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*
Laboratory of Host Defense, Research Institute for Disease Mechanism and Control, Nagoya University School of Medicine, Nagoya, Japan; and
Department of Bacteriology, National Institute of Infectious Diseases, Tokyo, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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level in serum was
significantly higher in IL-15-Tg mice than in non-Tg mice after BCG
infection. NK cells were remarkably increased, and Ag-specific T
cytotoxic 1 response mediated by CD8+ T cells
producing IFN- was significantly augmented in the IL-15-Tg mice
following BCG infection. Neutralization of endogenous IFN- by in
vivo administration of anti-IFN- mAb deteriorated the
clearance of the bacteria. Depletion of of NK cells or CD8+
T cells by invivo administration of
anti-asialo-GM1 Ab or anti-CD8 mAb hampered the
exclusion of bacteria. Thus, overexpression of IL-15 in vivo enhanced
protection against BCG infection via augmentation of NK and T cytotoxic
1 responses. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, as evidenced by severely disseminated tuberculosis in IFN-
knockout mice (1, 2). Although IFN- is produced by
mainly CD4+ Th1 cells (3),
Tc13
response mediated by CD8+ T cells producing
IFN- was evident in mycobacterial infection (4, 5, 6).
2-microglobulin or TAP knockout mice, both of
which lack functional CD8+ T cells, are
susceptible to infection with M. tuberculosis (7, 8). Recent studies suggest that CD8 CTL releasing perforin and
granulysin play a role in protection against M. tuberculosis
infection via cytolysis mechanism (9, 10). However, the
resistance to M. tuberculosis infection in both perforin-
and fas-deficient mice was unaltered, suggesting that the
cytotoxic function of CD8+ T cells may not be
critical in protection to tuberculosis (11, 12). On the
other hand, the critical function of CD8+ T cells
is mediated by IFN- (6). Taken together, it thus
appears that Tc1 response mediated by CD8+ T
cells producing IFN- plays a requisite role in resistance to
mycobacterial infection. M. bovis bacillus Calmette-Guérin (BCG) is the vaccine approved for prevention of M. tuberculosis (13). Although M. tuberculosis DNA vaccines can induce substantial protective immune responses, the current vaccine preparations are not as protective as BCG in a mouse model of human pulmonary M. tuberculosis (13, 14). Protection against pulmonary tuberculosis requires a sustained cellular immune response, while it is not clear that BCG is effective for inducing long-term cellular immunity sufficient for protection against pulmonary disease and it confers incomplete protection against tuberculosis, at least in adults (15). Therefore, it is required to develop immune adjuvants to increase the effectiveness of BCG vaccination.
IL-15 uses - and -chains of the IL-2R for signal transduction,
and thus shares many properties of IL-2, despite having no sequence
homology with IL-2 (16, 17, 18). IL-15 is reported to
stimulate NK cells and TCR
intestinal intraepithelial lymphocytes
(i-IEL) to produce IFN- and exhibit increased cytotoxicity
(19, 20, 21). In addition to these cells, memory phenotype
CD8+ T cells are reported to expand in response
to exogenous IL-15 or various infectious agents that can elicit IL-15
via IFN regulatory factor-1/NF-
B activation (22).
Recently, IL-15R
-/- mice and
IL-15-/- mice are reported to be deficient in
NK cells, NKT cells, and TCR i-IEL and memory phenotype
CD8+ T cells (23, 24). It thus
appears that IL-15 has potential roles in the development and
maintenance of significant fractions of lymphocytes, including NK, NKT,
TCR i-IEL, and memory phenotype CD8+ T
cells. There have been several lines of evidence for IL-15 production
in infection with various microbes, including M.
tuberculosis (25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35). We have demonstrated that
IL-15 is involved in protection against infections with
Escherichia coli (35) or avirulent
Salmonella choleraesuis (36) via activation of
T cell or NK cells. Recently, we have constructed transgenic
(Tg) mice expressing IL-15 cDNA encoding a secretable isoform and found
that the IL-15-Tg mice contained a large number of memory phenotype
CD8+ T cells expressing
CD44highCD62L+Ly-6C+
in the peripheral lymphoid tissues (37). IL-15-Tg mice may
be useful to elucidate the roles of IL-15-dependent memory phenotype
CD8+ T cells in host defense mechanism against
infection with various pathogens including mycobacteria.
With the aim to study the immunomodulatory effects of IL-15 in vivo, we
examined cell-mediated immunity against BCG in IL-15-Tg mice. We found
that purified protein derivative (PPD)-specific
CD8+ Tc1 responses producing IFN- were
significantly augmented in IL-15-Tg mice coincident with a marked
increase of NK cells. Neutralization of endogenous IFN- and
depletion of NK or CD8+ T cells by in vivo
administration of Abs deteriorated the clearance of the bacteria.
