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,§,§
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Third Department of Internal Medicine,
Department of Immunology and Medical Zoology,
Department of Biochemistry and
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Laboratory of Host Defenses Institute for Advanced Medical Sciences, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan;
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Fujisaki Institute, Hayashibara Biochemical Laboratories, Okayama, Japan;
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Center for Experimental Medicine, Institute of Medical Science, University of Tokyo, Tokyo, Japan; and
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Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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production, particularly in
collaboration with IL-12. IL-18, like IL-12, also augments NK activity.
Here we investigated the molecular mechanism underlying the
up-regulation of killing activity of NK cells by IL-18. IL-18, like
IL-12, dose dependently enhanced NK activity of splenocytes. This
action was further enhanced by costimulation with IL-12. Treatment with
anti-IL-2R Ab did not affect IL-18- and/or IL-12-augmented NK
activity, and splenocytes from IFN--deficient mice showed enhanced
NK activity following stimulation with IL-12 and/or IL-18. Splenocytes
from the mice deficient in both IL-12 and IL-18 normally responded to
IL-18 and/or IL-12 with facilitated NK activity, suggesting that
functional NK cells develop in the absence of IL-12 and IL-18. IL-18R,
as well as IL-12R mRNA, was constitutively expressed in splenocytes
from SCID mice, which lack T cells and B cells but have intact NK
cells, and in those from IL-12 and IL-18 double knockout mice. NK cells
isolated from SCID splenocytes expressed IL-18R on their surface.
IL-18, in contrast to IL-12, did not enhance mRNA expression of
perforin, a key molecule for exocytosis-mediated cytotoxicity. However,
pretreatment with concanamycin A completely inhibited this IL-18-
and/or IL-12-augmented NK activity. Furthermore, IL-18, like IL-12,
failed to enhance NK activity of splenocytes from perforin-deficient
mice. These data suggested that NK cells develop and express IL-12R and
IL-18R in the absence of IL-12 or IL-18, and that both IL-18 and IL-12
directly and independently augment perforin-mediated cytotoxic activity
of NK cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production by lymphocytes including T cells, B cells,
and NK cells (1, 2, 3, 4, 5). IL-18 up-regulates Fas ligand
(FasL)3 expression on cloned NK cells,
resulting in their facilitated killing activity against Fas-expressing
cells in a DNA-fragmenting manner (6). IL-18 also
up-regulates NK activity in a Fas/FasL-independent manner against
FasL-resistant target cells (1, 2). In vivo administration
of IL-18 augments NK activity in mice (7), and in vitro
treatment with IL-18 elevates NK activity of human PBMC and murine
splenocytes (2, 7). Although IL-18 is a potent factor to
induce IFN- production by T cells and B cells, IL-12 is prerequisite
to exert the action of IL-18 on these cells (1, 2, 3, 8, 9).
This is because IL-18R, which has recently been proved to be identical
to IL-1R-related protein (IL-1RrP) (10), is not
constitutively expressed but rather induced by the stimulation with
IL-12 on T and B cells (3, 9, 11). Thus, the function of
IL-18 on T cells and B cells completely depends on the coexistence of
IL-12. However, NK cells have the capacity to respond to IL-18 in a
distinct fashion. IL-18 induces IFN- production by cloned NK cells
without any help by IL-12 (5). Recently, we have
demonstrated that up-regulating action of IL-18 on NK activity, in
contrast to its capacity to induce IFN- production by T cells, does
not require endogenous IL-12, using IL-12 null mutant mice (7, 12, 13).
