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Departments of
* Medical Sciences and
Microbiology and Infectious Disease and Internal Medicine, University of Calgary, Calgary, Alberta, Canada; and
Department of Pediatrics, Stanford University, Stanford, CA 94305
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Studies of the role of granulysin have been thwarted by the inability to specifically deplete granulysin in human lymphocytes without affecting the other contents of the granules. Furthermore, blocking Abs to granulysin are ineffective, presumably because they do not gain access to the synapse between the cytotoxic lymphocyte and the target cell. Thus, it was necessary to apply a new technique to inhibit granulysin. For this purpose, RNA interference (RNAi)3 might hold promise. RNAi is a newly developed method of gene silencing that was initially applied with success in Caenorhabditis elegans (9, 10, 11), plants (12, 13), Drosophila melanogaster (14, 15), and Trypanosoma brucei (16). Until recently, mammalian cells were not amenable to RNAi, because in vitro transcribed long dsRNAs (>30 bp) led to activation of a nonspecific response that blocked initiation of protein synthesis and mRNA degradation (17). However, recently, it has been reported that dsRNA is processed to shorter fragments that mediate specific gene-silencing activity (18). This activity can be mimicked by double strand 21-nt RNA with 2-nt 3' overhang, which specially inhibit gene expression and bypass the sequence-independent response of mammalian cells to long dsRNA (19). These short RNA duplexes are referred to as small interfering RNA (siRNA) (18) and present an opportunity to use RNAi in mammalian cell systems.
Among the potential microbial pathogens, Cryptococcus neoformans is one of the most common life-threatening fungal infections in patients who have compromised cell-mediated immunity, including AIDS (20, 21, 22). This microbe has previously been shown to be susceptible to recombinant granulysin (7), and therefore, it is an excellent target for studies of expression and regulation of granulysin. Previous studies have shown that cytotoxic lymphocytes possess anticryptococcal activities, but these studies failed to identify the antimicrobial mechanism (23). We questioned whether granulysin might be the previously unidentified factor responsible for lymphocyte-mediated anticryptococcal activity (24). Furthermore, T cells are activated by the C. neoformans mitogen (CnM), which is contained in the cell wall of C. neoformans (25). Thus, C. neoformans provides a simple system, in which it can serve as both the stimulus and the target for studies of granulysin regulation and activity.
Although granulysin protein expression has been detected in CTL and NK cell lines, gene transcription and protein expression are induced after activation of PBL (26, 27). This suggests that activation is required for the expression and activity of granulysin in CD8 T cells. However, activation of primary CD8 T cells is a complicated process in which CD4 T cells are required. CD4 T cells are in turn activated by an accessory cell and a costimulation-dependent mechanism. The activated CD4 T cells provide T cell growth factors that activate CD8 T cells (28). Alternately, CD4 T cells can signal accessory cells to a heightened state of activation; the accessory cells then acquire the ability to stimulate CD8 T cells (29).
It has been demonstrated that lymphocyte-mediated antifungal activity was critically dependent on IL-15 (30). IL-15 is a member of the four-helix bundle cytokine family with growth factor activity for T cells and NK cells (31, 32). IL-15 is produced by many cell types including monocytes/macrophages (33, 34) and dendritic cells (35), but not by T cells (36). IL-15 also promotes the survival and proliferation of memory lymphocytes (36, 37, 38), and induces NK cells to be cytolytic effector cells (39). However, little is known about the mechanisms by which IL-15 induces direct antimicrobial activities of CD8 T cells. Furthermore, it was not known whether CD4 T cells are required or whether accessory cell signals, such as the accessory cell-derived IL-15, might be sufficient for expression of granulysin and antimicrobial activity.
