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An Antimicrobial Activity of Cytolytic T Cells Mediated by Granulysin

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Science  02 Oct 1998:
Vol. 282, Issue 5386, pp. 121-125
DOI: 10.1126/science.282.5386.121

Abstract

Cytolytic T lymphocytes (CTLs) kill intracellular pathogens by a granule-dependent mechanism. Granulysin, a protein found in granules of CTLs, reduced the viability of a broad spectrum of pathogenic bacteria, fungi, and parasites in vitro. Granulysin directly killed extracellularMycobacterium tuberculosis, altering the membrane integrity of the bacillus, and, in combination with perforin, decreased the viability of intracellular M. tuberculosis. The ability of CTLs to kill intracellular M. tuberculosis was dependent on the presence of granulysin in cytotoxic granules, defining a mechanism by which T cells directly contribute to immunity against intracellular pathogens.

Cytolytic T lymphocytes are required for protective immunity against intracellular pathogens such as Listeria monocytogenes and Trypanosoma cruzi, pathogens known to escape from the phagocytic vacuoles into the cytoplasm of infected host cells. CTLs have also been implicated in the control of organisms that are phagocytized by macrophages and remain localized within the phagosomes (for example, Salmonella typhimurium,Escherichia coli, and Mycobacterium tuberculosis). One mechanism by which immunity arises has been postulated to be the lysis of infected cells by the antigen-specific CTLs (1). This lysis is thought to be followed by the release of live bacteria, which are subsequently taken up and killed by newly immigrated and freshly activated macrophages (2). However, the increasing bacterial burden in the cells will eventually cause spontaneous lysis, which raises the intriguing question of why CTL-mediated lysis of the cell would be beneficial for the host. An explanation for the functional role of CTLs in immunity against intracellular infection was provided by the analysis of CTL in tuberculosis (2, 3). These experiments suggested that CTLs that kill infected cells through the granule-exocytosis pathway may release one or more effector molecules with the capacity to directly kill the intracellular microbial pathogen. We now show that granulysin is a critical effector molecule of the antimicrobial activity of CTLs.

Granulysin is a protein present in cytotoxic granules of CTL and natural killer (NK) cells (4, 5). Amino acid sequence comparison indicates that granulysin is a member of the saposin-like protein (SAPLIP) family. Granulysin is most similar to NK-lysin (43% identity and 67% similarity), a porcine protein with antibacterial activity (6). Granulysin is in the cytotoxic granules of T cells, which are released upon antigen stimulation (5). Two subsets of CTLs exist, which differ in phenotype, cytotoxic effector pathway, and antimicrobial activity (3). CD8+ CTLs lyse M. tuberculosis–infected macrophages by a granule-dependent mechanism that results in killing of the intracellular pathogen. In contrast, the cytotoxicity of CD4CD8[double negative (DN)] T cells is mediated by Fas-Fas ligand interaction and does not inhibit growth of the mycobacteria. The presence of granulysin in these two populations was therefore investigated (Fig. 1). By protein immunoblot analysis, granulysin was detected in three CD8+CTLs, two that were CD1-restricted and specific for M. tuberculosis and one that was major histocompatibility complex class I–restricted and specific for an influenza virus peptide (Fig. 1B). In contrast, three CD1-restricted, M. tuberculosis–specific DN CTLs expressed no granulysin. Confocal microscopy of CD8+ CTLs for granulysin revealed a punctate pattern consistent with granule localization (Fig. 1A). Double staining of the CD8+ T cells with antibodies to granulysin and perforin, a molecule known to be expressed in cytotoxic granules (7), showed a substantial colocalization. These data demonstrated the presence of granulysin in cytotoxic granules of CD8+ but not in DN CTLs.

Figure 1

The presence of granulysin correlates with the antimycobacterial effect of CTLs. (A) Detection of perforin and granulysin in CTLs by confocal laser microscopy (26). Cell lines recognizing either influenza virus (CD8.FP3) or M. tuberculosis (CD8.1) were double-labeled with anti-perforin (red vesicles) and anti-granulysin (green vesicles). Cells were visualized by differential interference contrast (DIC) (first panel of each row). Fluorescent confocal images were obtained for perforin expression (red, second panel of each row) and granulysin expression (green, third panel of each row). The two images were then superimposed (fourth panel of each row) to show vesicles expressing both perforin and granulysin (yellow). (B) Protein immunoblot analysis of CTLs for granulysin (27). Lysates of three DN (lane 1, DN.PT; lane 2, DN.OR; and lane 3, DN.AJW), three CD8+ (lane 4, CD8.TX; lane 5, CD8.FP3; and lane 6, CD8.2), and an NK cell line (20) (lane 7, YT) were separated by SDS–polyacrylamide gel electrophoresis. Granulysin was detected with a rabbit serum and visualized by chemiluminescence. The arrows indicate the two known forms of granulysin, 15 kD and 9 kD (5). The blot shown is a representative example of three independent experiments with similar results. (C) The ability to reduce the viability of intracellular M. tuberculosis correlates with the expression of granulysin. Granules of an M. tuberculosis–specific (CD8.TX) or influenza peptide–specific (CD8.FP3) line were released by treatment with strontium (28). CTLs were coincubated with macrophages (25), which had been infected with M. tuberculosis (29). After 18 hours, mycobacterial viability was determined (30). The number of cells expressing granulysin was determined by immunostaining (31). The results shown are representative for two independent experiments, each performed in triplicates. The data are given as CFU ± SEM (left) or percentage of cells expressing granulysin ± SEM (right). Scale bar represents 2 μm in all panels.