In this study, we investigated the precise mechanism of how IL-18
augments NK activity, particularly comparing with IL-12, a second
potent up-regulating factor for NK activity (14), because
both cytokines are occasionally secreted by activated macrophages at
the same time (1, 5). We confirmed that YAC-1 cells,
murine target cells of NK activity, were not susceptible to the
FasL-mediated killing. Thus, we addressed here the effect of IL-18
and/or IL-12 on Fas/FasL-independent killing activity of NK cells. We
found that IL-18 as well as IL-12 up-regulated NK activity in an IL-2-
or IFN--independent manner and that NK cells constitutively
expressed IL-12R and IL-18R, even in the absence of endogenous IL-12 or
IL-18. Furthermore, we investigated the molecular mechanism underlying
the up-regulation of NK activity by IL-18, and found that IL-18 did not
increase the expression of perforin, an essential molecule for
exocytosis-mediated killing action by NK cells and CTL, whereas IL-12
enhanced it. Surprisingly, up-regulating the effect of IL-18 on NK
cells nevertheless depended totally on perforin, because splenocytes
from perforin-deficient mice did not show NK activity even after
stimulation with IL-18.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female C57BL/6 mice (5–8 wk old) were purchased from Japan SLC
(Sizuoka, Japan), and female Fox Chase SCID C.B-17/ICR-scid
Jcl (SCID) mice 5–8 wk old (15, 16) were from Clea Japan
(Tokyo, Japan). IFN--deficient mice (17) were
backcrossed to C57BL/6 mice for F8 generations, and females 5–8 wk old
were used in this study. Mice deficient in both IL-12 and IL-18 and
their littermates with wild genotype were also used (7, 12, 13). Perforin-deficient mice with C57BL/6 backgrounds were
purchased from Taconic (Germantown, PA). Sprague Dawley rats were from
Charles River Japan (Yokohama, Japan). The animals were kept under
specific pathogen-free conditions.
Abs and reagents
Murine rIL-18 and rIL-12 were produced and purified as described
previously (1). Murine rIL-2 was purchased from Genzyme
(Boston, MA). mAbs against murine IL-2R 
-chain (IL-2R) (7D4, rat
IgG2b) and mAb against IL-2R ß-chain (IL-2Rß) (TM-ß1, rat IgG2b)
were purchased from PharMingen (San Diego, CA). PE-conjugated
anti-IL-2Rß mAb and FITC-conjugated anti-rat IgG1 mAb were
also from PharMingen. Purified rat IgG was from Cappel (West Chester,
PA). Concanamycin A, a potent inhibitor for perforin-mediated
cytotoxicity (18), was purchased from Wako Pure Chemical
Industries (Osaka, Japan). YAC-1, a Moloney virus-induced lymphoma of
A/Sn origin (NK-sensitive tumor cell line), was obtained from American
Type Culture Collection (ATCC, Manassas, VA). The murine T cell
lymphoma cell line L5178Y, which does not express Fas, and its subline
transfected with a full-length cDNA encoding murine Fas (A-1)
(6) were kind gifts from Dr. S. Yonehara of Kyoto
University (Kyoto, Japan). mFasL/L5178Y was a subline of L5178Y
transfected with a full-length cDNA encoding FasL from BALB/c mice
(19). The culture medium generally used in this study was
RPMI 1640 containing 10% FCS, 100 U/ml penicillin, 100 µg/ml
streptomycin, 50 µM of 2-ME, and 2 mM L-glutamine.
mAb against murine IL-18R
Soluble murine IL-18R was purified from the supernatant of COS-1
cells transfected with cDNA encoding murine IL-1RrP (11).
We confirmed that soluble murine IL-18R used in this study could
inhibit binding of [125I]IL-18 to murine
IL-18R-transfected COS-1 cells. Rats were i.p. immunized with 50 µg
of soluble murine IL-18R, followed by booster injections three times.
Spleen cells were prepared from the rats i.v. injected with soluble
IL-18R 4 days before and fused with Y3Ag1.2.3 cells (Lou rat myeloma
cell line) (10). Hybridoma supernatant reactivities were
assayed by the system of inhibition of binding of
[125I]-labeled murine IL-18 to the COS-1 cells
transfected with murine IL-18R. After a limiting dilution method, we
obtained cloned hybridoma-producing mAb against murine IL-18R,
mAb-MuIL-18R-Y38 (IgG1
). mAb-MuIL-18R-Y38 inhibited IFN-
production by IL-18-responsive cloned NK cells (5) in
response to IL-18 but not IL-12 and had no action like IL-18. mAb was
purified from hamster ascites fluid by protein G column
(20).