To determine whether granulysin is responsible for the antifungal activity of CD8 T cells, cells were stimulated with IL-15 and the expression of granulysin was detected by immunoblot with an Ab that recognizes both the 15- and 9-kDa forms of granulysin. The level of expression of granulysin was correlated with the anticryptococcal activity of CD8 T cells by assessing the number of viable organisms after incubation with the cytotoxic lymphocytes. To determine whether granulysin is necessary for the anticryptococcal activities of IL-15-activated CD8 T cells, granulysin was depleted with SrCl2, and gene silencing by siRNA was used to block granulysin. To determine whether perforin is required for the antifungal effect of granulysin, CD8 T cells were treated with concanamycin A and EGTA, which inhibit the perforin-mediated cytotoxicity pathway. Finally, the regulation of granulysin during activation by the CnM was examined. By comparing the activation in the presence of CD4 T cells to that in the absence of CD4 T cells, we assessed the contribution of CD4 T cells to the activation of CD8 T cells. Additionally, the requirement for IL-15 was assessed using a neutralizing Ab to IL-15.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C. neoformans CAP 67 (ATCC 52817, acapsular mutant of 3501) was obtained from the American Type Culture Collection (Manassas, VA) and was used in the present study. The organisms were maintained on Sabouraud dextrose slants (Difco, Detroit, MI) and passaged to fresh slants every month as previously described (40).
CnM was prepared as previously described (25, 41).
Briefly, C. neoformans was disrupted by rotating the
organisms with 0.5-mm diameter glass beads in a bead mill (Bead Beater;
Biospec Products, Bartlesville, OK). The cell wall of these disrupted
organisms was digested overnight by using 
-1,3-glucanase (ICN,
Aurora, OH) and solubilized in 50 mM phosphate buffer containing 1%
SDS (BDH, Toronto, Ontario, Canada), 100 µM pepstatin A
(Sigma-Aldrich, St. Louis, MO), and 100 mM EDTA (BDH) for 1
h. SDS was removed by acetone precipitation by centrifugation at 4°C.
The protein concentration was determined by the bicinchoninic acid
protein assay (Pierce, Rockford, IL).
Isolation and stimulation of PBMC
PBMC were isolated as described previously (25). Briefly, peripheral blood was obtained by venipuncture from healthy adults who had no history of cryptococcosis and had not worked with C. neoformans. PBMC were isolated by centrifugation (800 x g, 20 min) over a Ficoll-Hypaque density gradient (C-SIX Diagnostics, Mequon, WI). PBMC were harvested and washed three times in HBSS (Life Technologies, Burlington, Ontario, Canada) and then resuspended in medium containing RPMI 1640 (Life Technologies), 5% human AB serum (BioWhittaker, Walkersville, MD), 100 U of penicillin per ml, 100 µg of streptomycin per ml, 0.25 µg of amphotericin B per ml, 2 mM L-glutamine, 1 mM sodium pyruvate, and 0.1 mM nonessential amino acids (all from Life Technologies).
CD8 T cells were isolated by the MACS negative selection system using the CD8 T cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). The purity of CD8 T cells was >96% as determined by flow cytometry using FITC-labeled Abs to CD8, PerCp-labeled Abs to CD3, and FITC-labeled Abs to CD56 (BD Biosciences, Mountain View, CA). For CD4 T cell depletion experiments, CD4 T cells were depleted from PBMC by using CD4 microbeads (Miltenyi Biotec) (the remaining population was <2% CD4 T cells), followed by stimulation with CnM for 7 days. To isolate CD4 and CD8 cells that were depleted of accessory cells, CD8 T cells and CD4 T cells were purified from unstimulated or CnM-stimulated PBMC by CD8 T cell and CD4 T cell isolation kits, respectively (Miltenyi Biotec).
CD8 T cells were stimulated with IL-15 (50 ng/ml) (R&D Systems, Toronto, Ontario, Canada) for 48 h. In preliminary experiments, granulysin was detected when CD8 T cells were stimulated with 30 ng/ml IL-15. The amount of granulysin increased up to the maximum amount of IL-15 tested (100 ng/ml). However, 30 ng/ml IL-15 did not induce granulysin expression in CD8 T cells in all of the donors; thus, 50 ng/ml was used for all of the experiments. For some experiments, purified stimulated CD8 T cells were treated with 25 mM strontium chloride (Sigma-Aldrich) for 18 h (42, 43), or 10 nM concanamycin A (Sigma-Aldrich) for 2 h, or 4 mM EGTA (Sigma-Aldrich) for 12 h. The cells were washed three times in medium and placed in culture for the experiment. The viability of cells was not altered by these treatments as assessed by trypan blue exclusion.
For some experiments, PBMC were stimulated with PHA (1 µg/ml; Sigma-Aldrich) for 3 days or CnM (5 µg/ml) for 7 days before isolation of CD8 T cells. To block the function of IL-15, anti-IL-15 (1 µg/ml; R&D Systems) or an isotype-matched Ab (1 µg/ml; R&D) was added during the stimulation of CD8 cells.
