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The Journal of Immunology, 2006, 177: 5533-5539.
Copyright © 2006 by The American Association of Immunologists, Inc.

Release of LL-37 by Activated Human V{gamma}9V{delta}2 T Cells: A Microbicidal Weapon against Brucella suis1

Sherri Dudal, Chrystell Turriere, Stephanie Bessoles, Pascaline Fontes, Françoise Sanchez, Janny Liautard, Jean-Pierre Liautard and Virginie Lafont2

Institut National de la Santé et de la Recherche Médicale, Unité 431, Université Montpellier 2, Montpellier, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Human V{gamma}9V{delta}2 T cells play a crucial role in early immune response to intracellular pathogens. Moreover, in brucellosis, these cells are drastically increased in the peripheral blood of patients during the acute phase of infection. In vitro, V{gamma}9V{delta}2 T cells are capable of inhibiting Brucella growth and development through a combination of mechanisms: 1) cytotoxicity, 2) macrophage activation and bactericidal activity through cytokine and chemokine secretion, and 3) antibacterial effects. We previously described that antibacterial factors were found in supernatants from activated V{gamma}9V{delta}2 T cells. In this study, we show that V{gamma}9V{delta}2 T cells express the human cathelicidin hCAP18 and its mature form, known as LL-37, is released upon activation of V{gamma}9V{delta}2 T cells. We also show that LL-37 has an antibacterial effect on Brucella suis. Overall, our results demonstrate that LL-37 is a soluble factor responsible for a part of the bactericidal activity of V{gamma}9V{delta}2 T cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Control of infection by intracellular pathogens requires an orchestrated response by the immune system, involving complex interactions of immune cells with infected 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). 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. However, the number of V{gamma}9V{delta}2 T cells can dramatically increase in early response to infection by a number of intracellular pathogens of viral, bacterial, or parasitic origin (3). A particular feature of these V{gamma}9V{delta}2 lymphocytes is that they respond to nonpeptidic Ags, which are not processed and presented in an MHC-restricted manner. These Ags were originally isolated from mycobacteria and were subsequently found in various other intracellular pathogens as metabolic products from the isoprenoid synthesis pathway (4, 5). Following activation by nonpeptidic Ags, V{gamma}9V{delta}2 T cells proliferate, release large amounts of cytokines (particularly IFN-{gamma} and TNF-{alpha}), and acquire cytotoxic activity against tumor cells or infected cells (6, 7). Nevertheless, to date, the place and the specific role of V{gamma}9V{delta}2 T cells in the immune response and their impact on the development of infection are still not well understood and the mechanisms remain to be clarified.

Brucella is a Gram-negative bacteria. Following infection, most of patients undergo an acute infection phase with undulant fever, which can progress either to recovery or to a chronic form of the disease. Chronic infection is tissue specific (spleen, liver, heart, bones, and brain), which can cause endocarditis, arthritis, meningitis, and osteomyelitis. As Brucella infects and multiplies within host macrophages, establishment of persistent infection seems to be due to the ability of bacteria to survive in macrophages despite an immune response. Similar to most intracellular pathogens, Brucella produces nonpeptidic Ags that are derived from the isoprenoid synthesis pathway (such as (E)-4-hydroxy-3-methyl-but-2-enyl-pyrophosphate (HMB-PP)3), which can specifically activate V{gamma}9V{delta}2 T cells (8). Following activation, V{gamma}9V{delta}2 T cells produce cytokines and exert their cytotoxicity on Brucella-infected macrophages (6). More specifically, V{gamma}9V{delta}2 T cells are capable of inhibiting Brucella growth and development through a combination of mechanisms: 1) cytotoxicity, 2) macrophage activation and bactericidal activity through cytokine and chemokine secretion, and 3) antibacterial effects via granulysin and yet-unidentified antimicrobial factors (9).