Assay for killing activity
Spleen cells were prepared from C57BL/6, both IL-12- and
IL-18-disrupted mice, their wild-type littermates, IFN--deficient
mice, or perforin-deficient mice. Spleen cells were incubated with or
without various doses of IL-12 and/or IL-18 for 24 h. For some
experiments, spleen cells from C57BL/6 mice were incubated with various
combinations of IL-12 and IL-18, or 100 U/ml of IL-2 in the presence or
absence of 10 µg/ml of 7D4 plus 10 µg/ml of TMß-1 or 20 µg/ml
of rat IgG for 24 h. Cytotoxic activity of variously treated
spleen cells against YAC-1 cells was determined by 4-h
51Cr-release assay, and the percentage of cytotoxicity was
calculated (6). Briefly, YAC-1 cells were labeled with
[51Cr]sodium chromate and incubated with the variously
treated spleen cells at various E:T ratios for 4 h. The
radioactivity in the supernatant was measured by a gamma counter. The
killing activity of FasL transfectant against YAC-1 and A-1 was also
determined by the same method described above, using
51Cr-labeled YAC-1 cells and [51Cr]A-1 cells,
respectively (6).
Cell preparation from SCID mice
Spleen cells (5 x 106/ml) from 20 SCID mice were pooled and incubated in plastic dishes for 1 h at 37°C. Plastic nonadherent cells (5 x 106/ml) were additionally incubated in fresh plastic dishes for another 1 h, and nonadherent cells were collected and used as splenic lymphocytes. The cells obtained had almost no contamination with macrophages (data not shown) and contained no surface IgM+ cells and CD3+ cells. The splenic lymphocytes (2 x 106/ml) from SCID mice were incubated with various combinations of IL-12 and IL-18 for 24 h, and their total RNA was extracted. IL-2Rß+ cells were isolated by cell sorting and used as NK cells for the detection of their surface expression of IL-18R by immunohistochemistry.
FACS analysis and sorting
For determination of IL-18R expression on NK cells, after FcR
blocking using anti-FcR Ab (6), splenic lymphocytes
from SCID mice were incubated with anti-murine IL-18R mAb followed
by FITC-conjugated anti-rat IgG1 mAb and PE-conjugated IL-2Rß Ab.
Stained cells were analyzed using a dual laser FACScalibur (Becton
Dickinson, Mountain View, CA). Ten thousand cells were analyzed and
data were processed with CellQuest (Becton Dickinson). For sorting
experiments, an Epics XL Flow Cytometer (Coulter, Hialeah, FL) was
used.
Immunohistochemistry
IL-2Rß-positive cells were isolated from splenic lymphocytes from SCID mice by FACS sorting. Surface expression of IL-18R on the isolated NK cells were determined histochemically, according to the method described before except using 3,3'-diaminobenzidine (Sigma, St. Louis, MO) for coloring substrate (6). Isotype-matched control Ab instead of anti-IL-18R mAb was used for the control study.