Overexpression of IL-15 in vivo enhanced protection against BCG
infection via augmentation of NK and Tc1 responses.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IL-15-Tg mice, which were constructed using originally described
IL-15 cDNA under the control of an MHC class I promoter, have been
described previously (37). In brief, full-length cDNA
encoding the murine IL-15 gene with the originally described exon 5
(710 bp) was cloned between the BamHI and SalI
sites of a Tg expression vector, pHSE-3', which contains the H2-K
promoter and Ig enhancer, and -globin splice site and poly(A)
signal. Transgene DNAs were microinjected into the male pronucleus of
fertilized single cell embryos of C57BL/6 mice. Microinjected eggs were
transferred to pseudopregnant C57BL/6 foster mothers. IL-15-Tg mice
were identified by digesting genomic DNA with PstI, followed
by Southern blot analysis using an IL-15-specific probe. In each
experiment, age- and sex-matched C57BL/6 mice, purchased from Charles
River Japan (Hino, Japan), were used as a control. Mice were maintained
under specific pathogen-free conditions and offered food and water ad
libitum. All mice were used at 6–8 wk of age.
Microorganisms
Lyophilized M. bovis BCG were purchased from Kyowa Pharmaceuticals (Tokyo, Japan). BCG were dissolved in 7H9 medium (Difco, Detroit, MI) supplemented with albumin-dextrose-catalase enrichment (Difco). The viable bacterial numbers were determined by 7H10 (Difco) plate supplemented with oleic acid-albumin-dextrose-catalase enrichment (Difco). Small aliquots of BCG suspended in 7H9 medium containing 10% glycerol were stored in -80°C until use. The concentration of bacteria was quantified by plate counting. Before use, the bacteria were washed three times with PBS, and resuspended in PBS. Mice were inoculated i.p. with 2–10 x 106 CFU of BCG in a volume of 100 µl PBS.
Abs and reagents
FITC-conjugated anti-CD3
mAb (145-2C11), PE-conjugated
anti-TCR mAb (H57-597), PE-conjugated anti-NK1.1 mAb
(PK136), PE-conjugated anti-CD8 mAb (53-6.7), biotin-conjugated
anti-TCR mAb (GL3), CyChrome-conjugated anti-CD4 mAb
(GK1.5), FITC-conjugated anti-IFN- mAb (XMG1.2), FITC-conjugated
anti-IL-4 mAb (BVD4-1D11), FITC-conjugated rat IgG1 isotype control
Ig, and FITC-conjugated rat IgG2b isotype control Ig were purchased
from PharMingen (San Diego, CA). Staining with biotin-conjugated mAb
was followed by treatment with streptavidin-Cy-Chrome (PharMingen). The
2.4G2 (anti-FcRII/III-specific mAb, rat IgG1, producing
hybridoma) was obtained from American Type Culture Collection
(Manassas, VA). Anti-murine IL-15 sera were produced in Japan white
rabbit (Japan SLC, Hamamatsu, Japan) by immunization with an emulsion
containing approximately 0.5 mg E. coli-expressed murine
IL-15 protein in the form of an insoluble inclusion body preparation in
monophosphoryl lipid A, synthetic trehalose dicorynomycolate, and cell
wall skeleton emulsion (Corixa, Hamilton, MT). A total of three booster
injections was given each at 3-wk intervals after primary injection.
One week after the last immunization, blood was collected from
the heart. ELISA for IL-15 in individual sera was performed in
triplicate using purified anti-mouse IL-15 mAb (G277-3588;
Phamingen), anti-mouse IL-15 rabbit antiserum, and anti-rabbit
Ig, HRP-linked F(ab')2 (Amersham Pharmacia
Biotech, Uppsala, Sweden), or biotin-conjugated anti-mouse IL-15
mAb (G277-3960; BD PharMingen) and peroxidase-conjugated streptavidin
(Genzyme Diagnostics, Cambridge, MA). Murine rIL-15 was obtained from
Research Diagnostics (Flanders, NJ).
