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and VCAM-1 and Is Rapidly Down-Regulated by a Mechanism Involving T Cells and Expression of Fas1 ,2
*
Laboratory of Experimental Immunology, Division of Basic Sciences, and
Science Applications International Corp. Frederick, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD 21702;
Pediatric Oncology Branch, National Institutes of Health, Bethesda, MD 20892; and
§
Department of Oncology, Hoffmann-La Roche, Inc., Nutley, NJ 07110
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-/-
mice. In contrast, daily administration of IL-2 caused a sustained
increase in liver-associated NK cells that was not diminished in
IFN--/- mice. The IL-12-induced recruitment in both
hepatic NK and T cells was abrogated by in vivo treatment with
anti-VCAM-1 mAbs, while treatment with anti-ICAM-1 Abs
decreased only the recruitment of T cells in the IL-12-treated mice.
The rapid loss of newly recruited hepatic NK cells in IL-12-treated
mice did not occur in SCID mice or in
B.MRL-Faslpr
(Fas-) and
B6Smn.C3H-Faslgld
(FasL-) mutant mice, suggesting that T cells can actively
eliminate hepatic NK cells through a Fas-dependent mechanism. These
findings also imply that during the endogenous innate immune response
to infectious agents or tumors or in the host response induced by
cytokine therapies, the biologic effects of NK cells may be limited by
T cell-mediated effects. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The recruitment of leukocytes from the blood into sites of infection or
tumor development represents a critical early step in the development
of an effective immune response. Several recent reports have
contributed to a better understanding of the mechanisms involved in the
in vivo recruitment of NK cells from the vasculature into lymphoid
and/or nonlymphoid organ sites within the host (2, 10, 11, 12). A number of
biochemical and biologic agents have been shown to stimulate, in a
cytokine-dependent manner, the recruitment of NK cells from the
peripheral circulation in both lymphoid and nonlymphoid tissues (2, 11, 13). In particular, the treatment of mice with the potent
cytokine-inducing agent poly-ICLC leads to a TNF-dependent increase in
NK cell number and lytic activity within the liver (10), which is also
dependent on the expression of
VCAM-1/VLA-45 (11).
Similarly, infection of mice with murine CMV (MCMV) or induction of
IFN-
/ß results in a unique redistribution of NK cells to the
splenic marginal zone, and there is recruitment of NK cells to the
liver following infection of the liver with MCMV and lymphocytic
choriomeningitis virus (2, 14, 15). Depletion of NK cells has been
shown to increase the susceptibility of mice to MCMV (16) and herpes
simplex virus (17). More recently, new information has emerged that
some viruses (18) can induce an IFN-/ß-dependent inhibition of
IL-12 production that could contribute to the pathogenesis of such
viral infections.
These findings coupled with the documented effects of IL-12 on NK- and T cell-mediated functions in vitro (reviewed in 19 and the demonstrated ability of these cells to mediate anti-microbial or anti-tumor functions in vivo suggest that a better understanding of the effects of IL-12 on NK and T cell recruitment and function in a model organ such as the liver may provide new insight into the in vivo effector and immunoregulatory roles of these cells. In addition, recent evidence showing that IL-12 and IL-2 use parallel but distinct intracellular signaling pathways (20), reciprocally up-regulate the expression of each other receptors (19, 21), additively or even synergistically enhance the induction of cytokines and effector activity (22, 23, 24, 25), and possess synergistic antitumor activity (26, 27, 28, 29, 30) suggests that a better understanding of the in vivo biology of these cytokines could help optimize their therapeutic utility. The present studies were undertaken to investigate and compare the mechanisms by which exogenous administration of IL-2 or IL-12 causes the recruitment of NK vs T cells to the liver and to determine the potential cross-regulatory interactions that might occur between these two leukocyte subsets. The results demonstrate that the recruitment of NK vs T cells to the liver in response to IL-12 vs IL-2 differs quantitatively and qualitatively, and that the ultimate disappearance of newly recruited NK cells is dependent on T cells and a Fas/FasL-mediated mechanism. These findings suggest that the effectiveness of biologic therapies may be limited in some organ sites as a consequence of the induction of NK-T cell interactions that ultimately lead to the elimination of specific lymphocyte populations.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Specific pathogen-free C57BL/6 and C57BL/6 SCID mice (6–8 wk of
age) were obtained from the Animal Production Area, National Cancer
Institute-Frederick Cancer Research and Development Center (Frederick,
MD). The following spontaneous or induced mutant mouse strains were
purchased from The Jackson Laboratory (Bar Harbor, ME):
B6.MRL-Faslpr,
B6Smn.C3H-FasLgld, and
C57BL/6J-Icam1tm1Bay. C57BL/6
IFN--/- (GKO) mice were obtained from our
own mouse colony that was derived from mice donated by Dr. Dyana
Dalton, Genentech (South San Francisco, CA). Mice were kept under
specific pathogen-free conditions and were provided sterilized mouse
chow (Ziegler Brothers, Gardner, PA) and water ad libitum. Animal care
was provided in accordance with the procedures outlined in the Guide
for the Care and Use of Laboratory Animals (National Institutes of
Health Publication 86-23, 1985).