A group of molecules termed antimicrobial peptides has been described as possessing an antibacterial effect due to their positive charge and hydrophobic property (10). These cationic host defense peptides are a primitive and conserved component of the innate immune response. These peptides can be expressed either constitutively or induced in response to pathogens. The role of cationic peptides has been considered to be of primary significance in innate immunity and has even been suggested to be the link between innate and adaptive immunity (11). Antimicrobial molecules have been divided into several groups based on their length, secondary and tertiary structure, and the presence or absence of disulfide bridges. In mammals, two major families have been identified: defensins and cathelicidins. To date, three subfamilies, {alpha}, beta, and {theta}, of defensins exist, but only one molecule of the cathelicidin family has been identified in humans (12). Defensins and cathelicidins are synthesized as preproproteins. Preprodefensins contain a peptide signal sequence, an anionic peptide and a C-terminal cationic defensin. The removal of the anionic peptide is an activation step that converts the inert prodefensin to antimicrobial mature defensin (13). The human cathelicidin is stored in specific granules of cells as the proprotein hCAP18. Upon its release, unprocessed hCAP18 is cleaved into the C-terminal domain, LL-37, from the N-terminal cathelin domain (11). In addition to their antimicrobial properties, defensins and LL-37 have been shown to potently modulate the immune response with a wide variety of activities such as chemotaxis, reduction in proinflammatory cytokine production, and promotion of wound healing (14, 15). Even though they are mainly expressed in neutrophils and epithelial cells, defensins and cathelicidins have been found in monocytes and lymphocytes (16).

In a previous study, we have shown that supernatants from activated (A) V{gamma}9V{delta}2 T cells were capable of strongly reducing Brucella bacterial numbers (9). Therefore, we became interested in determining the soluble factor secreted by V{gamma}9V{delta}2 T cells that was capable of reducing bacterial numbers by either stopping Brucella growth or killing Brucella. In this study, we demonstrate that V{gamma}9V{delta}2 T cells express both {alpha}-defensins and, in greater quantities, the precursor of human cathelicidin, hCAP18. Furthermore, the mature form of human cathelicidin, LL-37, is released from activated V{gamma}9V{delta}2 T cells and has an antimicrobial effect on Brucella suis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Ags, Abs, and reagents

Nonpeptidic Ag HMB-PP was generously provided by J. L. Montero (Montpellier, France). Mouse monoclonal anti {alpha}-defensin Ab was purchased from Serotec, polyclonal rabbit anti-LL37 Ab used in flow cytometry analysis was a generous gift from Dr. B. Agerberth (Karolinska Institute, Stockholm, Sweden) (16). Anti-LL37 Ab used in Western blot analysis and immunoprecipitation was generously provided by Dr. R. L. Gallo (University of California, San Diego, CA) (17). Anti-LL-37 used in immunohistochemistry analysis was purchased from Phoenix Pharmaceuticals. Isotypically matched control mouse or rabbit Abs (conjugated or not) were all purchased from BD Biosciences. Recombinant human LL-37 was purchased from Phoenix Pharmaceuticals.

Cells

PBMC from healthy donors were prepared by density centrifugation on Ficoll-Paque (Eurobio). V{gamma}9V{delta}2 T cells were purified from nonadherent PBMC by positive immunoselection using an anti-{gamma}9 mAb (Beckman Coulter) and goat anti-mouse IgG-coated Dynal magnetic beads (Dynal) following the protocol described in Ref. 9 . After 1 wk of culture, flow cytometry analysis of cells double stained with anti-V{gamma}9/anti-V{delta}2 Ab demonstrated that viable T lymphocytes were >99% V{gamma}9V{delta}2 T cells. All cells used in our study come from pure V{gamma}9V{delta}2 T cell culture.

Preparation of V{gamma}9V{delta}2 T cell supernatants

Pure cultures of V{gamma}9V{delta}2 T cells in PBS with Ca2+ and Mg2+ (reference 14040; Invitrogen Life Technologies) were A or NA with HMB-PP (10 nM) at 37°C. At different time points (3, 6, and 18 h), cells were centrifuged, and the supernatants were harvested before being tested for their antimicrobial activity.

Antimicrobial effect of V{gamma}9V{delta}2 T cells on Brucella

HMB-PP-activated V{gamma}9V{delta}2 T cells (106/ml) or their supernatants were incubated directly with 104 B. suis bacteria (strain 1330; American Type Culture Collection (ATCC)) for 24 h in RPMI 1640 plus 10% FCS or PBS with Ca2+ and Mg2+ in a 48-well plate. Following this, serial dilutions of each well were performed and plated on tryptic soy broth (TS) agar plates to determine bacterial counts (CFUs). Data are expressed in CFU per milliliter or in percentage of CFU controls as indicated in the figures.

Antimicrobial effect of LL-37 on Brucella

Different concentrations of rLL-37 peptide were incubated directly with 104 B. suis bacteria (strain 1330; ATCC) for 24 h in PBS with Ca2+ and Mg2+ in 48-well plates. Following this, serial dilutions of each well were performed and plated on TS agar plates to determine bacterial counts (CFUs). Results were expressed in the percentage of CFU controls as indicated in the figures.