In vivo administration of Abs
Rat anti-mouse IFN- mAb (XMG1.2) and anti-CD8 mAb
(2.43) were purified from the supernatants of growing hybridoma cells
in serum-free medium (Nissui Pharmaceutical, Tokyo, Japan). Polyclonal
rabbit anti-asialo-GM1 Ab were purchased from
Wako Pure Chemical Industries (Osaka, Japan). To neutralize endogenous
IFN-, mice were treated with anti-IFN- mAb or control rat IgG
by an s.c. implanted ALZET osmotic pump (Model 2004; ALZA, Palo Alto,
CA) on day 0 after infection with BCG. For depletion of NK cells or
CD8+ T cells,
anti-asialo-GM1 Ab (200 µg), anti-CD8
mAb (200 µg), anti-rabbit IgG, or anti-rat IgG1 was
administrated 2 days before, and 5 and 12 days after infection. These
treatments resulted in a >95% depletion of NK cells or
CD8+ T cells, respectively, from spleens at 2 wk
after infection, as assessed by a FACSCalibur flow cytometer (BD
Biosciences, San Jose, CA).
Bacterial counts
Bacterial counts in the peritoneal cavity and liver, spleen, and lung on days 7, 14, and 28 after an i.p. infection with BCG were determined as described (38, 39). Briefly, peritoneal exudates were obtained from the peritoneal cavity by lavage with 4 ml HBSS (Nissui) and were serially diluted with HBSS. Serial dilutions of the exudate samples were plated on Middlebrook 7H10 medium supplemented with oleic acid-albumin-dextrose-catalaseenrichment and 20% (v/v) glycerol. For enumeration of viable counts in the liver, spleen, and lung, the organs were perfused with 20 ml sterile HBSS to wash out bacteria in the blood vessels immediately after mice were bled. Bacterial counts in the organs were measured as described above. The numbers of colonies were determined after incubation for 3 wk.
Cell preparation
Peritoneal exudate cells (PEC) were obtained by lavage of the peritoneal cavity with HBSS on days 7, 14, and 28 after inoculation. PEC were prepared by centrifugation and resuspended in RPMI 1640 containing 10% FBS, 100 U/ml of penicillin, 100 µg/ml streptomycin, and 10 mM HEPES. Cells were plated and allowed to adhere for 2 h at 37°C in a humidified atmosphere of 95% air and 5% CO2. Nonadherent cells were used as mononuclear cells, and adherent cells were washed several times with HBSS. Adherent cells were collected by scraping with a rubber policeman, washed, and counted. More than 95% of the cells retained by this procedure were macrophages.
In vitro culture
Plastic nonadherent cells of PEC were subjected to an Ag
stimulation assay for cytokine production. Nylon wool-passed plastic
nonadherent cells of PEC from BCG-infected non-Tg or IL-15-Tg mice on
days 0, 7, 14, and 28 were resuspended in RPMI 1640 and added to
96-well plates at a concentration of 2 x
105 cells/well. Cells were cultured without any
stimulation, or with 5 µg/ml PPD (Japan BCG Association, Tokyo,
Japan), or with 100 µg/ml anti-TCR mAb in the presence of
mitomycin-treated splenocytes (2 x 105)
from naive mice for 48 h at 37°C. Supernatants were collected
and stored at -80°C until the cytokine assay. In some experiments,
cells were pulsed with [3H]TdR 6 h before
harvesting; then [3H]TdR incorporation was
determined by liquid scintillation counting.
Cytokine ELISA
IFN- and IL-4 levels in the serum were determined by ELISA
(Genzyme Diagnostics). ELISA for IL-15 in individual sera was performed
in triplicate using purified anti-mouse IL-15 mAb (G277-3588;
PharMingen), anti-mouse IL-15 rabbit antiserum, and anti-rabbit
Ig, HRP-linked F(ab')2 or biotin-conjugated
anti-mouse IL-15 mAb (G277-3960; PharMingen) and
peroxidase-conjugated streptavidin. Murine rIL-15 was used as positive
control.