To assess whether the microbicidal effect of CTLs on infected macrophages was mediated by release of cytotoxic granules, we used human CTLs that effectively killed intracellular M. tuberculosis residing in macrophages (Fig. 1C). When the CTLs were pretreated with Sr++ to induce degranulation and deplete their cytotoxic granules, the ability of CTLs to kill the pathogen was abrogated (Fig. 1C, left). The ability to detect granulysin disappeared in parallel with the loss of microbicidal activity (Fig. 1C, right). In this system, the intracellular killing of mycobacteria was not attributable to a purinergic mechanism (8). Thus, the expression of granulysin and the ability to reduce the viability of intracellular M. tuberculosis correlated.

We used recombinant granulysin (9) to directly test its antimicrobial activity against several microbial pathogens. Culture conditions were adapted for the specific growth requirements of each organism, and growth inhibition induced by recombinant granulysin was initially screened by radial diffusion assay and confirmed by colony-forming unit (CFU) assay (Fig. 2). In these experiments, granulysin showed a dose-dependent growth inhibition of a broad spectrum of pathogens, including bacteria, fungi, and parasites. We found potent antibacterial activity in the micromolar range against gram-positive and gram-negative bacteria, causing a three orders of magnitude reduction in CFUs of S. typhimurium, L. monocytogenes, E. coli, and Staphylococcus aureus (Fig. 2, left). Granulysin also killed fungi and parasites, including Cryptococcus neoformans, Candida albicans, and Leishmania major (Fig. 2, right). The broad antimicrobial spectrum of granulysin is reminiscent of structurally unrelated defensins, which are nonspecifically released from cytoplasmic granules of polymorphonuclear leukocytes to kill phagocytized pathogens, including M. tuberculosis(10).

Figure 2

Dose-dependent cytotoxicity of granulysin against bacteria, parasites, and fungi. Microorganisms and granulysin were mixed and coincubated under appropriate conditions in a volume of 50 μl (bacteria and fungi: 3 hours at 37°C; L. major: 72 hours at 26°C). Samples were then diluted and spread on Trypticase soy agar (bacteria) or Sabouraud dextrose agar (fungi). After 24 hours, the number of CFUs was determined (32). Leishmania major parasites were quantitated by limiting dilution analysis performed on Novy-Nicolle-MacNeal blood agar slants, and the number of positive wells was determined microscopically after 14 days of incubation at 26°C. The results shown are representative for at least three experiments with similar results and are given as CFUs.

Mycobacterium tuberculosis is one of the most resistant pathogens to microbicidal mechanisms of mononuclear phagocytes. The only effector molecules clearly shown to be involved are reactive nitrogen intermediates (11). The effect of granulysin on the viability of virulent M. tuberculosis was examined by culturing M. tuberculosis in 7H9 media in the presence of various concentrations of granulysin (Fig. 3A, left). In five experiments, granulysin killed M. tuberculosis in a dose-dependent manner, with up to 90% of the bacteria killed within 72 hours, representing almost a logarithmic reduction in the number of CFUs. However, no antibacterial activity was detected when granulysin was added to M. tuberculosis–infected macrophages (Fig. 3A, right). Although the percentage reduction of M. tuberculosis CFUs was within an order of magnitude and less than that seen against E. coli, M. tuberculosisinfection in vivo can be produced by as few as 10 to 200 bacilli and is a slow process. In our experiments, the time of in vitro assay was only 72 hours, so that a cumulative antimicrobial effect mediated by these T cells over time could have a profound effect on the number of bacilli during the course of infection.

Figure 3

Granulysin delivered by perforin kills intracellular M. tuberculosis. (A) Effect of granulysin on the viability of extracellular (left) or intracellular (right) M. tuberculosis. Granulysin or HSP70 fragment (5) was added to 10,000 bacteria, and after 72 hours live bacteria were enumerated by determining the CFUs. Infected macrophages were incubated with granulysin to determine the effect on intracellular bacteria. After 72 hours, the number of CFUs was determined (30). The data show one representative result of five independent experiments, where the SD between the experiments was <5% for all protein concentrations. Data are given as percentage of killing of M. tuberculosis as compared with cultures incubated in the absence of protein. (B) Lytic activity of granulysin and perforin. Infected macrophages were incubated with granulysin or perforin. After 4 hours, the extent of cell lysis was determined (33). Results are given as percentage of specific lysis and show one typical experiment out of six. The SD between the single experiments was <7% for all concentrations. The spontaneous release of LDH by infected macrophages in the absence of protein was <10%. (C) Mycobactericidal activity of a combination of perforin and granulysin. Macrophages infected with M. tuberculosiswere incubated with granulysin (25 μM) and perforin (2000 U/ml). After 72 hours, the number of live bacteria was determined (30). The data are given as percentage of killing ofM. tuberculosis as compared with the bacteria cultured in diluent alone.

