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Departments of
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Surgery,
Medicine,
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Pathology, and
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Medical Microbiology and Immunology, Arthur G. James Comprehensive Cancer Center, Ohio State University, Columbus, OH 43210;
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Institute for Medicine, Microbiology, and Hygiene, Munich, Germany;
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Genetics Institute, Andover, MA 01810;
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Children’s Hospital, Columbus, OH 43205;
Lineberger Comprehensive Cancer Center, Curriculum in Genetics and Molecular Biology, Chapel Hill, NC 27514;
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Immunex Research and Development Corporation, Seattle, WA 98101;
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Vertex Pharmaceuticals, Cambridge, MA 02139; and
*
Roswell Park Cancer Institute, Buffalo, NY 14263
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, IFN-
,
macrophage-inflammatory protein-1, IL-1, IL-1-converting enzyme,
Fas, perforin, inducible nitric oxide synthase, and STAT1 did not
contribute to the observed toxicity, nor did B or T cells. However,
toxicity and death from treatment with IL-2 plus IL-12 could be
completely abrogated by elimination of NK cells. These results suggest
that the fatal systemic inflammatory response induced by this cytokine
treatment is critically dependent upon NK cells, but does not appear to
be mediated by the known effector molecules of this cellular
compartment. These data may provide insight into the pathogenesis of
cytokine-induced shock in humans. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-1,
IL-12, IL-15, and IL-18, which in turn induce NK cell production of
IFN- and TNF- (1, 2, 3, 4). IL-12 appears to be pivotal to the NK cell
response, as there is only modest NK cell cytokine production in the
absence of this factor (5, 6). We have demonstrated previously that the
combination of IL-15 and IL-12 exerts a profound synergy upon resting
NK cell production of IFN-, TNF-, and
MIP-13 (6, 7, 8). These
proinflammatory cytokines and chemokines play a critical role in the
clearance of obligate intracellular pathogens and, in some cases, the
promotion of sepsis, shock, and death (9). An identical profile of NK
cell cytokine and chemokine production can be induced by the
combination of IL-2 and IL-12 (5, 6). This reflects the fact that the
heterotrimeric IL-15R and IL-2R share the IL-2Rß and signaling
subunits and differ only in the specificity of their high affinity
-chains (10). The ability to obtain synergistic immunomodulatory
effects via activation of the IL-2/15R and the IL-12R expressed on NK
cells has led to investigations of this cytokine combination for the
immunotherapy of neoplastic disease (11, 12). In the current study, we
examined the effects of administering IL-2 or IL-15 in combination with
IL-12 in a murine toxicity model. While the dose of the individual
cytokines was well tolerated, the administration of IL-2 or IL-15 in
combination with IL-12 induced a lethal systemic inflammatory response
that did not require any of the major proinflammatory factors or
signaling pathways felt to be active in the induction of septic shock.
However, the lethal toxicity of this cytokine combination therapy was
critically dependent upon NK cells, but not B or T cells. |
Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Purified, yeast-derived rhuIL-2 (Chiron, Emeryville, CA)
or rhuIL-15 (Immunex, Seattle, WA) was administered at a dose of
3 x 105 U/day via the i.p. route. rIL-12 of murine
(mu) origin (Genetics Institute, Cambridge, MA) was administered i.p.
at a dose of 1 µg/day. rhuIL-2 and rmuIL-12 were administered daily
until the death of the animal. rmuIL-10 was supplied by Schering-Plough
(Kenilworth, NJ). Platelet-derived huTGF-ß1 (which has
activity in murine systems) was purchased from R & D Systems
(Minneapolis, MN) and reconstituted according to the manufacturer’s
recommendations in 4 mM HCl supplemented with 0.1% BSA (13). Rat IgG1
anti-muIFN- mAb (Endogen, Cambridge, MA), and hamster IgG
anti-muIFN- mAb (Genzyme Diagnostics, Cambridge, MA) were
administered at a dose of 100 µg per mouse via the i.p. route 12
h before cytokine therapy and then daily thereafter. Control Abs were
purchased from Sigma (St. Louis, MO). A dimeric rhuTNF receptor
p80/IgG1 Fc fusion protein (TNFR-Fc; Immunex) was used in TNF-
neutralization experiments (100 µg/mouse/day i.p. beginning 24 h
prior cytokine treatment) (14). Human IgG was used as a control for the
fusion protein (Baxter, Glendale, CA). Dexamethasone was
purchased from Moore Medical (New Britain, CT). Depletion of NK
cells was accomplished via i.p. administration of an anti-asialo
GM1 Ab (Wako BioProducts, Richmond, VA) every 3 days beginning 2
wk before the injection of cytokines (0.2 mg/mouse) (15). NK cell
numbers postdepletion were evaluated via flow-cytometric analysis of
murine splenocytes using a phycoerythrin (PE)-labeled pan-NK mAb (clone
DX5; PharMingen, San Diego, CA). Mice were depleted of
monocytes/macrophages via i.v. and i.p. injection of the F4/80 mAb (an
IgG2b mAb) 48 and 24 h before cytokine therapy (16). Macrophage
numbers postdepletion were evaluated via the enumeration of
plastic-adherent cells following a 2-h culture of PBMCs, splenocytes,
bone marrow cells, or peritoneal cells in 24-well plastic culture
dishes (1 x 106 cells/well in RPMI 1640 supplemented
with 10% FBS) (7). All cytokine reagents contained less than 0.015
EU/ml endotoxin, as measured by the E-Toxate system (Sigma).