Expression of the inducible isoform of NO synthase (iNOS) and IL-15 genes
Adherent PEC of IL-15-Tg or non-Tg mice on day 0 or 14 after BCG
infection were applied. Total RNA was extracted from adherent PEC at
specific times, basically according to the method of Chomczynski and
Sacchi (40). First strand cDNA was synthesized from 2 µg
RNA using reverse transcriptase and 20 pmol random primer in 20 µl
reaction buffer. Synthesized first strand cDNA were amplified by PCR
using 20 pmol of each primer with 2.5 U AmpliTaq Kit (Takara
Shuzo, Kyoto, Japan) in a total volume of 50 µl reaction buffer
consisting of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM
MgCl2, 0.01% gelatin, and 0.2 mM dNTP. PCR
cycles were run for 1 min at 94°C, 1 min at 54°C, and 30 s at
72°C. Before the first cycle, a denaturation step for 5 min at 94°C
was included, and after 35 cycles, the extension was prolonged for 2
min at 72°C. The specific primers were as follows: iNOS sense, 5'-AGC
TCC TCC CAG GAC CAC AC-3', and antisense, 5'-ACG CTG AGT ACC TCA TTG
GC-3'; IL-15 exon 7–8 sense, 5'-GTG ATG TTC ACC CCA GTT GC-3', and
antisense, 5'-TCA CAT TCT TTG CAT CCA GA-3'; and -actin sense,
5'-TGG AAT CCT GTG GCA TCC ATG AAA C- 3', and antisense, 5'-TAA AAC GCA
GCT CAG TAA CAG TCC G-3'. The PCR product was subjected to
electrophoresis on a 1% agarose gel and then was transferred to
GeneScreenPlus filter (Dupont NEN, Boston,
MA). The internal oligonucleotide probes were labeled with
[-32P]ATP using Megalabel 5'-labeling kit
(Takara Shuzo), according to the manufacturer’s instructions. The
internal oligonucleotide probes were as follows: iNOS, 5'-AAG CCA GCT
CAA GCC CGG AGA CCC TGT GCC CTG CTT CAT C-3'; IL-15 exon 7–8, 5'-GCA
ATG AAC TGC TTT CTC CT-3'; and -actin, 5'-TTC TGC ATC CTG TCA GCA
AT-3'. Prehybridization was performed by incubating the membrane in 1 M
NaCl, 1% SDS, and 10% dextran sulfate for 1 h. In hybridization,
the filters were incubated in 1 M NaCl, 1% SDS, 10% dextran sulfate,
and 100 mg/ml heat-denatured salmon sperm DNA with labeled probes for
18 h at 60°C, and then the filters were washed in 2x SSC, 1%
SDS for 15 min at 60°C. The radioactivity of each band of PCR product
was analyzed with the Fujix BAS2000 Bio-image analyzer (Fuji Photo
Film, Tokyo, Japan).
Flow cytometric (FCM) analysis
Plastic nonadherent cells of PEC were preincubated with a
culture supernatant from 2.4G2 to prevent nonspecific staining. After
washing, cells were stained with various combinations of mAbs. Staining
with biotin-conjugated mAb was followed by treatment with
streptavidin-Cy-Chrome. For three-color analysis of T cell subsets,
single-cell suspensions were stained with FITC-conjugated CD3,
PE-conjugated TCR, NK1.1 mAbs, or biotin-conjugated TCR,
and analyzed with a FACSCalibur flow cytometer (BD Biosciences). The
live lymphocytes were carefully gated by forward and side scattering.
The data were analyzed with CellQuest software (BD Biosciences).
Intracellular cytokine staining
Splenocytes (2 x 106 cells/ml)
were incubated without any stimulation, or with 25 ng/ml PMA and 1
µg/ml ionomycin, or with 5 µg/ml PPD (Japan BCG Association) and
100 pg/ml rIL-2 (Takeda Chemical, Tokyo, Japan) for 6 h at 37°C
and 5% CO2, with 10 µg/ml brefeldin A (Sigma)
added for the last 2 h in 24-well flat-bottom plates (Falcon; BD
Biosciences) in a volume of 1 ml RPMI containing 10% FCS. After 6
h of culture, the cells were harvested, washed once in HBSS containing
2.5% newborn horse serum and 0.1% NaN3
(staining buffer), and surface stained in staining buffer with
Cy-Chrome-conjugated anti-CD4 mAb and PE-conjugated anti-CD8
mAb. After surface staining, cells were subjected to intracellular
cytokine staining using the Fast Immune Cytokine System (BD
Biosciences), according to the manufacturer’s instructions. For
intracellular cytokine staining, we used FITC anti-IFN-, IL-4
mAbs, or FITC-conjugated rat IgG1 or IgG2b as isotype control. Samples
were acquired in a FACSCalibur flow cytometer and analyzed by CellQuest
software.
Statistical analysis
The statistical significance of the data was determined by Student’s t test A p value of <0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To elucidate the ability of the IL-15-Tg mice to limit the
bacterial growth against BCG infection, we examined the kinetics of
bacterial growth in the peritoneal cavity, the liver, the lung, and the
spleen of IL-15-Tg mice following an i.p. infection with 4.8 x
106 CFU of BCG. As shown in Fig. 1
, the bacterial number in each organ was decreased with time both in
non-Tg and IL-15-Tg mice. However, the bacterial numbers in the
peritoneal cavity on day 28 and in the liver or the lung on days 14 and
28 after BCG infection were significantly lower in IL-15-Tg mice than
in non-Tg mice (*, p < 0.05, **,
p < 0.005). Similar tendency in bacterial growth was
observed in the spleen (data not shown). Thus, IL-15-Tg mice were
resistant against BCG infection, as assessed by bacterial growth.