Reagents
Recombinant human IL-2 (Tecin; 3.2 x 107 U/mg)
and recombinant murine IL-12 (rmIL-12; 7 x 106 U/mg)
were provided by Hoffmann-La Roche (Nutley, NJ). Stock aliquots of
rmIL-12 in sterile Ca2+-, Mg2+-free Dulbecco’s
PBS were stored at -70°C until use. The rmIFN- (sp. act. =
4.7 x 106 U/mg) was provided by Genentech. For in
vivo administration the stock solutions were diluted as necessary with
PBS containing 0.1% sterile-filtered B6 mouse serum and used within
48 h. The following hybridomas were obtained from American Type
Culture Collection (Manassas, VA): YN1/1.7.4 (CRL-1878, rat
anti-mouse CD54) and M/K2.7 (CRL-1909, rat anti-mouse
CD106). The mAbs were isolated from clarified ascites by
affinity chromatography using GammaBind-Plus according to the
manufacturer’s instructions (Genex, Gaithersburg, MD), diafiltered
into sterile Ca2+-, Mg2+-free PBS, and stored
at -20°C. Control IgG was isolated from rat serum as described
above.
Isolation and analyses of hepatic mononuclear cells
Leukocytes were obtained from the livers of control and cytokine-treated mice by a modified version (31) of a previously described procedure (32). Briefly, livers were first perfused with 25 ml of Ca2+-, Mg2+-free PBS and excised. Cell suspensions were generated after a 2-min disruption in a stomacher (Tekmar, Cincinnati, OH). The resulting cell suspensions were centrifuged at 500 x g for 5 min, and the pellet was resuspended and filtered through 100-µm nylon mesh. The cellular filtrate was centrifuged at 500 x g for 5 min, and the pellet was resuspended to 35 ml in Ca2+-, Mg2+-free PBS. The cell suspension was then underlaid with 13 ml of Lympholyte-MJ (Cedar Lane Laboratories, Ontario, Canada) and centrifuged at 1400 x g for 30 min at 20°C. After centrifugation, 10 ml of the interface was aspirated, mixed with 40 ml of Ca2+-, Mg2+-free PBS, and centrifuged at 500 x g for 5 min. The resulting cell pellet was resuspended in 0.2% BSA in Ca2+-, Mg2+-free PBS for two-color cytometric analysis (FCA) or in RPMI supplemented with 5% FBS for NK activity as described below.
Cells were labeled with optimally titrated Abs, and cellular fluorescence was determined on 1.5 x 104 cells using a FACScan analyzer (Becton Dickinson, San Jose, CA). NK cells or T cells were detected using R-phycoerythrin-labeled NK1.1 (clone PK136) or FITC-labeled anti-CD3 (clone 145-2C11) obtained from PharMingen (San Diego, CA). In certain experiments the DX-5 Ab (provided by Lewis Lanier, DNAX, South San Francisco, CA) was substituted for NK1.1. The data were analyzed by the use of FACScan research and LYSYS software programs written for the Hewlett Packard Consort 30 microcomputer integral to the FACScan analyzer (Becton Dickinson). The percentage of cells bearing a particular phenotypic marker was determined on a pool of five livers per group, and the number of cells bearing that marker was then calculated by multiplying the percentage by the mean of the total number of cells isolated from the pooled livers. Lytic unit activity was assessed with a 4-h 51Cr release assay against YAC-1 target cells as described previously (11).