RT-PCR
Splenocytes from SCID mice (2 x 106/ml) or IL-12 and IL-18 double-knockout mice were prepared. For some experiments, they were incubated with 2 ng/ml IL-12 and/or 20 ng/ml IL-18 for 24 h. Total RNA was isolated by the method described previously (6), reverse transcribed, and amplified. The sense primer for murine IL-18R was CGT GAC AAG CAG AGA TGT TG and the anti-sense primer was ATG TTG TCG TCT CCT TCC TG (11). Thermocycle conditions for IL-18R were 95°C for 30 s for denaturing, 58°C for 30 s for annealing, and 72°C for 30 s for extension. The sense primer for murine ß1 chain of IL-12R (IL-12Rß1) was GCA AAC ACA TCA CCT TCC TCC TGC, and the antisense primer was GTG TGT CAC CAT CTT GGC AGG ATC (21). The sense primer for murine ß2 chain of IL-12R (IL-12Rß2) was GGC ACA GAC TGT AGA ATG CTC, and the antisense primer was TGC AGA AGC GCC TTT TGA GTT GGT (22). The sense primer for perforin was TGC TAC ACT GCC ACT CGG TCA, and the antisense primer was TTG GCT ACC TTG GAG TGG GAG (23). The sense primer for murine granzyme B was CTC CAC GTG CTT TCA CCA AA, and the antisense primer was GGA AAA TAG TAC AGA GAG GCA (24). Thermocycle conditions were 95°C for 30 s for denaturing, 64°C for 30 s for annealing, and 72°C for 1 min for extension. Thirty-five cycles were performed for IL-18R, IL-12Rß1, IL-12Rß2, and perforin. The primers and amplifying conditions for ß-actin were described previously (4). The PCR products were electrophoresed in 1.7% agarose gel and visualized by ethidium bromide fluorescence. For some experiments, densitometric analysis was performed with Densitograph (Atto, Tokyo, Japan). Relative expression of perforin mRNA or granzyme B mRNA was calculated as follows: Relative expression of molecule mRNA examined = ((densitometric units of IL-12- and/or IL-18-treated samples)/(densitometric units of their ß actin mRNA))/((densitometric units of untreated samples)/(densitometric units of ß-actin mRNA of untreated samples)).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IL-18 augmented NK activity of splenocytes. As shown in Fig. 1
A, IL-18 elevated killing
activity of spleen cells against YAC-1 cells in a dose-dependent
manner, reaching a plateau at 20 ng/ml of IL-18. The killing activity
of splenocytes was also enhanced when they were incubated with IL-12
(Fig. 1B). The maximal cytotoxicity of splenocytes after
incubation with IL-12 was almost similar to that after incubation with
IL-18. The minimum dose of IL-12 that was required to give maximal
cytotoxic activity was one-tenth of that of IL-18. Next, we examined
whether IL-18 collaborates with IL-12 to enhance NK activity, because
both IL-12 and IL-18 are produced by activated macrophages sometimes
simultaneously (1, 5). As shown in Fig. 1C,
IL-18 dose dependently increased NK activity of splenocytes in the
presence of optimal dose of IL-12 (2 ng/ml). Thus, IL-12 and IL-18
collaboratively augment NK activity of splenocytes. To investigate
whether YAC-1 cells were killed by the Fas/FasL-mediated killing
pathway, we incubated YAC-1 cells with FasL-transfected cells
(19). As shown in Fig. 1D, YAC-1 cells were not
killed by FasL-expressing effector cells that were able to kill
Fas-expressing A-1 target cells, indicating that up-regulating action
of IL-12, as well as IL-18, on YAC-1 killing by splenocytes was
independent of the Fas/FasL-mediated killing pathway.
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- or IL-2-independent
augmentation of NK activity by IL-12
and/or IL-18
We examined whether IL-12 (2 ng/ml) and/or IL-18 (20 ng/ml), at
their optimal concentration, augmented NK activity through the
induction of IL-2, another potent NK cell activator (25).
Splenocytes were cultured with IL-12 and/or IL-18 in the presence of
both anti-IL-2R Ab plus anti-IL-2Rß Ab. The combination of
these Abs completely canceled the cytolytic activity augmented by 100
U/ml of IL-2, but not that by IL-12 and/or IL-18 (Fig. 2A). The amount of mAbs used
completely inhibited the proliferative response of splenocytes to Con A
(data not shown). Furthermore, IL-2 was not detected in the supernatant
of the splenocytes incubated with IL-12 and/or IL-18, determined by
CTLL-2 proliferation assay (data not shown). Control Ab did not affect
the augmenting effect of IL-12 and/or IL-18 on NK activity (Fig. 2A). Next, we investigated whether IL-18 and/or IL-12
enhanced NK activity via the induction of IFN-, because both IL-18
and IL-12 are upstream cytokines of IFN-. We analyzed the effect of
IL-12 and/or IL-18 on spleen cells from IFN--deficient mice instead
of using anti-IFN- Ab, because production of a tremendous amount
of IFN- is induced by the combinational stimulation with IL-12 and
IL-18 (1, 2, 3, 4, 5) and may be much higher than the maximal
IFN- dose that can be neutralized by anti-IFN- Abs. As shown
in Fig. 2B, IL-18, as well as IL-12, enhanced NK activity of
spleen cells from IFN--deficient mice, excluding the contribution of
endogenous IFN- to the enhanced NK activity.