Mice
Female mice age 4–6 wk were utilized in all experiments. C.B-17
scid/scid (SCID) mice (BALB/c background), splenectomized
C.B-17 SCID mice, sham-operated C.B-17 SCID mice, and inbred BALB/c
mice were purchased from Taconic Farms (Germantown, NY).
Perforin-deficient (-/-) mice, CD3
transgenic mice,
IFN--/- mice, and
Faslpr/lpr mice were purchased from The
Jackson Laboratory (Bar Harbor, ME) (17, 18, 19, 20). TNFR p55-/-
mice, TNFR p75-/- mice, and TNFR
p55-/-/TNFR p75-/- mice were provided by
Immunex (21). Type I IL-1R-/- mice, type I
IL-1R-/-/TNFR p55-/- mice, and type I
IL-1R-/-/TNFR p75-/- mice were also
provided by Immunex (22). TNFR p55-/- and
Faslpr/lpr/TNFR p55-/- mice
were produced by Dr. Klaus Pfeffer (Munich, Germany) (20, 23).
MIP-1-/- mice were the gift of Dr. O. Smithies (Chapel
Hill, NC) (24). Mice deficient in the enzyme-inducible nitric oxide
synthase (iNOS-/-) were the gift of Dr. Ricardo
Gazzinelli (Bethesda, MD) (25). Mice deficient in the
IL-1ß-converting enzyme (ICE-/-) and
STAT1-/- mice were produced as described (26, 27). All
mice were housed in a specific pathogen-free environment and given food
and water ad libitum.
Analysis of cytokine-treated mice
Serum levels of IFN- and TNF- were measured using ELISAs
obtained from Endogen (Woburn, MA). IL-1ß and IL-6 levels were
measured using ELISAs obtained from Biosource International (Camarillo,
CA). KC and MIP-2 levels were measured using ELISAs from R & D Systems.
Serum chemistries were performed on mouse serum using a Vitros 500
analyzer (Johnson and Johnson, Raritan, NJ). Histopathologic
examination of cytokine-treated SCID mice and determination of mean
wet-to-dry lung ratios were performed as described (28). NK cells were
isolated from the spleens of cytokine-treated C.B-17 SCID mice and
analyzed for endonucleolytic cleavage of cellular DNA via a
flow-cytometric assay using propidium iodide and DNA gel
electrophoresis, as described (12, 29).
Measurement of acute phase proteins
Serially diluted serum was analyzed for haptoglobin and
1-acid glycoprotein by immunoelectrophoresis (30). The area
under the precipitation peak was quantitated in arbitrary units using
the National Institute of Health Image program 1.61. The data for each
peak were then converted into mg/ml values by comparison with the
values obtained with calibrated mouse acute phase plasma.
Inhibition of the NF-
B signaling pathways in mice receiving IL-2
and IL-12
An E1A-deleted recombinant adenovirus (rAd) expressing a
dominant-negative form of the I-B protein was constructed (31). A
rAd engineered to express the lacZ gene served as a control. A total of
109 PFUs of rAd/I-B or rAd/lacZ was administered to mice
via tail vein injection 48 h before treatment with IL-2 plus IL-12
(32). In vivo protein expression derived from these adenoviral vectors
was confirmed via immunohistochemistry, as described (7).