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To compare IL-15 synthesis between IL-15-Tg and non-Tg mice
following BCG infection, we next examined IL-15 expression at mRNA and
protein levels in IL-15-Tg and non-Tg mice infected with BCG (Fig. 2, A and C). Consistent with previous report
(37), IL-15 mRNA expression in the adherent PEC and IL-15
production in the serum were detected in naive IL-15-Tg mice before
infection. IL-15 expression was detected in non-Tg mice after BCG
infection, and the levels were much higher in IL-15-Tg mice than those
in non-Tg mice on days 14 and 28 after BCG infection (Fig. 2, A and C, p < 0.05).
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and IL-4 after BCG
infection. As shown in Fig. 2
B, the IFN- levels in serum
of IL-15-Tg mice were significantly higher on days 14 and 28 than those
in non-Tg mice. Serum IL-4 was not detected in either IL-15-Tg or
non-Tg mice at any stage of BCG infection.
High output NO production induced by iNOS is a major antimicrobial
mechanism, and iNOS mRNA induction is known to be regulated by IFN-
and TNF- (41, 42, 43). We compared iNOS mRNA in macrophages
from IL-15-Tg or non-Tg mice on day 14 after BCG infection. As shown in
Fig. 2C, the macrophages in the peritoneal cavity of
BCG-infected IL-15-Tg mice expressed a higher level of the iNOS mRNA as
compared with those of BCG-infected non-Tg mice.
Kinetics of peritoneal exudate lymphocytes in IL-15-Tg mice following infection with BCG
FCM analysis for expression of CD3, TCR, TCR,
NK1.1, CD4, and CD8 was conducted on nonadherent PEC on days 7, 14, and
28 after infection. A typical result was shown in Fig. 3
and the data were summarized in Table I. The proportions of CD3-
NK1.1+ cells in the peritoneal cavity of non-Tg
mice and IL-15-Tg mice before infection were 3.02 ± 0.03% and
3.14 ± 0.09%, respectively. The proportions of
CD3- NK1.1+ cells
increased and reached to 
9.2% in non-Tg mice on day 7 after BCG
infection and then rapidly decreased to 4.5% by day 28 after BCG
infection. Similar to non-Tg mice, CD3-
NK1.1+ cells were increased in IL-15-Tg mice on
day 7 after BCG infection, whereas, in contrast to non-Tg mice, the
proportions of CD3- NK1.1+
cells remained at a higher level on day 28 after BCG infection.
Similarly, NK1.1+ T cells were increased more in
IL-15-Tg mice than those in non-Tg mice on days 7 and 14 after BCG
infection, and the number of NK1.1+ T cells
remained at an increased level on day 28 in IL-15-Tg mice. The number
of CD3+ TCR+ T cells
in non-Tg mice and IL-15-Tg mice was significantly increased in
peritoneal cavity on days 7 and 14, and the increase was more prominent
in IL-15-Tg mice on these stages. The TCR T cells were decreased
in both non-Tg and IL-15-Tg mice on day 28 after BCG infection. As
reported previously (37), the number of
CD8+ T cells was larger in the peritoneal cavity
of naive IL-15-Tg mice without BCG infection compared with non-Tg mice.
The number of CD8+ T cells remained at a high
level during the course of BCG infection, although the proportion of
CD4+ T cells gradually increased in the
peritoneal cavity of IL-15-Tg mice by day 28 after BCG infection.
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To investigate whether Ag-specific T cells were generated more
efficiently in IL-15 mice during the course of BCG infection, T cells
were isolated from PEC of the mice before and on day 14 post-i.p.
infection with BCG, and were cultured with or without PPD in the
presence of APC, or on anti-TCR mAb-coated dishes, and the
culture supernatants were examined for IFN- release by ELISA. The
proliferative response and IFN- production of T cells from IL-15-Tg
mice were much the same level in response to TCR cross-linking
for 48 h as those from non-Tg mice (Fig. 4). On the other hand, T cells from IL-15-Tg mice on day 14 after
infection produced significantly higher level of IFN- in response to
PPD than those from non-Tg mice. Those results suggest that
BCG-specific Th1/Tc1 cells are more efficiently generated in IL-15-Tg
mice following BCG infection (p < 0.05). We
further examined IL-4 or IL-10 production by T cells from non-Tg mice
or IL-15-Tg mice infected with BCG. T cells from neither non-Tg mice or
IL-15-Tg mice produced IL-4 or IL-10 in response to PPD, suggesting
that PPD-specific Th2 or regulatory T cells (44) are not
generated in IL-15-Tg mice (data not shown).