Immunoblot analysis
Cells were placed in a lysis buffer (50 mM Tris (pH 6.8), 1%
SDS, 0.025% bromophenol blue, 10% glycerol, 20 mM DTT) and were
sonicated. The nuclei were removed by centrifugation at 10,000 x
g for 30 min. Protein from the lysates of 5 x
105 cells were loaded in each lane of an 16%
Tris-glycine gel (Invitrogen, San Diego, CA), separated by
electrophoresis, transferred to a nitrocellulose membrane (Bio-Rad,
Hercules, CA) and blotted with polyclonal anti-granulysin Ab
519/GST rabbit serum (1/1000 dilution) (7). The reactive
bands were visualized using HRP-conjugated anti-rabbit IgG (1/5000
dilution) (Santa Cruz Biotechnology, Santa Cruz, CA) with the ECL plus
Western blotting detection system (Amersham Pharmacia Biotech,
Piscataway, NJ). The membranes were then labeled with a mouse
monoclonal anti- actin Ab (Chemicon International, Temecula, CA)
and bands were detected as described above. For the detection of
perforin, the membranes were blotted with a polyclonal
anti-perforin Ab (Research Diagnostic, Flanders, NJ) (1/1000
dilution), and the bands were visualized as described above.
siRNA preparation and gene silencing assays
To design target-specific siRNA duplexes, we selected a sequence
of the type AA(N19) dTdT (N, any nucleotide) from
the open reading frame of the granulysin mRNA, to obtain a 21-nt sense
and 21-nt antisense strand with symmetric 2-nt 3' overhangs of
identical sequence. The selected siRNA sequence was also submitted to a
search by basic local assignment search tool against the human
genome sequence to ensure that only one gene was targeted. The siRNA
sequence targeting granulysin mRNA (GenBank accession number
NM-012483) was from position 476–498. dsRNA was
synthesized by Dharmacon (Dharmacon Research, Lafayette, CO). CD8 T
cells were transfected by electroporation (250 V, 800 µF) (Bio-Rad)
with granulysin siRNA (1 µg per well) or with CD20 siRNA (target
sequence: 5'-AACCACTCTTCAGGAGGATGT-3') (Dharmacon Research, Lafayette,
CO) or a nonsilencing siRNA (target sequence:
5'-AATTCTCCGAACGTGTCACGT-3') (Xeragon, Huntsvitte, AL), which
served as negative controls. It has been shown that CD20 siRNA could
decrease the constitutive expression of CD20 on Ramso B cell (a B cell
line that constitutively expresses CD20) by 
50% (H. D. Li and
Dr. J. P. Deans, personal communication). The treated cells
were stimulated with IL-15 as described above.
Semiquantitative RT-PCR
Total RNA was extracted from individual cell samples using the RNA extraction kit (Qiagen, Chatsworth, CA). The extracted total RNA was quantified fluorometrically using the SYBR Green II fluorescent RNA dye (Molecular Probes, Eugene, OR) on a LS-5 fluorescence spectrofluorometer (PerkinElmer, Wellesley, MA) with excitation at 468 nm and emission at 525 nm using a standard curve of rRNA (Sigma-Aldrich).
One microgram of total RNA from each sample was reverse transcribed using a Stratagene RT-PCR kit (Stratagene, La Jolla, CA), following the manufacturer’s instructions. The following granulysin-specific primers were used: sense, 5'-CATATGCATATGGGCCGTGACTACAGGACCTGTC-3'; and antisense, 5'-GGATCCGGATCCTTACCTGAGGTCCTCACAGATCTG-3' (44). The following human GAPDH-specific primers were used: sense, 5'-TCACCATCTTCCAGGAGCGA-3'; and antisense, 5'-AGTGATGGCATGGACTGTGG-3'. The PCR profile for granulysin and GAPDH was as follows: denaturation at 94°C for 1 min, followed by 25 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min. A final extension at 72°C for 10 min was used. The number of cycles was adjusted so that amplification occurred over the linear range. The PCR products were separated by electrophoresis on a 2% agarose gel and visualized by ethidium bromide staining. The gels were then photographed using Polaroid Type 55 Land film and analyzed by densitometry (MasterScan Interpretive Densitometer and RFLPscan; Scanalytics/CSPI, Fairfax, VA). Integrated density values were normalized to GAPDH values to yield a semiquantitative assessment of individual transcript levels.