Optimal concentrations of rLL-37 peptide (40 µg/ml) were incubated directly with 104 B. suis bacteria (strain 1330; ATCC) for 24 h in low (Tris, 10 mM; pH 7.9) or high (PBS with Ca2+ and Mg2+) salt buffer concentrations in 48-well plates. Serial dilutions of each well were performed, plated on TS agar plates to determine bacterial counts (CFUs) and expressed as number of CFU per milliliter.

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. Then, cells were permeabilized using a Cytofix/Cytoperm plus kit (BD Biosciences) for intracellular staining. For defensin expression analysis, cells were incubated with mouse anti-{alpha}-defensin mAb (IgG1 isotype) or mouse control IgG1 and followed by incubation with an FITC-conjugated mouse anti-Ig. For cathelicidin expression analysis, cells were incubated with polyclonal rabbit anti-LL-37 Ab or polyclonal rabbit Ig and then with a PE-conjugated rabbit anti-Ig Ab. Each step of Ab incubation was 30 min, and then the cells were washed once and analyzed on FACScalibur (BD Biosciences) using CellQuest software.

Microscopy analysis

V{gamma}9V{delta}2 cells (1 x 106/well) were fixed with 2% formaldehyde for 20 min. After two washes in washing buffer (PBS plus 2% FCS), cells were incubated with 5 µg of primary Ab (a rabbit anti-LL-37 Ab or rabbit IgG control) in washing buffer supplemented with 0.1% saponin for 30 min. After two washes, secondary Ab (Alexa Fluor 546-conjugated anti-rabbit IgG; Molecular Probes) was diluted 1/200 in washing buffer with saponin (0.1%) and incubated for 30 min. Then, cells were washed twice before being mounted with Mowiol mounting solution and viewed by microscopy.

Immunoprecipitation of hCAP18/LL-37

After 18 h of V{gamma}9V{delta}2 cell activation (4 x 107), supernatants were harvested and cells were lysed in 1 ml of lysis buffer containing 50 mM HEPES (pH 7.4), 150 mM NaCl, 10 mM NaF, 10 mM iodoacetamide, 1% Nonidet P-40, 1 mM PMSF, 1 mM Na2VO3, and 1 µg/ml of each protease inhibitor (leupeptin, aprotinin, and chymostatin). hCAP18/LL-37 were immunoprecipitated from NA or A V{gamma}9V{delta}2 cell lysates or their supernatants with 2 µg of anti-LL-37 Ab. Immune complexes were collected using protein A-Sepharose (Amersham Biosciences) and washed twice with lysing buffer or PBS before resuspending in 50 µl of reducing sample buffer. Purified proteins were further separated by 15% SDS-PAGE, and transferred to polyvinylidene difluoride membranes (Millipore). Western blot analysis was performed with an anti-LL-37 Ab, and the corresponding HRP-conjugated Ab was used. Immunoreactive bands were visualized with the chemiluminescence Western blotting system (Amersham Biosciences).

Statistical analysis

The mean of triplicate samples from the same experiment are shown for each data point with their SEM and is representative of a minimum of three experiments performed with separate human blood donors. The value of 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
 Disclosures
 References
 
Antimicrobial factors are present in supernatant from activated V{gamma}9V{delta}2 T cells

We previously showed that V{gamma}9V{delta}2 T cells as well as supernatants from activated V{gamma}9V{delta}2 T cells contain factor(s) that were able to affect growth and/or survival of pathogenic bacteria Brucella in the absence of host cells, indicating a potent antimicrobial effector function of these cells (9) (Fig. 1A). However, because the experiments were performed in nutrient-rich medium allowing bacterial proliferation, it was not possible to know whether V{gamma}9V{delta}2 T cells have a bacteriostatic or bactericidal effect on Brucella. To assess the putative antimicrobial activity of V{gamma}9V{delta}2 T cells, we replaced the complete medium by PBS, which does not contain nutrients that are required for growing cells and bacteria, but allow their survival for the duration of the experiment. A or NA V{gamma}9V{delta}2 T cells were placed in 48-well plates containing 104 bacteria per well in PBS for 18 h. Then, the number of viable bacteria present in wells was quantified by plating serial dilutions on TS agar. Data expressed in CFU per milliliter showed that the number of bacteria was decreased in wells that contain activated V{gamma}9V{delta}2 T cells (2.6 x 107 ± 3.5 x 106 CFU/ml) vs NA V{gamma}9V{delta}2 T cells (2.2 x 106 ± 1.9 x 105 CFU/ml). To underline the specificity of the antibacterial activity of V{gamma}9V{delta}2 T cells, we performed the same experiment with {alpha}beta T cells. No significant decrease in the number of bacteria was observed between NA and activated {alpha}beta T cells (1.4 x 107 ± 6.9 x 105 CFU/ml vs 1.2 x 107 ± 1.3 x 106; data not shown).