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in response
to PPD, we utilized cytokine FACS analysis for expression of CD4, CD8,
and intracellular IFN-. A typical result is shown in Fig. 5
and data were summarized in Table II. As shown in Fig. 5, most of the IFN--producing T cells in response
to PPD and rIL-2 were of CD4+ phenotype in non-Tg
mice infected with BCG 14 days previously, and the relative number of
the CD4+ Th1 cells was significantly higher in
IL-15-Tg mice on day 14 after BCG infection (p
< 0.05, Table II). Notably, large number of CD8+
T cells from the BCG-infected IL-15-Tg mice produced IFN- in
response to PPD and rIL-2 (Fig. 5, Table II). Such Tc1 cells were not
detected in non-Tg mice on day 14 after BCG infection. These results
indicated that the generation of CD8+ Tc1 cells
was significantly increased in IL-15-Tg mice infected with
BCG.
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on bacterial growth
in IL-15-Tg mice during BCG infection
IFN- is known to play a critical role in protection against
mycobacterial infection (1, 2). We examined the effects of
neutralization of endogenous IFN- by in vivo administration of
anti-IFN- mAb on mycobacterial growth in the peritoneal cavity,
the liver, the lung, and the spleen of IL-15-Tg mice after BCG
infection. IL-15-Tg and non-Tg mice were treated with anti-IFN-
or control rat IgG by a s.c. implanted ALZET osmotic pump on day 0
after an i.p. infection with 5 x 106 CFU of
BCG. The numbers of bacteria recovered from the peritoneal cavity; the
liver, or the lung of infected mice on day 14 were determined. As shown
in Fig. 6
, the bacterial numbers in the peritoneal cavity, the liver, or the lungof non-Tg mice and IL-15-Tg mice, both of which were treated with
anti-IFN- mAb, were significantly higher on days 14 after BCG
infection than those in isotype control Ab-treated non-Tg and IL-15-Tg
mice, respectively. Similar tendency in bacterial growth was observed
in the spleen (data not shown). Thus, these results indicated that
IFN- was a key cytokine in control of mycobacterial infection both
in non-Tg mice and IL-15-Tg mice.
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Both NK cells and CD8+ cells capable of
producing IFN- were increased in IL-15-Tg mice during BCG infection.
To investigate the contribution of NK cells and/or
CD8+ T cells to protection against BCG infection
in IL-15-Tg mice, anti-asialo-GM1 Ab or
anti-CD8 mAb were administered i.p. 2 days before, and 5 and 12
days after an i.p. inoculation with 5 x 106
CFU of BCG. We confirmed by FACS analysis that NK or
CD8+ T cells were almost depleted in the spleen
and the liver of IL-15-Tg mice on day 14 after BCG infection (data not
shown). Bacterial counts in the peritoneal cavity and the liver of the
Ab-treated IL-15-Tg mice were determined on day 14 after BCG infection.
As shown in Fig. 7A, the numbers in the peritoneal cavity and the liver of
IL-15-Tg mice treated with anti-asialo-GM1 Ab
were significantly larger on day 14 after infection than those in
control Ab-treated IL-15-Tg mice (p < 0.01).
On the other hand, the number was only marginally increased in the
liver of non-Tg mice treated with
anti-asialo-GM1 Ab. When IL-15-Tg mice were
treated with anti-CD8 mAb, the numbers of BCG were markedly greater
in the peritoneal cavity and the liver on day 14 than those in isotype
control Ab-treated IL-15-Tg mice (p < 0.01,
Fig. 7B). Non-Tg mice depleted of CD8+
T cells showed a slight increase in number in the peritoneal cavity or
the liver on day 14 after BCG infection. These results suggested that
both NK cells and CD8+ T cells contributed to the
enhanced protection against BCG infection in IL-15-Tg mice.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. IFN- level in
serum was significantly higher in IL-15-Tg mice than in non-Tg mice
after BCG infection, and neutralization of endogenous IFN-
deteriorated the clearance of the bacteria in IL-15-Tg mice. The
peritoneal macrophages expressed the higher amounts of iNOS mRNA,
suggesting that macrophages in IL-15-Tg mice are more immunologically
activated following BCG infection. Taken together, overexpression of
IL-15 in vivo may enhance protection against BCG infection via
increased IFN- production and macrophage activation. IL-15 is known to play important roles in proliferation, accumulation, and maintenance of NK cells (23, 24). The results of the present study revealed that IL-15-Tg mice have much the same numbers of NK cells in the peripheral lymphoid tissues as those in non-Tg mice, but NK cells were markedly increased in IL-15-Tg mice on day 7 after BCG infection and remained high on day 28 after BCG infection. Depletion of NK cells hampered the exclusion of bacteria in IL-15-Tg mice after BCG infection. We have previously reported that NK cells increased at the earlier stage after avirulent strain of S. choleraesuis and neutralization of endogenous IL-15 by in vivo administration with anti-IL-15 mAb inhibited the appearance of NK cells and exacerbated the infection (25, 36, 37). Taken together, it appears that IL-15 may serve to induce proliferation and/or accumulation of NK cells during BCG infection, and the increase in number of NK cells is at least partly responsible for the enhanced resistance against BCG infection in IL-15-Tg mice.