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Next, we measured the net responsiveness of spleen cells to
exogenous IL-18 and/or IL-12 in NK activity. Splenocytes from wild-type
mice used so far might have been contaminated with the cells that had
been previously stimulated with endogenous IL-12 and/or IL-18 in vivo.
Furthermore, spleen cells used in these experiments contained
macrophages that are able to produce IL-12 and IL-18 in vivo or in
vitro upon stimulation. Thus, we used spleen cells from IL-12 and IL-18
null mice. The mice deficient in both IL-12 and IL-18 showed very weak
NK activity (7, 12), suggesting that there are few in vivo
activated NK cells in these mice. Using these unstimulated NK cells, we
examined their capacity to gain NK activity following stimulation with
IL-12 and/or IL-18. As shown in Fig. 3,
compared with splenocytes from wild-type mice, those from
IL-12-/-IL-18-/- mice showed rather higher
NK activity in response to stimulation with IL-12 and/or IL-18,
suggesting that functional NK cells develop even in the absence of
IL-12, IL-18, or IFN- (Figs 2
B and 3).
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The responsiveness of NK cells to IL-12 and IL-18 observed above
suggested that NK cells should express IL-18R and IL-12R
constitutively. We then investigated the expression of IL-18R and
IL-12R mRNA in NK cells by RT-PCR. To demonstrate this, we freshly
isolated nonadherent spleen cells from SCID mice as NK cells, because
SCID mice lack both T cells and B cells but have mature NK cells
(26, 27, 28). IL-18R mRNA was constitutively expressed in
splenic nonadherent cells from SCID mice (Fig. 4A). Surface IL-18R was also
constitutively expressed on NK cells from SCID mice. As shown in Fig. 4B, highly enriched NK cell fraction contained the cells
whose surface was stained by anti-IL-18R mAb. The cells were not
stained with isotype-matched control Ab (data not shown). To measure
the proportions of IL-18R-expressing NK cells, we stained splenic
nonadherent cells from SCID mice with both anti-IL-18R mAb and
anti-IL-2Rß mAb. As shown in Fig. 4C, calculated
proportion of IL-18R-expressing IL-2Rß+ cells is about
35%. This was also the case for spleen cells from euthymic wild-type
mice (data not shown). These cells also constitutively expressed mRNA
for IL-12Rß1 and IL-12Rß2, which constitute functional IL-12R
(Fig. 4).
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), were used. As
shown in Fig. 4A, both splenocytes from the double-mutant
mice and from wild-type mice constitutively expressed IL-18R as well as
IL-12R. Since naive T cells and B cells have no responsiveness to IL-18
unless they have been stimulated (3, 4, 11), IL-18R
expression in splenocytes from the double-knockout mice might be due to
that in NK cell fraction. In a separate experiment, we examined the
expression of IL-18R mRNA in spleen cells from IFN--deficient mice.
We found that they also constitutively expressed IL-18R mRNA (data not
shown). IL-18 enhances perforin-dependent cytotoxicity of splenocytes
Next, we examined the molecular mechanism by which IL-18 and/or
IL-12 enhanced NK activity. To investigate whether IL-18 and/or IL-12
augment perforin-mediated killing activity, we incubated the IL-18-
and/or IL-12-treated spleen cells with concanamycin A, a potent
inhibitor for perforin-mediated cytotoxicity (18). As
shown in Fig. 5A, pretreatment
with concanamycin A completely abrogated the enhancing effects of IL-12
and/or IL-18 on NK activity.
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B, stimulation with either IL-12 and/or IL-18 did not
induce NK activity in splenocytes from perforin-knockout mice.