Isolation of total cellular RNA and Northern blot analysis
Total RNA was isolated from the liver using Trizol reagent (Life Technologies, Gaithersburg, MD). Five micrograms of total RNA was separated in a 1% agarose gel in 1x MOPS buffer. After the electrophoresis, the gel was treated with 0.05 N NaOH for 20 min and then neutralized in 5x MOPS buffer. The RNA was transferred overnight to a Hybond-N nylon membrane (Amersham, Arlington Heights, IL) with 10x SSC. The hybridization and detection were performed either with 32P-labeled probe, according to the Hybond-N manufacturer’s protocol or with digoxigenin-labeled antisense riboprobe, as follows. The murine ICAM-1 cDNA clone Ly-47 in pBluescript SK- and murine VCAM-1 cDNA clone Ly-59 were purchased from American Type Culture Collection. Clone L-59 was used as a template for the PCR with the sense (5'-AAGGTGAGGACGGAGGGGT-3') and the antisense (5'-CAGAGATCGTTGTATTCCTGG-3') primers to amplify the 481-bp murine VCAM-1 cDNA. The plasmid, pmVCAM-1, was constructed by subcloning the PCR product into the vector pCR2.1 with a TA cloning kit (Invitrogen, San Diego, CA) such that the T7 promoter produces the antisense strand. This plasmid was capable of detecting and discriminating both full-length murine VCAM-1 as well as an alternative splice variant, truncated VCAM-1 (tVCAM-1). The riboprobes for murine ICAM-1 (Ly-47) and VCAM-1 (pmVCAM-1) were generated by T3 and T7 RNA polymerase with digoxigenin, respectively. The digoxigenin-labeled RNA probe was diluted to 100 ng/ml with DIG Easy Hyb buffer (Boehringer Mannheim, Indianapolis, IN) and was incubated with the membrane at 65°C overnight after prehybridization. The membrane was then washed twice in 0.1x SSC/0.1% SDS at 65°C for 15 min. The detection of bound digoxigenin-labeled probes was performed according to the DIG System User’s Guide for Filter Hybridization (Boehringer Mannheim) with disodium 3-(4-methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.13,7]decan}-4-4yl)phenyl phosphate (CSPD) as a substrate.
Statistical analyses
Experimental results were analyzed for their statistical significance by Student’s t test. Results were considered statistically significant for p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Initial studies were performed to determine the unstimulated, homoeostatic levels of resident hepatic NK and T cells in C57BL/6 mice. The results show that the percentage of NK1.1+, CD3-, and NK1.1-, CD3+ cells were 16 and 38%, respectively (data not shown). In four separate determinations, the mean ± SD numbers of resident hepatic NK and T cells were 2.9 ± 1.3 x 105 and 1.9 ± 0.3 x 106, respectively.
IL-12-induced alterations in hepatic NK and T cells were investigated
in C57BL/6 mice injected i.p. with 0.5 µg of rmIL-12 daily for 7
consecutive days, and the livers were processed for the ex vivo
determination of NK1.1+, CD3-, or
NK1.1-, CD3+ cells on days 1, 4, and 7 of
treatment. Twenty-four hours after IL-12 administration, the number of
NK1.1+, CD3- cells recovered from the liver
was increased approximately 10-fold over homoeostatic levels to
24.4 x 105 (p < 0.01; Fig. 1
A). The observed increase in
hepatic NK cells was due largely to an increase in both lymphoid cell
number as well as the frequency of NK1.1-expressing cells from 16% on
day 0 to 24% on day 1 (Fig. 1C). Coinciding with the
observed rmIL-12-induced increase in liver-associated NK cells was a
corresponding increase in NK lytic activity by 24 h (Fig. 1A). The initial increase in hepatic NK cell number and
lytic activity following rmIL-12 administration was not sustained by
continued IL-12 administration, since the number of liver-associated NK
cells declined to 8.3 and 0.8 x 105 by days 4 and 7,
respectively (Fig. 1A). This decrease in NK1.1+,
CD3- NK cells occurred largely because of a dramatic
decrease in frequency from 24% on day 1 to 4% on day 4 (Fig. 1C). Concomitant with the decrease in hepatic NK cell number
and lytic activity was an increase in liver-associated
CD3+, NK1.1- cells to 7.2 and 18.8 x
106 on days 4 and 7, respectively, of rmIL-12
administration (p < 0.01; Fig. 1A).
