate host cells, which in turn, can release an arsenal of antimicrobial agents to inhibit the multiplication of bacteria. Several families of molecules could be involved in this antimicrobicidal activity, including chemokines, defensins, or cathelicidins [44 , 45 ]. Macrophage-inflammatory protein-1{alpha} (MIP-1{alpha}), for example, is produced under certain conditions by activated V{gamma}9V{delta}2 T cells and has been shown to activate macrophages and thus, could help them to eliminate bacteria [46 , 47 ]. A recent study has shown that several chemokines such as CC chemokine ligand 20/MIP-3{alpha} can display antimicrobial activity [48 ]. Antimicrobial products present in the supernatant could also include {alpha}-defensins or cathelicidins (LL-37), which are expressed in lymphocytes. Cathelicidins such as LL-37 also appear to be a good candidate, as they exhibit potent chemotactic and antimicrobial activities [49 ]. We are currently exploring the soluble factors responsible for the antimicrobial effects by V{gamma}9V{delta}2 T cells on B. suis.

Another point that is important to clarify is whether the effects described are specific to V{gamma}9V{delta}2 T in response to infection by intracellular pathogens or whether other subsets of immune cells can act similarly. To investigate this, we used our in vitro infection model to test two other subsets of cells (NK cells or polyclonal populations of {alpha}ß T cells). {alpha}ß T cells do not modify intramacrophagic Brucella development, but NK cells impair it at the same level as V{gamma}9V{delta}2 T cells. However, we have previously demonstrated that the mechanisms used by NK cells to reduce intracellular Brucella development are different than those described here with V{gamma}9V{delta}2 T cells. For NK cells, the main effect is a contact-dependent mechanism with a release of lytic granules leading to lysis of infected macrophages. Therefore, the effects of V{gamma}9V{delta}2 T cells and more particularly, the mechanisms that they use to inhibit or block Brucella development appear to be specific.

To summarize, we have demonstrated that V{gamma}9V{delta}2 T cells use a combination of mechanisms to limit the spread of intracellular bacteria. Contact-dependent mechanisms are used to lyse infected host cells and remove the pathogen’s protective niche, and soluble factors produced by V{gamma}9V{delta}2 T cells affect the viability of the bacteria itself. Understanding the mechanisms involved in the early immune response by V{gamma}9V{delta}2 T cells will aid in development of therapeutic treatments or effective vaccines that use nonpeptide antigens to target these cells and harness their potent antimicrobial effects.


   ACKNOWLEDGEMENTS
 
This study was supported in part by a European Commission grant (#QLK2-1999-0014), an Ecos-Anuies program (Franco-Mexico) grant (Action Number PM990S01), and the Foundation pour la Recherche Medicale. We acknowledge Dr. Jacques Dornand for helpful discussion.

Received July 27, 2004; accepted January 4, 2005.


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