Since it has been shown that IL-12 as well as IL-2 augments NK activity
by up-regulating the expression of perforin (23, 29), we
examined whether IL-18 also augments perforin mRNA expression in NK
cells. To exclude the possible contamination of T cells, we used
splenic nonadherent cells from SCID mice. IL-18 did not increase the
expression of perforin mRNA, while IL-12 did increase it (Fig. 5C). This was also the case for the mRNA expression of
granzyme B, another cytotoxic molecule responsible for
exocytosis-dependent cytotoxicity of NK cells and CTL
(29).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(30), a powerful effector cytokine, as well as adhesion
molecule-inducing factor and IFN- (30), a potent
macrophage activating factor and a modulating factor for acquired
immunity. NK cells also participate in innate immunity as effector
cells, which eliminate virus-infected cells in an MHC-unrestricted
manner and also play a role in tumor surveillance (35, 36). Indeed, severity of some bacterial and viral infections
have been reported to be reversely related to NK cell functions
(28, 30, 37, 38, 39). Since NK cells are an essential
component of the innate immunity, regulation of NK cell activity via NK
receptors is now under extensive studies (40, 41).
Cytokine-induced activation of NK cells, particularly by IL-18 and
IL-12, is also of great interest.
We here demonstrated that NK cells constitutively express functional
IL-18R as well as IL-12R. Recently, Torigoe et al. reported that
N-terminal and internal amino acid sequences of affinity-purified
IL-18R were identical to those predicted from cDNA encoding the IL-1RrP
(10). They also confirmed that COS-1 cells transfected
with the cDNA were able to respond to human IL-18 with nuclear
translocation of NF-B (10), indicating that the cDNA
they cloned encodes functional IL-18R. COS-1 cells transfected with
cDNA encoding murine IL-1RrP (11) bind murine rIL-18,
indicating that IL-1RrP is also the receptor for IL-18 in murine system
(M. Kurimoto, unpublished observations). Recently, we have shown that
splenic T cells isolated from NK cell-depleted mice did not respond to
the stimulation with IL-18, whereas those cells exhibited proliferation
and IFN- production in response to IL-18 once they were preincubated
with IL-12, suggesting that T cells require the costimulation with
IL-12 for their acquisition of the responsiveness to IL-18 (3, 4, 9, 11). Indeed, T cells incubated with IL-12 express IL-18R, but
naive T cells do not (11). In contrast, splenic
nonadherent cells freshly isolated from SCID mice express IL-18R
without any stimulation (Fig. 4). The splenic nonadherent cells from
SCID mice are predominantly constituted by NK cells (15, 42). Furthermore, NK cells isolated from splenocytes of SCID
mice by FACS sorting were able to be stained with anti-murine
IL-18R mAb (Fig. 4, B and C). Therefore, NK cells
appear to constitutively express IL-18R, at least in SCID mice.
Functional IL-12R is composed of two subunits, IL-12Rß1 and
IL-12Rß2 (21, 22). IL-12Rß1 is expressed in NK cells
and T cells and is equally expressed in Th1 and Th2 subpopulations
(43, 44). The former is defined by particularly skewed
cytokine production profile of IL-2 and IFN-, and the latter of
IL-4, IL-5, IL-10, and IL-13 (45). Recently, it has been
reported that IL-12Rß2 is expressed only in Th1 cells but not in Th2
cells (43, 44). In the case of splenic nonadherent cells
freshly isolated from SCID mice, IL-12Rß2 as well as IL-12Rß1 was
expressed under normal condition (Fig. 4A). In fact, splenic
nonadherent cells from SCID mice showed enhanced NK activity by the
stimulation with IL-18 and/or IL-12, but with a little weaker activity
than did those from C57BL/6 (data not shown). To exclude the
possibility that endogenous IFN- contributed to the constitutive
expression of IL-12R or IL-18R in NK cells, we examined the
responsiveness of splenocytes from IFN--deficient mice to the
stimulation with IL-12 and/or IL-18, and we observed that IFN- null
splenocytes also similarly respond to IL-12 and/or IL-18 (Fig. 2B), indicating that both IL-12R and IL-18R expressions in
NK cells do not require the intrinsic IFN-. Constitutive expression