IL-15 also plays important roles in proliferation, accumulation, and
maintenance of NKT cells (24, 45). Our current results
reveal that NKT cells were increased and remained at a higher level in
IL-15-Tg mice compared with non-Tg mice following BCG infection (Fig. 3
and Table I). NKT cells are known to recognize glycolipids, including
phospatidylinositol containing compound from bacteria in the context of
CD1d molecules (46, 47). CD1 gene knockout mice that are
deficient in NKT cells are not susceptible to M.
tuberculosis infection, suggesting that NKT cells made no
contribution to immunity (8). However, it remains unknown
whether NKT cells play protective role in infection with less virulent
mycobacteria, such as BCG. Studies with TCR gene knockout mice
suggested that TCR T cells play a role in granuloma formation to
mycobacteria (48). We previously reported that TCR T
cells increased on day 7 after BCG infection well before Ag-specific
TCR T cells appeared (49). Consistent with this
finding, TCR T cells increased drastically on day 7 and
thereafter rapidly decreased in IL-15-Tg mice following BCG infection.
We have previously reported that TCR T cells proliferated in
response to IL-15 during the course of Salmonella
(25) or E. coli (35). IL-15 may
serve to induce accumulation and expansion of TCR T cells in the
inflamed sites, but may not be able to maintain TCR T cells in
contrast to the cases of NK cells and CD8+ T
cells. TCR T cells are reported to be easily subjected to
activation-induced cell death by apoptosis (50, 51).
Therefore, it is possible that activated TCR T cells may be
subjected to activation-induced apoptosis even in the presence of
IL-15, whereas IL-15 may protect NK cells and
CD8+ T cells from activation-induced cell
death.
A notable finding in the present study is that IL-15-Tg mice developed
a significant level of Ag-specific Tc1 response after BCG infection and
the CD8+ T cells contribute to protection against
BCG infection in IL-15-Tg mice. IL-15 is known to be important for
proliferation and maturation of memory-type CD8+
T cells (22, 23, 24, 52). We previously reported that
CD8+ T cells expressing memory markers,
CD44+, Ly-6C+, and
CD69- of the phenotype, were increased in naive
IL-15-Tg mice (37). Furthermore, we found that IL-15-Tg
mice preferably developed Ag-specific CD8+ Tc1
responses, producing IFN-, and generated a significantly lower level
of IgE, but a higher level of IgG2a specific for OVA after OVA
sensitization (53). Thus, overexpression of IL-15 helps to
induce Ag-specific CD8+ Tc1 cells and maintain
the CD8+ T cells, exerting a strong antibacterial
activity in IL-15-Tg mice. Recent studies suggest that CD8 CTL
releasing perforin and granulysin play a role in protection against
M. tuberculosis infection via cytolysis mechanism (9, 10). We do not know the relative contribution of cytotoxicity
and IFN- production by CD8+ T cells to
protection against BCG infection in IL-15-Tg mice. The resistance to
M. tuberculosis infection in perforin-deficient mice was
unaltered, suggesting that the cytotoxic function of
CD8+ T cells may not be critical in protection to
tuberculosis (11, 12). Studies with IFN- knockout mice
revealed that protection against infection with M.
tuberculosis depends on IFN- (1, 2). Our results
also showed that neutralization of endogenous IFN- hampered the
resolution of BCG infection. Taken together, we speculate that
IL-15-dependent Tc1 cells play a critical role in enhanced protection
against BCG infection in IL-15-Tg mice via IFN- production. Maeurer
et al. (54) have recently reported that administration of
exogenous IL-15 3 wk after M. tuberculosis infection
prolonged the survival rate of BALB/c mice, although apparent Tc1
response was not detected in these mice. IL-15-Tg mice, which
constitutively produced IL-15 during the course of mycobacterial
infection, exhibited an enhanced Tc1 response, whereas administration
of IL-15 only at effector phase during infection might not affect the
generation of Tc1 cells. Alternatively, M. tuberculosis is
known to suppress in vitro response of T cells partly via NO induction
(55), which may inhibit Tc1 response against M.
tuberculosis-infected APC in vitro in their study.