One explanation for the inability of granulysin to kill intracellular as compared with extracellular M. tuberculosis was the possible failure of the purified recombinant protein to gain access to the intracellular compartment in which mycobacteria reside. Such a defect may be overcome by the pore-forming agent perforin, which colocalized with granulysin (Fig. 1A). Experiments were therefore designed to compare relative activities of granulysin and purified perforin (12) in lysing human macrophages infected with virulent M. tuberculosis (Fig. 3B). Granulysin, when added in a concentration range that efficiently killed extracellular bacteria, showed little lytic activity against infected macrophages. In contrast, purified perforin, which is known to lyse various hematopoietic targets (13), exhibited substantial lytic activity against identically infected macrophages but was ineffective in reducing the viability of M. tuberculosis either in culture or intracellularly in macrophages. Thus, granulysin is a powerful mycobactericidal agent but may not be effectively delivered to the phagolysosomal compartment. In contrast, perforin is an ineffective antimicrobial agent, although it has the capacity to lyse infected target cells. A combination of perforin and granulysin resulted in macrophage lysis and decreased viability of intracellular mycobacteria (Fig. 3C). Thus, granulysin could kill intracellular M. tuberculosis if perforin, or possibly other pore-forming molecules of T cell granules, provided access to the intracellular compartment. The bacteria may be killed intracellullarly or extracellularly in tuberculosis, but this issue may not be of great biological importance. We know that M. tuberculosis grows both intracellularly or extracellularly, such that a CTL granule protein that can reduce the viability of the pathogen within infected cells or in the vicinity of infected or dying cells could have profound protective activity.

Both perforin and members of the amoebapore family are thought to mediate their biological effects by formation of microscopic pores leading to damage of target cell membranes (14). Consequently, we sought to ascertain whether granulysin killed M. tuberculosis by altering the structure or integrity of the bacteria. Scanning electron microscopy revealed that granulysin induces discrete lesions and distortions in the bacterial surface of M. tuberculosis (Fig. 4). Mycobacteria, singly and in clusters, were found to contain multiple small protrusions that were almost entirely absent from control samples. The reduction in viable CFUs in conjunction with evidence of bacterial surface alteration and distortion indicates that granulysin is directly cytotoxic to M. tuberculosis and other bacteria.

Figure 4

Granulysin induces lesions in the mycobacterial cell surface. Mycobacterium tuberculosis was incubated with or without granulysin (30 μM) for 80 hours. 1 × 106 bacteria were fixed and processed for scanning electron microscopy (34). (A) Mycobacterium tuberculosis incubated for 80 hours in medium alone (×23,000; scale bar represents 1 μm in all panels). (B) Mycobacterium tuberculosis coincubated for 80 hours with granulysin results in the formation of marked lesions in the bacterial surface (×23,000). (C) Granulysin-treated bacteria singly and in clusters reveal surface lesions (×40,000). (D) Percentage of granulysin-treated M. tuberculosis with lesions. Results shown represent the mean ± SEM of more than 400 bacilli per group enumerated per treatment.

These findings define a pathway by which antigen-specific T cells directly contribute to the death of microbial pathogens, specifically microorganisms residing in intracellular compartments. Perforin has a role in delivery of granule-associated proteins into subcellular compartments (15). We hypothesize that this mechanism allows granulysin to gain access to intracellular pathogens and destroy them. Although initial studies with perforin gene knockout mice had suggested that perforin is not required for the early control of tuberculosis (16), more recent studies indicate that perforin is in fact required for long-term protection (17). The perforin-granulysin microbicidal pathway is likely to complement non–antigen-specific mechanisms by which monocytes kill pathogens, including the generation of nitric oxide and oxygen free radicals.

A number of biologic functions have been proposed for the SAPLIP family, which includes granulysin, NK-lysin, saposins, surfactant-associated protein B, amoebapores, and plant aspartic proteinases. The present results provide evidence for the antimicrobial activity of granulysin. The presence of granulysin in NK cells (18) and of related peptides in cytoplasmic granules ofEntamoeba histolytica, the amoebapores (19), suggests that the SAPLIP family represents an ancient yet highly conserved form of antimicrobial host defense, likely contributing to innate immune responses. The importance of this pathway is reflected by the presence of granulysin and other family members in CTLs, indicating that the adaptive immune response has evolved to include antimicrobial peptides for effective immunity. We propose that the role of CTLs in protection against intracellular pathogens is not merely to lyse target cells and disperse the intracellular pathogens but in addition to deliver granulysin, a lethal weapon by which CTLs can directly reduce the viability of a variety of bacteria, fungi, and parasites genetically selected to evade host defense.

  • * Current address: Institut für Klinische Mikrobiologie, Immunologie und Hygiene, Universität Erlangen, D-91054 Erlangen, Germany.

  • To whom correspondence should be addressed: rmodlin{at}medicine.medsch.ucla.edu

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