Anticryptococcal activity of CD8 T cells
A CFU assay was performed as previously described (45, 46, 47). C. neoformans (2 x 103/well) were incubated with or without 106 lymphocytes. The number of CFU of C. neoformans per well was determined at 0, 24, or 48 h by lysing the effector cells with 0.1% Triton X-100 followed by diluting and spreading onto Sabouraud dextrose agar plates. Preliminary experiments established that this concentration of Triton X-100 lysed effector cells without affecting fungal viability.
Results are expressed as the percentage of growth according to the following formula: ((CFU experimental/CFU inoculum) - 1) x 100. Thus, a value of zero indicates that the number of CFU at the start and conclusion of the incubation were the same, and fungistasis was obtained. A value of +100 indicates that the number of C. neoformans doubled, while values less than zero indicate killing.
Statistics
Values are expressed as mean ± SE. Each experiment was performed with different donors on different days. Statistical analysis was performed by using the Fisher least-squares difference, when allowed by the F value (Statview; Brain Power, Calabasas, CA). For these tests, a p of <0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine whether IL-15 could enhance the anticryptococcal
activities of CD8 T lymphocytes, purified peripheral blood CD8 T cells
were stimulated with IL-15. The ability of stimulated CD8 T cells to
inhibit the growth of C. neoformans was compared with that
of unstimulated CD8 T cells. When C. neoformans was placed
in culture without CD8 T cells, there was a >400% increase in the
number of organisms after 48 h (Fig. 1
A). When C.
neoformans was cultured with CD8 T cells stimulated with IL-15,
there was a significant anticryptococcal effect. In these cultures, the
number of organisms at 24 and 48 h was significantly lower than
the starting inoculum (Fig. 1A), indicating that the
stimulated CD8 T cells had acquired fungicidal activity and killed
C. neoformans. By contrast, the growth of C.
neoformans in the presence of unstimulated CD8 T cells was not
significantly different from wells containing C. neoformans
alone (Fig. 1A), indicating that unstimulated CD8 T cells
had not acquired anticryptococcal activity. These results indicate that
CD8 T cells acquired anticryptococcal activity after IL-15
stimulation.
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B).
Hence, increased anticryptococcal activity correlated with
up-regulation of granulysin in CD8 T cells. Granulysin was necessary for CD8 T cell-mediated anticryptococcal activity
After demonstrating a correlation with increased activity,
experiments were performed to determine whether depletion of granulysin
correlates with a reduction in antifungal activity in activated CD8 T
cells. The currently available technique uses
SrCl2, which depletes the granule components of
the cells without causing cellular toxicity (48, 49).
Unstimulated and IL-15-stimulated CD8 T cells were treated with
SrCl2. The CD8 T cells expressed granulysin after
stimulation by IL-15, and this expression was abrogated after treatment
with SrCl2 (Fig. 2A). In parallel, the
IL-15-stimulated CD8 T cells that had been
SrCl2-treated were challenged with C.
neoformans. Treatment of IL-15-stimulated CD8 cells with
SrCl2 abrogated the anticryptococcal activity
(Fig. 2B). Thus, depletion of granules containing granulysin
correlated with a marked reduction in anticryptococcal activity.
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A).
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B).
After demonstrating that expression of granulysin was blocked at both
the level of mRNA and protein expression, experiments were performed to
determine the effect of blocking granulysin expression on the
antifungal activity. CD8 T cells were transfected with granulysin siRNA
and control siRNA, stimulated with IL-15, and challenged with live
C. neoformans. The growth of C. neoformans was
inhibited in the presence of IL-15-stimulated CD8 T cells. By contrast,
anticryptococcal activity was abrogated by transfection of granulysin
siRNA in IL-15-stimulated CD8 T cells (Fig. 3C). This
suggests that granulysin is necessary for CD8 T cell-mediated
anticryptococcal activity.