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B). However, in
marked contrast to the results of rmIL-12 injection, continued IL-2
administration resulted in a further increase in hepatic NK cell
number, and by day 3 and 4 of treatment was approximately 26-fold
greater than that in unstimulated mice (p <
0.01; Fig. 1B). The increase in liver-associated NK cells
induced by rhIL-2 was reflected in the marked augmentation of hepatic
NK lytic activity (Fig. 1B). In addition, by 48 h of
administration rhIL-2 induced a transient 2-fold increase in the number
of NK1.1-, CD3+ cells recovered from the liver
(p < 0.01), which returned to homoeostatic
levels by 96 h (Fig. 1B). Taken together these data
demonstrate that compared with rhIL-2, rmIL-12 induces a rapid, but
unsustained, increase in liver-associated NK cells and lytic activity.
Moreover, the data suggest that concurrent with the observed decrease
in hepatic NK cell number induced by 4 days of rmIL-12 administration,
there is a marked increase in the number of liver-associated T cells.
IL-12 induction of hepatic NK cell, but not T cell, infiltrates is
dependent on IFN-
Regulatory events that could contribute to the observed pattern of
IL-12-induced liver-associated lymphocyte alterations were next
investigated. Previous studies have shown that many, but not all, the
in vivo effects of IL-12 can be attributed to the induction of IFN-
production (33, 34). To study the role of IFN- in mediating the
observed IL-12-induced changes in hepatic leukocyte subsets, C57BL/6
and B6 mice deficient in IFN- (IFN--/-)
were injected i.p. with 0.5 µg of rmIL-12 daily for 4 consecutive
days, and the livers were processed for the determination of NK and T
cell numbers on days 1 and 4, respectively. In agreement with the data
summarized in Fig. 1A, 24 h after rmIL-12
administration the number of NK1.1+, CD3-
cells recovered from the livers of wild-type C57BL/6 mice was augmented
approximately ninefold (to 4.9 x 106 cells/liver)
over the unstimulated level (5.5 x 105 cells/liver;
p < 0.01; Fig. 2). In
contrast, under identical treatment conditions, the injection of
rmIL-12 into B6 IFN--/- mice did not result
in a significant increase in liver-associated NK cells (3.8 x
105, 4.2 x 105, and 4.8 x
105 cells/liver on days 0, 1, and 4, respectively).
However, by day 4 of continued rmIL-12 administration, a comparable
increase in the number of liver-associated T cells was observed in both
the C57BL/6 and B6 IFN--/- strains of mice
(Fig. 3). These data demonstrate that the
IL-12-induced increase in hepatic NK cells, but not T cells, was
dependent on endogenous IFN-. This conclusion was supported by
additional experimental groups showing that comparable levels of
IL-12-induced liver-associated NK cell numbers were observed 24 h
following the i.p. injection of 2.5 x 105 U of
rmIFN- to both C57BL/6 and B6 IFN--/-
mice, and this effect was not further enhanced in the
IFN--/- strain by rmIL-12 (Fig. 2). The role
of endogenous IFN- in the rhIL-2-induced hepatic NK cell
infiltration was determined using C57BL/6 and B6
IFN--/- mice that were treated i.p. with
1 x 105 U of rhIL-2 twice daily. Twenty-four hours
later the livers were processed for the determination of NK cells.
Under these experimental conditions a similar fold increase in the
number of NK1.1+, CD3- cells was obtained from
the livers of C57BL/6 and B6 IFN--/- treated
with rhIL-2 (Fig. 2). Taken together, these data demonstrate that
endogenous IFN- was both critical and specific for hepatic NK cell
infiltration induced by rmIL-12.