of IL-18R also does not require endogenous IL-12 or IL-18, because
lymphocytes from IL-18, IL-12 double-deficient mice expressed IL-18R
under normal condition (Fig. 4A) and their NK activity was
equally or rather more strongly augmented by IL-12 and/or IL-18 as
compared with the lymphocytes from wild-type mice (Fig. 3). IL-18 and
IL-12 do not appear to cooperatively activate killing of NK cells from
wild-type mice with a background of both C57BL/6 and 129/SvJ (Fig. 3),
whereas these stimuli cooperatively activated that of those from
C57BL/6 mice (Figs. 1 and 5B). This may imply that
sensitivity of NK cells to IL-12 and/or IL-18 differ between C57BL/6
mice and 129/SvJ mice. Constitutive expression of IL-12R on NK cells is
also independent of endogenous IL-12 or IL-18 because of the same
reasons (Figs. 3 and 4). These data strongly suggested that immune
competent NK cells can develop without IL-12 or IL-18 and that
peripheral NK cells are ready to respond immediately and independently
to both IL-18 and IL-12. This may account for the fact that NK cells
have the capacity to make a first line of host defenses (37, 38) together with macrophages that produce IL-12 and IL-18 in
response to microbes and their products (1, 5).
Perforin and granzyme B have been shown to be key molecules for
cytolytic action of NK cells (29). Perforin is localized
in the cytoplasmic granules of NK cells and CTL and is accumulated at
the site where target cells interact, followed by exocytosis of these
molecules onto target cells leading to cell lysis (29).
IL-12, like IL-2, has been reported to up-regulate production of
perforin by cytotoxic lymphocytes (25). This was also the
case for nonadherent splenocytes from SCID mice (Fig. 5C) or
splenocytes from C57BL/6 mice (data not shown). In contrast, IL-18 did
not up-regulate the perforin mRNA expression (Fig. 5B). We
also measured mRNA expression of granzyme B, which facilitates target
cell lysis in concert with perforin (25), in similarly
treated nonadherent splenocytes from SCID mice and found that IL-18,
unlike IL-12, did not enhance its expression (Fig. 5B). In
separate experiments, we did time course study from 3 h to 48
h and found no obvious elevation of either perforin or granzyme B mRNA
in IL-18-stimulated, nonadherent SCID splenocytes at any time point
(data not shown). However, IL-18, like IL-12, did not enhance cytotoxic
activity of splenocytes from perforin-deficient mice (Fig. 5B). Taken together, these data indicate that IL-18 enhances
perforin-dependent cytotoxic activity of NK cells without up-regulating
perforin or granzyme B expression. This is in clear contrast with IL-12
that enhances NK activity by up-regulating these molecules and may be
responsible for the cooperative effect of IL-12 and IL-18 (Fig. 1). The
unique effect of IL-18 on NK activity may be explained by several
possibilities. First, IL-18 may up-regulate an undefined effector
molecule other than granzyme B, which functions in perforin-dependent
cytotoxicity. Second, IL-18 may activate some intracellular mechanism
leading to exocytosis. Third, IL-18 may facilitate the processing of
perforin from an inactive precursor to an active form (18)
and the intracellular transport. Further studies are needed to address
these possibilities.
In summary, we here demonstrated that NK cells constitutively express functional IL-18R as well as IL-12R and up-regulate perforin-dependent cytotoxic activity in response to IL-18 and IL-12 by apparently distinct mechanisms. We are now investigating the physiological roles of IL-18 in NK cell-dependent host defenses.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Kenji Nakanishi, Department of Immunology and Medical Zoology, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, 663-8501 Japan.
3 Abbreviations used in this paper: FasL, Fas ligand; IL-1RrP, IL-1R-related protein; SCID, Fox Chase SCID C.B-17/ICR-scid Jcl; IL-2R, IL-2R -chain; IL-2Rß, IL-2R ß-chain; IL-12Rß1, IL-12Rß1 chain; IL-12Rß2, IL-12Rß2 chain; PE, phycoerythrin.
Received for publication June 29, 1998. Accepted for publication October 20, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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