CD8+ T cells from BCG-infected IL-15-Tg mice
produced IFN- in response to exogenous PPD in the presence of APC.
Exogenous particulate Ags such as PPD are usually present in
association with MHC class II, whereas recent reports have suggested
that there is a degree of degeneracy in the processing pathways and
that Ags in the extracellular milieu can also be processed and
presented in association with MHC class I (56, 57, 58, 59), which
are usually required for activation of CD8+ T
cells. Therefore, it is possible that PPD may be presented by MHC class
I on APC in alternative processing pathway. However, TAP-deficient mice
are reported to be susceptible to M. tuberculosis infection,
indicating that TAP-dependent Ag presentation, a major pathway for
activation of CD8+ T cells, is critical for
protection against M. tuberculosis infection
(8). PPD may contain Ag that can bind directly MHC class I
on APC, which activate Tc1 cells in IL-15-Tg mice infected with
BCG.
The relative contribution of NK cells and CD8+ T
cells to protection against BCG infection in IL-15-Tg mice remains to
be elucidated. Either in vivo administration of
anti-asialo-GM1 Ab or anti-CD8 mAb
abrogated the antibacterial activity, suggesting that both NK cells and
CD8+ T cells are required for protection against
BCG infection in IL-15-Tg mice. Th1 cells, into which naive
CD4+ T cells differentiate in the presence of
IL-12 and IFN-, secrete IL-2, IFN-, and TNF- for induction of
cell-mediated immunity (60, 61, 62). Thus, early production of
IFN- by NK cells may be responsible for development of Tc1 responses
in IL-15-Tg mice. Alternatively, asialo-GM1 is
expressed not only by NK cells, but also some subset of T cells such as
native CD8+ T cells, antiviral
CD8+ T cells, and alloreactive
CD8+ T cells (63). In vivo treatment
with anti-asialo-GM1 thereby may deplete
asialo-GM1+
CD8+ Tc1 cells, which are responsible for
antibacterial activity in IL-15-Tg mice. This speculation warrants
further examination of the characteristics of Ag-specific
CD8+ T cells in IL-15-Tg mice.
Infection of mice with the less virulent BCG consistently found
that CD8+ T cells made no contribution to
immunity in normal mice (64, 65). Our results also showed
that depletion of CD8+ T cells in non-Tg mice was
less effective on the bacterial clearance compared with IL-15-Tg mice.
Although BCG has been used as a vaccine, it confers incomplete
protection against tuberculosis, at least in adults (14).
Therefore, it is important to establish effective vaccination to induce
strong and long lasting immunity against M. tuberculosis
infection. It is likely that host cytokine cascade may have the most
pronounced and long-lasting effects on the T cells’ responses.
Recently, Freidag et al. (66) reported that IL-12 improved
the efficacy of BCG vaccination in mice challenged with M.
tuberculosis. Kremer et al. (67) showed that
intradermal injection with DNA encoding IL-18 led to increased in vitro
PPD-dependent IFN- production in mice infected with BCG. These
results imply that these cytokines can be used as immune adjuvant given
with BCG vaccination to increase efficacy of BCG vaccination. Since
CD8+ Tc1 cells, besides
CD4+ Th1 cells, are critical in protection
against virulent M. tuberculosis infection (5, 8, 68), our current data with IL-15-Tg mice may also suggest that
IL-15 can be used as immune adjuvant to increase the efficacy of BCG
vaccination via enhancing Tc1 response. Additional experiments with
aerosol challenge with M. tuberculosis might allow us to
define conclusively whether IL-15 is useful for development of new
immunoprotective approaches against mycobacterial infection.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Yasunobu Yoshikai, Laboratory of Host Defense, Research Institute for Disease Mechanism and Control, Nagoya University School of Medicine, Nagoya 466-8550, Japan. E-mail address: yyoshika{at}tsuru.med.nagoya-u.ac.jp
3 Abbreviations used in this paper: Tc1, T cytotoxic 1; BCG, bacillus Calmette-Guérin; FCM, flow cytometric; i-IEL, intestinal intraepithelial lymphocyte; iNOS, inducible NO synthase; PEC, peritoneal exudate cells; PPD, purified protein derivative; Tg, transgenic.
Received for publication July 20, 2000. Accepted for publication May 15, 2001.
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