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Previous studies from our laboratory have shown that the biologic
response modifier poly-ICLC induces the recruitment of NK cells to
lungs and liver via a TNF--dependent mechanism (10), and that this
is at least partly mediated through VLA-4/VCAM-1 interaction (11). The
involvement of VLA-4 and VCAM-1 interaction was also observed for the
IL-2-induced NK cell infiltration into lungs and liver (11). To
investigate the potential regulatory role of VCAM-1 in mediating the
observed IL-12-induced alterations in hepatic leukocyte subsets,
C57BL/6 mice were injected i.p. with 0.5 µg of rmIL-12 daily for 4
consecutive days and with 500 µg of either -VCAM-1 mAb or control
rat IgG immediately before the start of rmIL-12 treatment and again on
day 3. The livers were then processed for the quantitation of NK and T
cells on days 1 and 4, respectively. Twenty-four hours after the
initial injection of rmIL-12 alone or in combination with control rat
IgG, the number of NK1.1+, CD3- cells
recovered from the liver was increased approximately eight- to ninefold
over the control homoeostatic level to 23.3 and 19.2 x
105, respectively (p < 0.01; Fig. 4). Coinjection of -VCAM-1 mAb
inhibited the rmIL-12-induced NK cell infiltration of liver such that
the number of NK1.1+, CD3- cells was not
increased over the level observed in control mice (Fig. 4). Similar
results were obtained on day 4 for the inhibition of rmIL-12-induced
hepatic T cell infiltration by treatment of mice with -VCAM-1 mAb
(Fig. 4).
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-ICAM-1 mAb on IL-12-induced hepatic NK and T cell
infiltration. Using an experimental design identical with that outlined
above, coinjection of -ICAM-1 mAb resulted in a 75% inhibition of
the rmIL-12-induced increase in liver-associated T cells
(p < 0.01), but did not alter the NK cell
infiltration pattern (Fig. 4). To investigate whether the apparent
differential regulation of NK and T cell infiltration patterns by
ICAM-1, compared with VCAM-1, was due to a difference in the kinetics
of their induction by rmIL-12, total hepatic RNA was purified from
unstimulated C57BL/6 mice and from mice treated i.p. with 0.5 µg of
rmIL-12 for 1, 4, and 7 days for assessment of ICAM-1 and VCAM-1 gene
expression by Northern blot analyses. The data presented in Fig. 5 show that the expression of both VCAM-1
and ICAM-1 in the liver is not detected in unstimulated mice. However,
within 24 h following rmIL-12 administration the genes for both
ICAM-1 and VCAM-1 were expressed, and their expression further
increased with continued treatment. Taken together these data
demonstrate that IL-12-induced recruitment of hepatic NK cells is
strictly dependent on the VLA-4/VCAM-1 interaction, while T cell
infiltration proceeds via both VCAM-1- and ICAM-1-dependent pathways.
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and VCAM-1 expression for
the induction of hepatic NK cell infiltration by rmIL-12 was next
investigated. Total hepatic RNA was purified from unstimulated C57BL/6
and B6 IFN--/- mice 24 h after the i.p.
administration of 0.5 µg of rmIL-12 for assessment of VCAM-1 gene
expression by Northern blot analyses. The data shown in Fig. 6 confirm that there was no detectable
endogenous expression of VCAM-1 in the livers of unstimulated C57BL/6
or B6 IFN--/- mice. Following administration
of rmIL-12, expression of full-length VCAM-1 was detected by 24 h
in C57BL/6 mice but remained undetectable in B6
IFN--/- mice (Fig. 6). At this time point,
the induction of hepatic NK cell infiltration was observed in C57BL/6,
but not B6, IFN--/- mice (Fig. 2). By 4 days
of IL-12 administration there was a pronounced induction of both VCAM-1
and tVCAM-1 mRNA expression in C57BL/6 wild-type mice, with a lower
level of expression in IFN--/- mice.
Overall, these data demonstrate that the regulatory pathway for the
rmIL-12-induced recruitment of hepatic NK cells involves both the
production of endogenous IFN- and the subsequent expression of the
VCAM-1 adhesion molecule.
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Because the increase in IL-12-induced hepatic NK cell number is
rapidly lost, we speculated that some active down-regulating event had
been induced. Since there was an increase in the appearance of hepatic
T cells that coincided with the decline in hepatic NK cells, we further
speculated that T cells might be contributing to the loss of the NK
cells. This hypothesis was tested using T cell-immunodeficient C57BL/6
SCID mice (Fig. 7). These SCID mice were
injected i.p. with 0.5 µg of rmIL-12 daily for 4 consecutive days,
and the number of liver-associated NK cells was determined on days 1
and 4. Twenty-four hours after the initial injection of IL-12, an
approximately threefold increase in the number of hepatic NK cells was
observed in B6 SCID mice (p < 0.01). However,
unlike the results obtained in wild-type C57BL/6 mice, in which a rapid
loss of these cells then occurs, there was instead a further increase
in the number of liver-associated NK cells in B6 SCID mice. Since SCID
mice are devoid of functional T or B cells, these results demonstrate
that either T or B cells are involved in the loss of hepatic NK cells.