Figure 1
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  		FIGURE 1. Presence of antimicrobial factors in supernatants of activated V{gamma}9V{delta}2 T cells. A, B. suis culture was diluted in nutrient-rich medium (RPMI 1640 plus 10% FCS) and added to a 48-well plate at a final concentration of 1 x 104 bacteria/well and incubated with either medium alone (M), HMB-PP-activated V{gamma}9V{delta}2 T cells ({gamma}{delta} T cells), or supernatant prepared from an overnight culture of HMB-PP-activated V{gamma}9V{delta}2 T cells (SN). After 18 h, bacteria were recovered from the wells, and the number of CFUs was assessed by serial plating on TS medium. Data are shown as the mean of triplicate wells plus SEM, and significant differences between the conditions studied and medium are indicated as follows: *, p < 0.001. These results are representative of three experiments performed with pure population of V{gamma}9V{delta}2 T cells from different human donors. B, B. suis culture was diluted in PBS and added to a 48-well plate at a final concentration of 1 x 104 bacteria/well and incubated with either HMB-PP (10 nM)-activated 0.5 x 106 V{gamma}9V{delta}2 T cells ({gamma}{delta} T cells) or with supernatants prepared from V{gamma}9V{delta}2 T cells that were activated for 3, 6, or 18 h (SN). Activated (A) conditions were compared with their NA controls. After 18 h, bacteria were recovered from the wells and the number of CFUs was assessed by serial plating on TS medium. Data are shown as the mean of triplicate wells plus SEM expressed in percentage of CFU control ((CFU of activated cells or supernatants from activated cells/CFU of NA cells or supernatants from NA cells) x 100). Significant differences between NA and A conditions are indicated as follows: *, p < 0.01; **, p < 0.001. These results are representative of three experiments performed with pure populations of V{gamma}9V{delta}2 T cells from different human donors.

 
Then, we analyzed the presence of factors in supernatants of V{gamma}9V{delta}2 T cells. A or NA V{gamma}9V{delta}2 T cells or their supernatants, were placed in 48-well plates containing 104 bacteria per well in PBS for 18 h (Fig. 1B). The number of viable bacteria present in wells was quantified by plating serial dilutions on TS agar and normalized to their appropriate nonactivated control as indicated in the figures. The number of bacteria was strongly decreased in wells that contain activated V{gamma}9V{delta}2 T cells or their supernatants. The supernatants collected from V{gamma}9V{delta}2 T cells that were activated for 3, 6, or 18 h with HMB-PP, were increasingly effective in reducing the number of bacteria. Therefore, upon activation, V{gamma}9V{delta}2 T cell release soluble factors that are able to strongly reduce the number of B. suis, showing a bactericidal effect.

Expression of {alpha}-defensins and cathelicidin by V{gamma}9V{delta}2 T cells

Following this, we characterized the soluble factors responsible for this potent antimicrobial activity. We have previously shown that TNF-{alpha}, IFN-{gamma}, and granulysin, are not the soluble factors involved in the reduction of extracellular Brucella (9). Because the precursors of {alpha}-defensins and LL-37 are found in NK cells and some subpopulations of T cells (16), we determined whether V{gamma}9V{delta}2 T cells express these molecules using intracellular staining for {alpha}-defensins (1, 2, 3) and LL-37 and flow cytometry analysis. As shown in Fig. 2A, {alpha}-defensins are only moderately expressed within V{gamma}9V{delta}2 T cells, whereas LL-37 is strongly expressed in V{gamma}9V{delta}2 T cells. Through microscopy analysis, we confirmed that LL-37 is expressed in V{gamma}9V{delta}2 T cells (Fig. 2B). The Abs used in flow cytometry and microscopy analysis did not differentiate between precursors and/or mature forms of these cationic peptides. Therefore, we used Western blot analysis to show that LL-37 (Fig. 3) is mainly present and stored in its precursor form inside nonactivated and HMB-PP-activated V{gamma}9V{delta}2 T cells. Furthermore, we immunoprecipitated LL-37 from supernatants prepared from nonactivated and activated V{gamma}9V{delta}2 T cells and found that only the supernatants from activated V{gamma}9V{delta}2 T cells contained LL-37, which was present in its mature form (4.5 kDa) and not as the precursor hCAP18 (18 kDa), as confirmed by the rLL-37 peptide control. Therefore, the secreted form from activated V{gamma}9V{delta}2 T cells is LL-37, as expected, and the precursor form hCAP18 is present in both nonactivated and activated V{gamma}9V{delta}2 T cells, suggesting that regeneration of intracellular stores with hCAP18 occurs as LL-37 is released.