Granulysin did not require perforin to function
It has been demonstrated that perforin is required for the
antimycobacterial activity of granulysin (7). For this
reason, experiments were performed to determine whether perforin was
required for the anticryptococcal activity of granulysin. CD8 T cells
were stimulated with IL-15 in the presence or absence of concanamycin
A, an inhibitor of vacuolar ATPase that is required to maintain
perforin in lytic granules (50). Immunoblot analysis
revealed that perforin was decreased by concanamycin A treatment but
the granulysin level was not affected in IL-15-stimulated CD8 T cells
(Fig. 4A). We could not detect
perforin expression in peripheral blood unstimulated CD8 T cells by
Western blot. Our results are consistent with previous studies in which
perforin is not detected in unstimulated peripheral blood CD8 T cells
(51, 52). Correspondingly, experiments were performed to
determine the effect of concanamycin A treatment on the
anticryptococcal activities of CD8 T cells. IL-15-stimulated CD8 T
cells treated with concanamycin A showed similar levels of antifungal
activity to those of concanamycin A-untreated IL-15-stimulated CD8 T
cells (Fig. 4B). Similar results were obtained with EGTA,
which chelates calcium and blocks perforin polymerization (Fig. 4C).
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Our previous work showed that CnM causes activation and
proliferation of peripheral blood T lymphocytes (25). This
provided an opportunity to study the events involved in the activation
of CD8 T cells that was more similar to the activation during an
infection than the activation provided by recombinant IL-15. To
determine whether CD8 T cells could be activated by CnM to express
granulysin and anticryptococcal activity, PBMC were stimulated and the
CD8 T cells were isolated. Immunoblot analysis showed that a small
amount of the inactive 15-kDa form of granulysin was detected in CD8 T
cells isolated from unstimulated PBMC but failed to detect the 9-kDa
active form (Fig. 5A). CD8 T
cells isolated from PHA- or CnM-stimulated PBMC expressed a greater
amount of the 15-kDa form, and the 9-kDa form of granulysin was easily
detected (Fig. 5A). Correspondingly, CD8 T cells isolated
from CnM-stimulated PBMC acquired anticryptococcal activity, while CD8
T cells isolated from unstimulated PBMC did not (Fig. 5B). A
similar effect was observed when other encapsulated strains of C.
neoformans were used as targets (data not shown).
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Although CD4 cells are critically important for immune responses
and host defense to C. neoformans (53), both
CD4 and CD8 T lymphocytes are involved in the generation of protective
immune responses to C. neoformans (54, 55, 56, 57).
Furthermore, CD8 cells can be activated independently of CD4 T cells in
response to C. neoformans (55, 56, 57, 58). Therefore,
experiments were performed to determine whether CD4 T cells were
required for the activation of CD8 cells that resulted in granulysin
expression and anticryptococcal activity. CD8 T cells were isolated
from CnM-stimulated PBMC that had been depleted of CD4 T cells. These
cells were compared with CD8 T cells isolated from CnM-stimulated PBMC
that had not been depleted of CD4 T cells. Immunoblot analysis
confirmed the previous observation that CD8 T cells stimulated in the
presence of CD4 cells expressed both the 15- and 9-kDa forms of
granulysin (Fig. 6A). However,
CD8 T cells that had been isolated from CD4 T cell-depleted,
CnM-stimulated PBMC expressed only a trace amount of the 15-kDa form of
granulysin and undetectable levels of the 9-kDa granulysin (Fig. 6A), which was similar to the expression of unstimulated CD8
T cells. Thus, CD4 T cells were required for the up-regulation of
granulysin in CD8 T cells.
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B). By contrast, when C.
neoformans was cultured with CD8 T cells isolated from CD4 T
cell-depleted PBMC, the CD8 T cells had not acquired antifungal
activity (Fig. 6B), indicating that CD4 T cells were
necessary. IL-15 was required for granulysin-mediated anticryptococcal activity
Having observed that IL-15 was sufficient and CD4 T cells were
required to induce granulysin expression and antifungal activity (Figs. 1 and 6), it was necessary to determine whether IL-15 was required for
the CD4-dependent expression of granulysin and anticryptococcal
activity. CD8 T cells isolated from PBMC stimulated with CnM in the
presence of a neutralizing Ab to IL-15 were compared with CD8 T cells
isolated from CnM-stimulated PBMC in the presence of an isotype-matched
Ab. The results of the immunoblot showed that CD8 T cells isolated from
CnM-stimulated PBMC expressed both forms of granulysin while
anti-IL-15 Ab treatment abrogated the expression of granulysin
(Fig. 7A). The
anticryptococcal activity of CD8 T cells acquired after CnM stimulation
was also abrogated when an anti-IL-15 Ab was added to the culture
(Fig. 7B). Thus, IL-15 was necessary for the expression of
granulysin and antifungal effect of CD8 T cells.