Since the repeated administration of IL-12 results in an increase in T
cells but no change in hepatic B cell numbers in immunocompetent mice,
the results implicate a role for T cells in NK cell disappearance.
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Since the loss of IL-12-induced hepatic NK cells occurred coincident with a recruitment of hepatic T cells and did not occur in SCID mice, we then speculated that because activated T cells often express FasL, these infiltrating T cells might be eliminating the IL-12-recruited NK cells from the liver by a Fas/FasL-dependent mechanism. Such a mechanism could be somewhat analogous to the known involvement of Fas/FasL interactions in restricting immigration of activated lymphocytes into the testes, the anterior chamber of the eye, and the placenta (35, 36, 37), except that in this case the effect could be mediated by the recruited T cells.
To investigate whether Fas/Fas ligand interaction also was involved in
mediating the decrease in hepatic NK cells following continuous IL-12
administration, C57BL/6,
B6.MRL-Faslpr (B6
Fas-), or
B6Smn.C3H-Faslgld (B6
FasL-) mice were injected i.p. with 0.5 µg of
rmIL-12 daily for 4 consecutive days, and the number of
liver-associated NK cells was determined on days 1 and 4 of treatment.
Twenty-four hours after IL-12 administration into C57BL/6, B6
Fas-, or B6 FasL- mice, the number of hepatic
NK cells was increased approximately 7-, 3.5-, and 2.9-fold,
respectively (p < 0.01; Fig. 8). In agreement with Fig. 1A,
the induction of hepatic NK cell infiltration by rmIL-12 in C57BL/6
mice was transient, and continued treatment resulted in a decrease in
newly recruited hepatic NK cells such that by day 4 the number of
liver-associated NK cells approximated that observed in unstimulated
mice. In contrast, continuous
treatment of B6 Fas- or B6 FasL- mice with
rmIL-12 for 4 days resulted in the sustained induction of
liver-associated NK cell infiltration and an approximately 5- or
4.7-fold increase, respectively, in NK cell number compared with that
in unstimulated mice (p < 0.01). These effects
occurred despite similar fold increases in hepatic T cells and only
minimal changes in the CD4+/CD8+ ratio on days
1 and 4 in both Fas- and FasL- mutant mice
(Fig. 9). These results demonstrate that the T cell-coincident loss of
IL-12-induced hepatic NK cells is also dependent on a Fas/FasL-mediated
event.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and VCAM, followed later by infiltration of T
cells and concurrent loss of NK cells via a Fas/FasL-dependent
mechanism. Moreover, we have shown that the observed influx of T cells
occurs via a IFN--independent, VCAM-1- and ICAM-1-dependent
mechanism. Previous studies that have investigated the hepatic
lymphocyte compartment in IL-12-treated mice have noted an increased
number of CD8+ T cells and NK cells, but not of
CD4+ T cells (38). This is the first report showing that
the regulatory pathways involved in the IL-12-mediated induction of
liver-infiltrating NK cells and T cells are distinct, and further
suggest that once in the hepatic parenchyma, these lymphocyte
populations may undergo a dynamic interaction that determines their
tissue life span.
The pattern of IL-12-induced lymphocyte infiltration of the hepatic
parenchyma was in marked contrast to that obtained following injection
of mice with rhIL-2, in which an IFN--independent increase in the
intrahepatic lymphocyte population was primarily restricted to the NK
cell compartment and was of long duration. The immunomodulatory effects
mediated by IL-12 or IL-2 on T and NK cells are in part overlapping and
involve induced production of cytokines (39, 40, 41), enhanced
transcription and production of several granule-associated proteins
(42, 43), increased expression of cell surface adhesion molecules (44, 45), enhancement of cytotoxicity, and increased proliferation of
activated cells (1, 2, 3, 4, 5, 19). Although these observations suggest that
IL-12 and IL-2 may share common activation pathways, differences in
their effects have also been reported. Only IL-2 is mitogenic for
resting NK cells, inducing their proliferation in vitro and in vivo,
whereas IL-12 induces proliferation only in preactivated NK and T
cells, and does so less effectively than IL-2 (46, 47). Recent studies
have suggested that the functional differences observed in IL-2- and
IL-12-stimulated NK and T cells may depend at least in part on
differential gene regulation (48). The markedly contrasting impact of
IL-12 or IL-2 treatment on liver-associated NK and T cells as seen in
our studies suggests that the nonoverlapping effects of these two
cytokines predominate during the induction, infiltration, and tissue
residence of these lymphocyte populations.