Figure 2
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  		FIGURE 2. Expression of {alpha}-defensins and LL-37 in V{gamma}9V{delta}2 T cells. A, Flow cytometry analysis. V{gamma}9V{delta}2 T cells were permeabilized before staining for intracellular LL-37 or defensins. For LL-37 staining, V{gamma}9V{delta}2 T cells were stained with rabbit polyclonal anti-LL-37 Ab (black line) or control rabbit polyclonal Ig (gray-filled shape) and then with a PE-conjugated anti-rabbit Ig Ab secondary Ab. For defensin staining, V{gamma}9V{delta}2 T cells were stained with a mouse anti-defensin mAb (black line) or mouse control IgG1 (gray-filled shape), and then with an FITC-conjugated anti-mouse Ig Ab. B, Immunohistochemical analysis. After fixation and permeabilization, V{gamma}9V{delta}2 T cells were stained with a rabbit polyclonal anti-LL-37 Ab or control rabbit polyclonal Ig and then with a Alexa Fluor 546-conjugated anti-rabbit Ig Ab. Positive staining is shown in red.

 

Figure 3
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  		FIGURE 3. Detection by Western blotting of hCAP18 in V{gamma}9V{delta}2 T cells and LL-37 in supernatants prepared from activated V{gamma}9V{delta}2 T cells. V{gamma}9V{delta}2 T cells were A or NA with HMB-PP for 18 h. Then, supernatants were harvested and cells were lysed. hCAP18/LL-37 was immunoprecipitated from cell lysates and separately from their supernatants. Immunocomplexes were loaded on 15% SDS-PAGE gels and the presence of hCAP18 or LL-37 was shown by Western blotting.

 
Antibacterial effects of rLL-37 on B. suis

The spectrum of microbicidal activity of cationic peptides is as diverse as the variation in the number of mature peptides within the family. Cationic peptides are microbicidal against Gram-negative and/or Gram-positive bacteria, fungi, parasites, and enveloped viruses (14). Their antimicrobial activity has often been assessed by determining the minimal inhibitory concentration of peptide required to affect bacterial viability in vitro. The minimal inhibitory concentration of peptides vary depending on the function of peptide sequences, bacterial species, and concentrations of salt in antimicrobial medium assays. Moreover, it has been reported that some Brucella strains are resistant to cationic peptides (18). To determine the antimicrobial activity of LL-37 on B. suis, various doses (10, 20, and 40 µg/ml) of rLL-37 were added to B. suis cultures diluted to 1 x 104 CFU/ml in PBS. A dose-dependent decrease in bacterial numbers was observed after 18 h of incubation with increasing concentrations of LL-37 (Fig. 4). Hence, LL-37 was demonstrated to have an antimicrobial activity against B. suis.


Figure 4
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  		FIGURE 4. rLL-37 has an antimicrobial effect on Brucella. B. suis culture containing 1 x 104 bacteria/well was aliquoted into 48-well plates and incubated with either PBS alone or PBS plus different concentrations of rLL-37. After 24 h, bacteria were recovered from the wells and the number of CFUs was assessed by serial plating on TS medium. Data are shown as the mean of triplicate wells plus SEM, expressed in percentage of CFU control ((CFU in PBS alone or with LL-37/CFU in PBS alone) x 100). Significant differences between conditions studied and controls are indicated as follows: *, p < 0.1; **, p < 0.01. These results are representative of three experiments.

 
Because salt and cation concentrations modify antimicrobial activity of cationic peptides (19), we have compared the activity of LL-37 (40 µg/ml) on B. suis in medium containing low (Tris buffer; Fig. 5B) or high (PBS buffer; A) salt concentrations. In these experiments, bacterial levels are expressed as number of CFU per milliliter to facilitate analysis. PBS or Tris buffers are poor in nutrients and do not allow the growth of Brucella but maintain its survival for at least 24 h (~105 bacteria/ml are present in controls at all time points). Stronger antimicrobial activity of LL-37 was observed at low salt concentrations with a 2 log decrease in bacterial number in Tris buffer vs a 1 log in PBS buffer. Moreover, kinetic analysis showed that the strongest effects of LL-37 on Brucella were observed after 18 h of incubation. Moreover, the decrease in absolute number of Brucella in the presence of LL-37 after 18 and 24 h of incubation shows that LL-37 has a bactericidal effect on Brucella.