Statistical analysis
Statistical significance was analyzed by the Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Intraperitoneal administration of rhuIL-2 (3 x
105 U/day) plus rmuIL-12 (1 µg/day) was lethal to C57BL/6
mice within 4 to 6 days (Fig. 1
). The
combination of IL-15 plus IL-12 elicited identical results, which was
expected given that IL-15 signals through components of the IL-2R (6, 33). No deaths were observed in control mice receiving daily injections
of IL-2, IL-15, or IL-12 alone. Similar results were obtained with IL-2
or IL-15 plus IL-12 in several different species of mice, including
BALB/c, 129, B6 x 129, CD-1, and C.B-17 mice bearing the
scid/scid (SCID) mutation (data not shown). Indeed, SCID
mice, which lack B and T cells (34), exhibited 100% mortality within 3
to 5 days of the initiation of treatment and were utilized extensively
in the majority of experiments.
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SCID mice receiving IL-2, IL-12, or IL-2 plus IL-12 were subjected
to histopathologic evaluations. IL-2-associated findings included
mononuclear cell infiltrates in the portal areas of the liver, splenic
extramedullary hemopoiesis, and pulmonary interstitial mononuclear cell
infiltrates. IL-12-associated changes included mild hyperplastic and
degenerative changes of the gastrointestinal mucosa, and scattered foci
of apoptotic lymphoid cells within the lymphoid organs. These IL-2- and
IL-12-induced lesions were mild, of late onset (72–96 h), and
consistent with those previously described for these cytokines (35, 36). Changes in SCID mice treated with IL-2 plus IL-12 included both
significant exacerbation of IL-2- and IL-12-associated lesions as well
as novel changes such as fibrinoid necrosis of lymphoid tissue in the
spleen and lymph nodes, confluent foci of necrosis in pancreatic
exocrine tissue, macrophage activation and proliferation, and fibrinoid
necrosis of pulmonary arterioles. Apoptosis of SCID lymphocytes (i.e.,
NK cells) within the lymphoid tissues (spleen and lymph nodes) was
markedly enhanced (Fig. 2A).
Analysis of nonadherent splenic NK cells from cytokine-treated SCID
mice by DNA gel electrophoresis and propidium iodide staining (12)
confirmed this observation (Fig. 2B and data not shown).
Lesions of the gastrointestinal tract associated with IL-12
administration were also markedly exacerbated by the combination of
IL-2 and IL-12 (Fig. 2C). Pulmonary pathology was prominent
and included perivascular and septal mononuclear cell infiltrates
associated with multifocal hemorrhage and alveolar edema. The formation
of pulmonary edema at the 72-h time point was significantly greater in
mice receiving the combination of IL-2 plus IL-12
(p < 0.05, Fig. 2D) (28). Analysis
of serum chemistries revealed significant increases in the liver
enzymes ALT, AST, and LDH (5.3-, 9.9-, and 2.4-fold increases over
baseline, respectively) as well as acute phase proteins beginning
24–48 h after treatment with IL-2 plus IL-12, but not following the
administration of IL-2 or IL-12 alone (Fig. 2E) (37).
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Serum levels of IFN- and TNF- rose rapidly in SCID mice
treated with IL-2 plus IL-12, peaked at approximately 24 h, and
remained elevated until death (Fig. 3,
A and B). Anti-asialo GM1 Ab-treated SCID mice
did not exhibit elevated serum levels of IFN- or TNF- after
receiving IL-2 plus IL-12, suggesting that this cytokine combination
acted directly on NK cells to induce production of IFN- and TNF-
(not shown). Serum levels of IL-1ß, IL-6, KC, and MIP-2 were also
found to be elevated during combined administration of IL-2 with IL-12
(summarized in Fig. 3C). The elevated levels of
proinflammatory cytokines seen with the combination of IL-2 plus IL-12
were not the result of a simple additive effect, since administration
of IL-2 or IL-12 alone did not elicit significant cytokine production
in SCID mice (with the exception of IFN- production in mice
receiving IL-12). Of note, elevations in TNF- and IL-1ß occurred
early in the course of treatment (i.e., within 1–3 h), whereas peak
levels of IL-6, KC, and MIP-2 appeared later in the disease course, as
is observed in animal models of septic shock (Fig. 3, B and
C, and data not shown) (9, 38).