Many of the in vivo immunomodulatory effects of IL-12 have been
ascribed to its ability to induce IFN- production. Therefore, the
various activities ascribed to IL-12 represent the sum of activities
attributable to IFN--dependent and IFN--independent effects of
this cytokine. For example, IL-12 is pivotal for the differentiation of
naive CD4+ Th0 cells to cells with a Th1 phenotype through
regulation of IFN-, and although both IL-2 and IL-12 induce IFN-
production, IL-2 is relatively inefficient for inducing Th1 cell
differentiation (49). The use of mice deficient in IL-12 (50), IFN-
(51, 52) or IFN-R (33) have proven instrumental in studying this
complex relationship. In our studies the induction of the hepatic T
cell infiltrate by rmIL-12 was similar in
IFN--/- mice and wild-type mice. These data
imply that although a regulatory circuit may exist between IL-12 and
IFN- for T cell differentiation, induction of IFN- following
IL-12 administration is not involved in the subsequent recruitment and
expansion of the intrahepatic T cell compartment.
In contrast to that caused by IL-2, the IL-12-mediated increase in the
intrahepatic NK cell population was abrogated in
IFN--/- mice, indicating a requirement for
endogenous IFN- production in NK cell mobilization and infiltration.
In this regard, it is important to note that the administration of
rmIFN- was sufficient to induce increased hepatic NK cellularity to
levels comparable to those observed with IL-12 alone and was not
further augmented by concomitant IL-12 treatment. The importance of
IFN- in mediating the antiviral effects of NK cells in the liver has
been previously reported Tay and Welsh (53), who demonstrated that
production of IFN- by hepatic NK cells was the predominant mechanism
for regulating MCMV synthesis in the liver. However, the mechanism
through which IL-12 induction of IFN- leads to the initial NK cell
infiltration of hepatic parenchyma is currently unknown, but may
involve the up-regulation of specific cell surface adhesion molecules.
NK cells can be induced to extravasate from peripheral blood and
infiltrate into both lymphoid and nonlymphoid organs in response to a
variety of pathologic conditions or specific biologic response
modifiers (1, 2, 3, 4). An initial event in this process involves NK cell
adhesion to endothelial barriers. Previous studies from our laboratory
have shown that the VCAM-1/VLA-4-dependent interaction is critical for
the induction of NK cell infiltration by poly-ICLC- and IL-2 into the
lungs, liver, and tumor lesions after mobilization from the bone
marrow, and that the principal site of interaction during this critical
event is the interface between peripheral blood and endothelium (11).
In the current study we have shown that 1) the expression of the genes
encoding VCAM-1 and ICAM-1 in the liver is augmented by IL-12
treatment; and 2) the initial increase in IL-12-induced hepatic NK
cells is attenuated by blocking Abs directed against VCAM-1, but not
ICAM-1. Further, the induction of hepatic VCAM-1 RNA is minimal in
IFN--/- mice 24 h after IL-12
treatment, and this correlates with a concurrent lack of NK cell
infiltration in these mice as well. Taken together these results
demonstrate a link among IL-12, IFN-, and VCAM-1 in the regulation
of NK cell infiltration into the hepatic parenchyma.