Figure 5
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  		FIGURE 5. Comparison of antimicrobial effect of LL-37 in medium containing low or high salt concentrations. B. suis culture containing 1 x 104 bacteria/well was aliquoted into 48-well plates and incubated with either medium alone, or medium plus rLL37 (40 µg/ml) for the time indicated. A, Experiments are performed in high salt concentration conditions (PBS buffer). B, Experiments are done in low salt concentrations (Tris buffer, 10 mM (pH 7.9)). At the time indicated, bacteria were recovered from the wells, and the number of CFUs was assessed by serial plating on TS medium. Data are shown as the mean of triplicate wells plus SEM, and significant differences between the conditions studied and medium are indicated as follows: *, p < 0.1; **, p < 0.05. These results are representative of three separate experiments performed.

 
Impact of LL-37 removal from supernatants of V{gamma}9V{delta}2 T cells

Finally, to formally establish that LL-37 was involved in the antimicrobial effect of V{gamma}9V{delta}2 T cells, LL-37 was eliminated from activated V{gamma}9V{delta}2 T cell supernatants by immunoprecipitation. Absence of LL-37 in supernatants after immunoprecipitation was verified by Western blot analysis (data not shown). Fig. 6 shows that a partial reduction of antimicrobial effect is observed after removal of LL-37.


Figure 6
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  		FIGURE 6. Removal of LL-37 in supernatants of V{gamma}9V{delta}2 T cells. B. suis culture containing 1 x 104 bacteria/well was aliquoted into 48-well plates and incubated with either supernatants from nonactivated V{gamma}9V{delta}2 T cells (SN), activated V{gamma}9V{delta}2 T cells (SNA), or SNA with the removal of LL-37 by immunoprecipitation (SNA + IP). After 24 h, bacteria were recovered from the wells and the number of CFUs was assessed by serial plating on TS medium. Data are shown as the mean of triplicate wells plus SEM, expressed in percentage of CFU control ((CFU from activated conditions/CFU from NA condition) x 100). Significant differences between conditions studied and controls are indicated as follows:*, p < 0.01; **, p < 0.001. These results are representative of three separate experiments.

 
Together, these results demonstrate novel findings on V{gamma}9V{delta}2 T cell antimicrobial activity against B. suis, which strongly implicates the cationic peptide, LL-37. Nevertheless, because immunoprecipitation did not remove all of the antimicrobial activity of supernatants on B. suis, we cannot exclude that, in addition to LL-37, activated V{gamma}9V{delta}2 T cells release other soluble factors that take part in their bactericidal activity against B. suis.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In our previous study (9), we demonstrated that cell contact mechanisms played an important part in reducing the levels of intracellular B. suis in infected macrophages. However, we also observed that some unidentified soluble factors released from activated V{gamma}9V{delta}2 T cells, were capable of strongly reducing bacteria in medium and, to a lesser extent, in infected macrophages separated from V{gamma}9V{delta}2 T cells by a filter. These soluble factors could not be attributed to effects of proinflammatory cytokines (IFN-{gamma} and TNF-{alpha}) nor granulysin, an antibacterial protein contained in lytic granules (9). Studies from Dieli et al. (20) have shown that granulysin from V{gamma}9V{delta}2 T cells can kill both intracellular and extracellular bacteria, Mycobacterium tuberculosis, but further studies showed that granulysin requires cell-to-cell contact and the observed extracellular effects came from recombinant granulysin that was added to supernatant or from strontium chloride treatments (21, 22). Therefore, granulysin is not a potential soluble factor. Thus, we pursued our study to determine which soluble factors were secreted by V{gamma}9V{delta}2 T cells and able to reduce the level of extracellular B. suis.