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, IFN-, and IL-1 in death induced by IL-2 plus
IL-12
We investigated the mechanism of this fatal cytokine-induced
inflammatory response treatment using cytokine neutralization
strategies and genetically altered mouse strains. Results are
summarized in Table I. Neutralization of
IFN- or TNF- did not afford protection to SCID mice treated with
IL-2 plus IL-12, nor did genetic deficiencies in IFN- or the TNFR
complex (p55 and/or p75). We noted somewhat prolonged survival within
both the control and experimental groups in our initial experiments
with TNFR p55-/- mice. However, in subsequent experiments
with the identical strain of TNFR p55-/- mouse and an
independently generated strain, we found that all background mice and
TNFR p55-/- mice died within 5 to 7 days (Table I and
data not shown). These data in conjunction with the results of our
TNF- neutralization experiments led us to conclude that TNF- was
not solely responsible for the toxicity seen in this model.
STAT1-/- mice were also susceptible to the toxicity of
IL-2 plus IL-12, which is significant because STAT1 is critical for
IFN--induced gene regulation and also for the induction of apoptosis
by TNF- (39, 40). IFN- and TNF- were simultaneously
neutralized using both an anti-IFN- mAb and a TNFR-Fc soluble
receptor construct (14, 41). Mortality rates of the experimental and
control groups were identical. TNF- was neutralized in
IFN--/- mice, and IFN- was neutralized in TNFR
p55-/-/TNFR p75-/- mice, without effect.
Type I IL-1R-/- mice, IL-1R-/-/TNFR
p55-/- mice, and IL-1R-/-/TNFR
p75-/- mice all succumbed to the lethal effects of IL-2
plus IL-12, whereas mice receiving IL-2 alone or IL-12 alone exhibited
minimal toxicity (not shown). In addition, IL-1R-/-/TNFR
p55-/- mice were treated with a neutralizing Ab to
muIFN- during treatment with IL-2 plus IL-12, yet this intervention
did not ameliorate toxicity or prolong survival (not shown). Several
alternative effector molecules were considered as possible mediators of
toxicity; however, mice with targeted genetic deficiencies in MIP-1,
ICE, perforin, Fas, and iNOS remained completely susceptible to the
toxic effects of IL-2 plus IL-12.
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Dexamethasone, TGF-ß1, and ibuprofen have been used effectively
to prevent morbidity and death in animal models of septic shock and
other inflammatory processes (42, 43, 44). These agents were administered
in pharmacologically relevant doses before the start of cytokine
treatment and then daily thereafter; however, none was capable of
ameliorating the toxicity of IL-2 plus IL-12 (Table II). Inhibition of NF-B signaling in
vivo via overexpression of I-B in the liver was also ineffective in
preventing mortality in this model, in contrast to its ability to
protect mice from the lethal effects of endotoxin (45). Indeed, mice
expressing the I-B protein were actually more susceptible to the
toxic effects of IL-2 plus IL-12 than were mice treated with the
control vector (p < 0.05, Table II). This
experiment was repeated in TNFR p55-/- mice because
of the role I-B has in potentiating TNF-mediated apoptosis (31, 46)
(M. Karin, unpublished observation). The toxicity of IL-2 plus
IL-12 in TNFR p55-/- mice overexpressing I-B was
essentially identical in the experimental and control groups (Table II).
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SCID mice that lack T and B lymphocytes undergo massive NK cell
apoptosis following administration of IL-2 plus IL-12 (Fig. 2
, A and B). To determine whether the toxicity of
this model was mediated by NK cells, we administered IL-2 plus IL-12 to
SCID mice depleted of NK cells by pretreatment with an anti-asialo
GM1 Ab (Fig. 4A). IL-2 (or
IL-15) plus IL-12 elicited minimal toxicity when administered to SCID
mice depleted of NK cells, and 100% of mice in this group survived,
whereas control mice receiving IL-2 plus IL-12 all died within 5 days
of the initiation of treatment (Fig. 5A). To confirm this
observation, we administered IL-2 and IL-12 to SCID mice that had been
depleted of NK cells by splenectomy and observed 100% survival (47).
Sham-operated SCID mice treated with IL-2 plus IL-12
exhibited 100% mortality at 5 days (Fig. 5B). Furthermore,
CD3 transgenic mice that completely lack mature NK cells (and T
cells) due to a developmental block (18) showed absolutely no
toxicity when treated with IL-2 plus IL-12. Control mice of the
appropriate background all died between 4 and 8 days (Fig. 5C).