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A). A
similar effect was observed when CD40-CD40 ligand interactions
were blocked with an anti-CD40 Ab during stimulation (data not
shown). Correspondingly, purified CD4 plus CD8 T cells that had been
depleted of accessory cells failed to display anticryptococcal
activity. These data suggest that accessory cells are required for the
expression of granulysin by CD8 T cells and anticryptococcal
activity. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies have attempted to define the mechanism of the lymphocyte-mediated anticryptococcal activity without success. It had been observed that CD8 T cells and NK cells form conjugates with C. neoformans, and predicted that the organisms were killed by granule exocytosis (23). Hydroxyl radical scavengers (diethyl urea, propyl gallate), cyclooxygenase inhibitors (piroxicam, indomethacin), PGE2, and mAbs or ligands reactive with receptors on human lymphocytes failed to demonstrate a correlation with the anticryptococcal activity of CD8 T cells (24). Although catechin (a hydroxyl radical scavenger) and salicylic acid (a cyclooxygenase inhibitor) inhibited the antifungal activity, the authors (Levitz et al. (24)) did not make a general conclusion because other related agents did not have similar effects.
It has been shown that recombinant granulysin showed a dose-dependent cytotoxicity against a variety of organisms including Escherichia coli, Staphylococcus aureus, Mycobacterium tuberculosis, C. neoformans, and Leishmania major in vitro (6, 7). Increased expression of granulysin by cytotoxic lymphocytes correlated with cytotoxicity to M. tuberculosis and Mycobacterium leprae (8, 59, 60), and strontium ions abrogated the activity (59, 61). To confirm this correlation using C. neoformans, the expression of granulysin was established and SrCl2 was used in the current studies. SrCl2 depletes all of the granule components including granulysin without causing cellular toxicity (48, 49). However, because of the inability to specifically inhibit granulysin, definitive evidence of granulysin-mediated cytotoxicity has heretofore been lacking. When granulysin expression was blocked by granulysin-specific siRNA at the level of mRNA and protein expression, the anticryptococcal activities of CD8 T cells were abrogated. The current study shows that granulysin is necessary for the anticryptococcal activity of CD8 T cells. Further studies will be required to establish whether granulysin is required for antimycobacterial and other antimicrobial activities.
The current studies provide strong support for the utility of siRNA. The discovery that dsRNA could induce gene silencing in organisms as diverse as plants and parasitic protozoans raises the possibility that RNAi might be a nearly universal mechanism of gene silencing. This notion has been supported by the identification of homologs of proteins that participate in the silencing process in virtually all genomes examined to date, with the exception of Saccharomyces cerevisiae (62). The first indications that this response might also extend to mammals came from the observation that injection of dsRNA into early mouse embryos induced sequence-specific silencing (63, 64). Recently, RNAi mediated by 21- and 22-nt RNA has been observed in numerous mammalian cell lines, such as 293, NIH/3T3, Hela S3, Hela SS6, COS-7 cells (19), and rat fibroblast F5 and FR (wt648) (65). As an extension of previous studies, we have demonstrated that siRNA can induce potent and specific gene silencing in human primary cells. In the present study, 21-nt siRNA duplexes were used to block granulysin expression in human primary CD8 T cells. Granulysin mRNA expression was suppressed in the presence of siRNA. Granulysin protein expression was also dramatically reduced by siRNA. By contrast, granulysin mRNA and protein expression were not affected by two control siRNA, CD20 siRNA, and nonsilencing siRNA, suggesting that the effect was not due to a nonspecific effect of siRNA. Thus, siRNA may hold promise in human primary cells and provides optimism for studies of dsRNA-mediated silencing in mammalian systems.