Perhaps even more interesting than the mechanism(s) governing the initial recruitment of hepatic NK cells by IL-12 is the very rapid subsequent loss of this population. Our experimental results performed in various normal and mutant mouse strains demonstrate that the loss of these NK cells occurs in conjunction with marked local T cell recruitment, is absent in T-deficient mice, and depends on Fas/FasL-mediated events. There is precedent for the Fas/FasL-mediated loss of leukocytes in several model systems. A major area of interest in this regard derives from studies that reported the killing of Fas+ T cells by FasL+ tumor cells (54, 55, 56). These results provide evidence that tumor cells can in some situations protect themselves from effector T cells attempting to infiltrate and destroy neoplasms. Further, immune privilege in some normal tissues is maintained via expression of FasL that serves to initiate Fas-mediated apoptosis of potentially dangerous infiltrating leukocytes (35, 36, 37, 57, 58). There also is some evidence that one leukocyte population may under some conditions destroy another, as highly Ag-specific CTL (anti-H-2k) have been reported to lyse syngeneic bystander BALB/c (H-2d) Con A blasts in a Fas/FasL-dependent, perforin-independent manner (59). Other studies have reported that CD4+ T cells stimulated with anti-CD3 mAb or PMA and ionomycin induced apoptosis in cocultured inactivated CD8+ T cells, and this effect was inhibited by addition of soluble Fas mAb (60).
To our knowledge, these results provide the first evidence for a unique Fas/FasL-dependent regulatory event in which where there is a Fas/FasL-mediated elimination of newly recruited NK cells in vivo and show that this effect occurs in the absence of any overt Ag stimulation of the T cell compartment. These results support the hypothesis that Ag-independent, cytokine-mediated events are sufficient in vivo for the loss of a leukocyte subset. We speculate that this effect is T cell dependent because it occurs coincident with the appearance of T cells in the liver and is absent in T cell-deficient SCID mice. Such a down-regulatory process may be vital for preventing tissue damage or other undesirable effects during the evolution of an innate immune response and may provide a self-limiting safeguard mechanism for unrestrained effects of activated leukocytes. The technology to formally investigate the role of Fas and FasL through detection on the appropriate leukocyte subsets is now under development, and future studies associating the expression of these molecules with apoptotic functions should soon be possible. Alternatively, such effects might also result in a premature down-regulation of a beneficial immune response. Studies supporting these suggestions have been reported by Welsh and colleagues (61), who have shown that T cells activated by a viral infection become susceptible to apoptosis after repeated stimulation through the TCR.
Another intriguing possibility is that in some cases the Fas/FasL-dependent elimination of one leukocyte subset by another could be deleterious to a more beneficial role of some effector cells, and therefore limit overall therapeutic efficacy. For example, some studies investigating the antitumor activity of IL-12 have concluded that NK cells are not the primary cell type involved in this process (62). However, one interpretation of our results could be that in some organ sites in T cell-competent mice, treatment with IL-12 can result in a relatively rapid depletion of NK cells, thereby severely limiting their potential contribution to any beneficial therapeutic effect. The absence of such cells might contribute to the observed difficulties in the treatment of diseases such as various chronic infections or metastatic disease in organ sites such as the liver. Therefore, it remains possible that the antitumor activity of IL-12 could be further augmented in some anatomical sites by the continued presence of an NK cell infiltrate. A broader implication of these findings is that the transition between an initial innate immune response and a subsequent adaptive response may not always proceed in a manner that takes full advantage of all available effector cells. In particular, while the rapid down-regulation of an early innate response by a subsequent adaptive response may serve a protective role in limiting organ damage during the immune response to acute infections, it may hamper the full, sustained engagement of mechanisms required to induce complete regression of cancers or some chronic infectious diseases.
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Footnotes |
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2 This work was supported by the Werner Kirsten Student Intern Program at the Frederick Cancer Research and Development Center, Frederick, MD (to P.R.), and by a Visiting Fellowship (to M.W.) from Japan through the National Institutes of Health Visiting Fellow Program administered by the Fogarty International Center.
3 Current address: EntreMed, Inc., 9610 Medical Center Dr., Suite 200, Rockville, MD 20850.
4 Address correspondence and reprint requests to Dr. Robert H. Wiltrout, Laboratory of Experimental Immunology, National Cancer Institute-Frederick Cancer Research and Development Center, Building 560, Room 31–93, Frederick, MD 21702-1201. E-mail address:
5 Abbreviations used in this paper: VLA-4, very late antigen-4; MCMV, murine cytomegalovirus; FasL, Fas ligand; rmIL-12, recombinant murine IL-12; FCA, two-color cytometric analysis; tVCAM-1, truncated VCAM-1; rhIL-2, recombinant human IL-2.
Received for publication April 7, 1998. Accepted for publication August 6, 1998.
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