In this study, we have shown that activated V{gamma}9V{delta}2 T cells rapidly secrete soluble factors, which have a strong bactericidal effect on Brucella and are secreted within 3 h of activation, lasting up to 18 h after activation (Fig. 1). This shows that V{gamma}9V{delta}2 T cells are capable of sustaining their antimicrobial activity. Among known antimicrobial molecules, two families have been found to be present in mammals: defensins and cathelicidins. Most of these molecules are synthesized and stored in cells in their immature forms and are processed into mature forms by proteases (12, 15). We have shown by flow cytometry and Western blot analysis that intracellular stores of V{gamma}9V{delta}2 T cells consist primarily of cathelicidin hCAP18 and, to a lesser extent, of {alpha}-defensin, and that V{gamma}9V{delta}2 T cells secrete LL-37 upon activation (Figs. 2 and 3). Interestingly, hCAP18 does not colocalize with perforin and is not stored in lytic granules containing perforin and granulysin in the V{gamma}9V{delta}2 T cells (data not shown).

It has been reported that, in neutrophils, hCAP18 is processed extracellularly by proteinase 3 into the N-terminal cathelin domain, and the C-terminal domain (mature LL-37) (23). Proteinase 3 can be found in azurophil granules in neutrophils along with cationic peptides and can be produced by monocytes and lymphocytes (24). To date, no data has been reported concerning the presence and expression of proteinase 3 in V{gamma}9V{delta}2 T cells. We have not been able to detect the expression of proteinase 3 in V{gamma}9V{delta}2 T cells by flow cytometry (data not shown). However, more recent studies have shown that other proteases can be involved in the processing of hCAP18 into active antimicrobial peptides. In the vagina, it has been reported that gastricsin, another serum protease, can process hCAP18 (25). Also, other serine proteases (not yet identified) are responsible for processing hCAP18 into multiple cathelicidins at the skin surface (17). Moreover, in bovine and porcine species, the processing of cathelicidin precursor is conducted by elastase (26, 27). Although, proteinase 3 does not seem to be expressed in V{gamma}9V{delta}2 T cells (or too weakly to be seen in flow cytometry), other proteases may be responsible for processing of hCAP18 into LL-37.

The next step was to show that LL-37 was capable of decreasing the numbers of B. suis bacteria. LL-37 has a broad antimicrobial spectrum and is active on both Gram-positive and Gram-negative bacteria (28, 29). Nevertheless its antimicrobial activity can vary considerably from one species to another and even from one strain to another. For example, pathogenic bacteria such as Listeria monocytogenes, Staphyloccoccus aureus, Salmonella typhimurium, and M. tuberculosis are sensitive to cationic peptides, thus making cationic peptides an important innate defense mechanism (30, 31). However, some bacteria such as Pseudomonas aeruginosa, Enterococcus faecalis, and Streptococcus pyogenes are capable of evading cationic peptides such as LL-37 through their ability to produce proteases, which degrade LL-37 (32). Hence, it was important to show that B. suis was sensitive to LL-37. We demonstrated in Fig. 4 that LL-37 is a potent antimicrobial weapon against B. suis. It acts in a dose-dependent manner and the concentrations of LL-37 used (10–40 µg/ml corresponding about to 2–8 µM) are in accordance with already published studies on other bacterial species (28, 29).

It has been reported that salt and cation concentrations influence the antimicrobial activity of cationic peptides as in the case of LL-37, and that the effects of salt and cation concentrations can vary from one bacterial species to another (19). We have tested the effects of salt concentrations on the antimicrobial activity of LL-37 against Brucella (Fig. 5). LL-37 showed more potent activity against Brucella in low salt concentration conditions. To determine whether LL-37 has bacteriostatic or bactericidal effects on Brucella, we performed kinetic studies and expressed the data as CFU per milliliter. These studies showed that 1) both media used (PBS and Tris) do not allow Brucella growth but maintain its survival, 2) absolute numbers of bacteria decrease in the presence of LL-37 demonstrating the bactericidal activity of LL-37, and 3) the most effective bactericidal activity of LL-37 is observed after 18 h of incubation with Brucella.

To confirm that the antimicrobial effects of LL-37 in supernatants from activated V{gamma}9V{delta}2 T cells were due to LL-37, we removed LL-37 by immunoprecipitation and obtained a decrease in the antimicrobial potency of the supernatants from activated V{gamma}9V{delta}2 T cells (Fig. 6). Because only one-third of the supernatant antimicrobial activity was eliminated, it is possible that shorter forms of LL-37 that would not be detected by our LL-37 Ab or other soluble factors, are secreted from V{gamma}9V{delta}2T cells. A recent study shows that postsecretory processing of LL-37 can occur, leading to shorter peptides with different potencies, and in particular, some of them display an enhanced antimicrobial action on bacteria (17). These shorter peptides may not be removed by immunoprecipitation and, thus, remain active in supernatants. Furthermore, human cathelicidin is secreted on the proprotein hCAP18, which is extracellularly processed into two molecules, LL-37 and a cathelin-like protein. A recent report (33) showed that cathelin-like proteins possess an antimicrobial activity in addition to its inhibitory protease activity. Thus, the cathelin-like domain could play a part in the antimicrobial effect present in supernatants of activated V{gamma}9V{delta}2 T cells. Moreover, {alpha}-defensins are also expressed by V{gamma}9V{delta}2 T cells and could intervene in the antimicrobial activity.