Previous studies have shown that granulysin-mediated killing of intracellular M. tuberculosis was dependent on perforin (7). After establishing that granulysin was required for CD8 T cell-mediated anticryptococcal activity, experiments were performed to determine whether the granulysin-mediated antifungal effect is perforin dependent. CD8 T cells were treated with EGTA, a chelator of calcium, or concanamycin A, which has been shown to selectively block the perforin and granzyme cytotoxicity pathway (50, 66). The anticryptococcal activity of CD8 T cells was not affected. This observation suggests that the granulysin-mediated antimicrobial activity of CD8 T cells is perforin independent. A critical difference between the perforin-independent killing of Cryptococcus and the perforin-dependent killing of Mycobacteria (7) is that the cryptococci were extracellular while mycobacteria were intracellular. This is supported by the observation that perforin acts as a gateway for granzymes through the plasma membrane. Both of these events might require intimate contact of the T lymphocyte and the target, and previous studies have shown that intimate contact between the T lymphocytes and C. neoformans is required for the anticryptococcal activities (23, 46, 67). Thus, it may be that perforin is required for granulysin to access the compartment containing mycobacteria, but granulysin is directly active on extracellular C. neoformans at the conjugation region.
It has been established that CD4 T cells are required for CD8 T cell
proliferation in response to C. neoformans
(58). Likewise, CD4 T cells are required for the CD8 T
cells to produce granulysin and to become cytotoxic cells with
anticryptococcal activity. CD4 cells could participate in a number of
different ways. CD4 T cells secrete T cell growth factors such as IL-2,
which activate CD8 T cells (28, 32, 68). Additionally, CD4
T cells signal accessory cells, such as dendritic cells and
monocytes/macrophages, to express important cytokines such as IL-1,
IL-6, IL-15, or TNF-
(69, 70, 71), or surface-expressed
ligands, such as CD40, LFA-1, or ICAM-1 (72, 73), so that
CD8 T cells can be activated (29). The studies of IL-15
shed some light on these two possibilities.
We considered two possibilities that might explain how both CD4 T cells and IL-15 might be required. IL-15 is required for lymphocyte-mediated anticryptococcal activity, and IL-15 is produced by cells of the monocyte and macrophage lineage (30). The first possibility was that IL-15 would be necessary for the response of CD4 T cells. This would be consistent with the requirement for IL-15 in the response to CnM. The CD4 cells would then provide T cell help for the CD8 cytotoxic T cells. In this circumstance, we would anticipate that IL-15 would be required to stimulate CD4 T cells, and CD4 T cells would then be required to stimulate CD8 T cells. However, we observed that CD4 T cells did not need to be present when IL-15 was used to stimulate CD8 cells. Indeed, IL-15 was sufficient to stimulate CD8 T cells. Thus, the data is most consistent with a second model in which CD4 T cells are activated, but because CD4 T cells do not release IL-15 (36), they stimulate accessory cells of the myeloid lineage to produce IL-15. The IL-15 in turn stimulates CD8 T cells to express granulysin and become cytotoxic.
In summary, we have examined granulysin function and regulation in primary CD8 T cells. Using siRNA-mediated RNAi, we found that granulysin was necessary for CD8 T cell-mediated antimicrobial activity. The granulysin-mediated antifungal effect was independent of perforin. In response to CnM, CD4 T cells were required to activate accessory cells that secrete IL-15, which up-regulated granulysin expression and enhanced the antifungal effect in CD8 T cells.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Christopher H. Mody, Room 273, Heritage Medical Research Building, University of Calgary, Calgary, Alberta, Canada T2N 4N1. E-mail address: cmody{at}ucalgary.ca
3 Abbreviations used in this paper: RNAi, RNA interference; siRNA, small interfering RNA; CnM, Cryptococcus neoformans mitogen.
Received for publication June 20, 2002. Accepted for publication September 10, 2002.
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 A. Nakashima, A. Shiozaki, S. Myojo, M. Ito, M. Tatematsu, M. Sakai, Y. Takamori, K. Ogawa, K. Nagata, and S. Saito Granulysin Produced by Uterine Natural Killer Cells Induces Apoptosis of Extravillous Trophoblasts in Spontaneous Abortion Am. J. Pathol., September 1, 2008; 173(3): 653 - 664. [Abstract] [Full Text] [PDF]
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 A. P. G. da Silva, D. Unks, S.-c. Lyu, J. Ma, R. Zbozien-Pacamaj, X. Chen, A. M. Krensky, and C. Clayberger In vitro and in vivo antimicrobial activity of granulysin-derived peptides against Vibrio cholerae J. Antimicrob. Chemother., May 1, 2008; 61(5): 1103 - 1109. [Abstract] [Full Text] [PDF]
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U. Muller, W. Stenzel, G. Kohler, C. Werner, T. Polte, G. Hansen, N. Schutze, R. K. Straubinger, M. Blessing, A. N. J. McKenzie, et al. IL-13 Induces Disease-Promoting Type 2 Cytokines, Alternatively Activated Macrophages and Allergic Inflammation during Pulmonary Infection of Mice with Cryptococcus neoformans J. Immunol., October 15, 2007; 179(8): 5367 - 5377. [Abstract] [Full Text] [PDF]
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J. C. D. Wiseman, L. L. Ma, K. J. Marr, G. J. Jones, and C. H. Mody Perforin-Dependent Cryptococcal Microbicidal Activity in NK Cells Requires PI3K-Dependent ERK1/2 Signaling J. Immunol., May 15, 2007; 178(10): 6456 - 6464. [Abstract] [Full Text] [PDF]
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 C. F. Zheng, L. L. Ma, G. J. Jones, M. J. Gill, A. M. Krensky, P. Kubes, and C. H. Mody Cytotoxic CD4+ T cells use granulysin to kill Cryptococcus neoformans, and activation of this pathway is defective in HIV patients Blood, March 1, 2007; 109(5): 2049 - 2057. [Abstract] [Full Text] [PDF]
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L. P. Huang, S.-C. Lyu, C. Clayberger, and A. M. Krensky Granulysin-Mediated Tumor Rejection in Transgenic Mice J. Immunol., January 1, 2007; 178(1): 77 - 84. [Abstract] [Full Text] [PDF]
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 B. Liu, S. Liu, X. Qu, and J. Liu Construction of a eukaryotic expression system for granulysin and its protective effect in mice infected with Mycobacterium tuberculosis. J. Med. Microbiol., October 1, 2006; 55(Pt 10): 1389 - 1393. [Abstract] [Full Text] [PDF]
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 A. A. Siddiqui, R. J. Shattock, and T. S. Harrison Role of Capsule and Interleukin-6 in Long-Term Immune Control of Cryptococcus neoformans Infection by Specifically Activated Human Peripheral Blood Mononuclear Cells Infect. Immun., September 1, 2006; 74(9): 5302 - 5310. [Abstract] [Full Text] [PDF]
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Q. Zhou, R. A. Gault, T. R. Kozel, and W. J. Murphy Immunomodulation with CD40 Stimulation and Interleukin-2 Protects Mice from Disseminated Cryptococcosis Infect. Immun., April 1, 2006; 74(4): 2161 - 2168. [Abstract] [Full Text] [PDF]
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F. McAllister, C. Steele, M. Zheng, J. E. Shellito, and J. K. Kolls In Vitro Effector Activity of Pneumocystis murina-Specific T-Cytotoxic-1 CD8+ T Cells: Role of Granulocyte-Macrophage Colony-Stimulating Factor Infect. Immun., November 1, 2005; 73(11): 7450 - 7457. [Abstract] [Full Text] [PDF]
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L. L. Ma, C. L. C. Wang, G. G. Neely, S. Epelman, A. M. Krensky, and C. H. Mody NK Cells Use Perforin Rather than Granulysin for Anticryptococcal Activity J. Immunol., September 1, 2004; 173(5): 3357 - 3365. [Abstract] [Full Text] [PDF]
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J. J. Endsley, J. L. Furrer, M. A. Endsley, M. A. McIntosh, A. C. Maue, W. R. Waters, D. R. Lee, and D. M. Estes Characterization of Bovine Homologues of Granulysin and NK-lysin J. Immunol., August 15, 2004; 173(4): 2607 - 2614. [Abstract] [Full Text] [PDF]
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G. G. Neely, S. Epelman, L. L. Ma, P. Colarusso, C. J. Howlett, E. K. Amankwah, A. C. McIntyre, S. M. Robbins, and C. H. Mody Monocyte Surface-Bound IL-15 Can Function as an Activating Receptor and Participate in Reverse Signaling J. Immunol., April 1, 2004; 172(7): 4225 - 4234. [Abstract] [Full Text] [PDF]
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