V{gamma}9V{delta}2 T cells have been clearly implicated in innate immunity and in host defense against infections. In this study, we have shown that secretion of antimicrobial peptides such as LL-37 could be involved in host defense. Although it has been clearly demonstrated that antimicrobial peptides are involved in host defense by killing bacteria directly, there is also evidence that these peptides may reduce bacterial numbers by activating other immune cells. For example, in the case of Brucella infection, it has been demonstrated that cationic peptides with no direct antimicrobial activity can still reduce bacterial numbers in animal infections by specifically killing infected macrophages (34). In other animal infection models (S. aureus and Salmonella), cationic peptides play a role in innate immunity to bacterial invasion not only by eliminating extracellular bacteria but also by modulating the responses of a variety of effector cells of the innate immune response, including epithelial cells, monocytes, macrophages, and dendritic cells (DC) (35). Notably, LL-37 has chemotactic effects, inducing selective migration of monocytes and neutrophils, and it induces the release of inflammatory mediators and degranulation of mast cells (14, 35). Also, LL-37 directly modifies the activity of monocytes and macrophages by reducing the LPS-stimulated production of proinflammatory cytokines, inducing production of chemokines (MCP-1 and IL-8) and amplifying respiratory burst response (36, 37). In animals, Brucella infects and survives inside macrophages. Therefore, we could not exclude that, in addition to its direct antimicrobial effect on extracellular Brucella, LL-37 also has an effect on the intracellular development of Brucella through the activation of macrophages. Using an in vitro macrophage infection model, we have shown that LL-37 has no effect on the multiplication and survival of intracellular Brucella (data not shown). This is not surprising because LL-37 would not greatly modify stimulation with LPS of Brucella origin because it is a weak activator of proinflammatory responses, and furthermore, the mechanism that Brucella uses to invade macrophages involves inhibiting respiratory burst response. Thus, these two intracellular protective immune mechanisms, which are triggered by LL-37, would not affect Brucella.

Recently, LL-37 was shown to modulate DC differentiation and thus influence T cell polarization induced by DCs (38). Thus, LL-37 appears to function as a bridge between the innate and adaptive immune systems. Through release of cytokines and LL-37, activated V{gamma}9V{delta}2 T cells certainly play a part in the polarization of adaptive immune response and take a particular place in the immune response at the interphase between innate and acquired responses.

Finally, our results clearly demonstrate that V{gamma}9V{delta}2 T cells use LL-37 as an antimicrobial defense mechanism in innate immunity and, more particularly, against B. suis. Brucella is still a public health problem in developing countries, and the importance of studying this bacteria has increased because Brucella spp. are categorized in the B group in the National Institute of Allergy and Infectious Diseases Biodefense Research Agenda. Brucella is capable of causing infection following the inhalation of a very small number of organisms, and, to date, no human vaccine exists. Understanding mechanisms of the anti-Brucella immune response will provide insight in vaccine research.


   Acknowledgments
 
We thank Dr. R. L. Gallo and Dr. B. Agerberth for their gifts of LL-37 Abs. We are also grateful to Dr. Jacques Dornand for critical reading of the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This study was supported in part by European Commission Grant QLK2-1999-0014 and an Ecos-Anuies program (Franco-Mexico) grant (Action No. PM990S01). Back

2 Address correspondence and reprint requests to Dr. Virginie Lafont, Institut National de la Santé et de la Recherche Médicale, Unité 431, Université Montpellier 2, Place Eugene Bataillon, CC 100, 34095 Montpellier Cedex 05, France. E-mail address: vlafont{at}univ-montp2.fr Back

3 Abbreviations used in this paper: HMB-PP, (E)-4-hydroxy-3-methyl-but-2-enyl-pyrophosphate; TS, tryptic soy broth; DC, dendritic cell; A, activated; NA, nonactivated. Back

Received for publication March 30, 2006. Accepted for publication July 26, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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