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Given the ability of NK cell-derived cytokines to potentiate
macrophage effector functions (1, 2, 3, 48), the role of macrophages in
the toxicity of this model was investigated. SCID mice were depleted of
monocytes and macrophages by approximately 50% in the peripheral
blood, spleen, and bone marrow, and by >95% in the peritoneal cavity
by injecting the F4/80 mAb (16) via the i.v. and i.p. routes 48 and
24 h before the administration of IL-2 plus IL-12 (Fig. 4B). Mice receiving the F4/80 mAb tolerated the
administration of IL-2 plus IL-12 significantly better than mice
receiving the control Ab and exhibited a 50% survival rate
(p < 0.05). Control mice all died within 3 to
4 days (Fig. 5D). IL-10 is a potent macrophage deactivator
(49). Pretreatment of SCID mice with rmuIL-10 afforded significant
protection from the toxicity of IL-2 plus IL-12 (66% survival overall,
p < 0.02, Table II). Taken together, these data
suggest that monocytes/macrophages, in addition to NK cells, have a
role in mediating the lethal toxicity of IL-2 plus IL-12.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and TNF-, and production of these
cytokines was dependent upon the presence of NK cells. However, neither
IFN- nor TNF- was required for the fatal inflammatory reaction
induced by IL-2 plus IL-12. Other effector molecules of the NK cell and
macrophage compartments, namely MIP-1, IL-1, ICE, Fas, perforin,
iNOS, and the STAT1 pathway of signal transduction, were eliminated as
critical mediators of toxicity in this model.
Our histopathologic findings and analysis of serum cytokine levels
indicated that the administration of IL-2 plus IL-12 had induced a
severe systemic inflammatory response. High circulating levels of
IL-1ß, IL-6, and the IL-8 homologues (KC and MIP-2) appeared only in
the serum of mice receiving the combination of IL-2 plus IL-12, which
is significant in that the production of these cytokines is limited to
periods of inflammation, tissue injury, and immunologic challenge (38).
Indeed, the sequence of cytokine induction was highly reminiscent of
that observed in experimental models of endotoxemia and in humans
diagnosed with septic shock (9, 38). The presence of high serum levels
of IL-6 at the time of death is particularly significant in that they
represent the net effect of biologically active IL-1ß and TNF-,
and have been found to correlate inversely with survival in patients
with septic shock (50). Thus, treatment of mice with IL-2 plus IL-12
induces many of the same proinflammatory mediators that are active in
sepsis. The presence of increased pulmonary edema, multiple organ
system toxicities, and an acute phase response in mice receiving the
combination of IL-2 and IL-12 lends additional support to this
observation (9, 51). Induction of proinflammatory mediators has also
been implicated in the pathogenesis of the shocklike states associated
with high dose cytokine therapy in humans, and we initially
hypothesized that the lethal reaction to IL-2 plus IL-12 might be the
result of additive toxicities induced by the overlapping actions of
known proinflammatory factors (52, 53). However, no protection was
afforded by simultaneous neutralization of TNF- and IFN- or
utilization of IL-1R-/-/TNFR p55-/- and
IL-1R-/-/TNFR p75-/- mice. The data also
suggest that the fatal toxicity of IL-2 plus IL-12 was not mediated via
NF-B signaling within the liver; however, it is possible that
NF-B was only partially inhibited and therefore still able to
activate the transcription of NF-B-responsive genes during this
intense inflammatory response (45). We cannot exclude the possibility
that other cytokines, cytokine receptors, or effector molecules might
combine with IL-1, TNF-, or IFN- following administration of IL-2
plus IL-12 to induce a lethal inflammatory response. However, the data
suggest that stimulation of the NK cell compartment with IL-2 plus
IL-12 might result in the production of novel factors or factors that
have yet to be characterized as proinflammatory. Such a
hypothesis is supported by our characterization of several unique
pathologic lesions that have not been observed in other models of shock
(9, 51).
The appearance of IL-1ß, IL-6, and other macrophage-derived cytokines
in the circulation, and the presence of activated and proliferating
macrophages in the splenic bed following the administration of IL-2
plus IL-12 implied that this treatment had directly or indirectly
activated the macrophage compartment (54). The importance of
macrophages in the toxicity of this model was confirmed by studies in
which partial depletion of the macrophage compartment resulted in a
50% survival rate for mice receiving IL-2 plus IL-12. The protection
afforded by IL-10 pretreatments also suggested a role for the
macrophage compartment in the toxicity of this model; however, we
cannot rule out the possibility that IL-10 may exert its protective
effects via some other pathway (49). The prevention of death by the
congenital absence or depletion of NK cells and the improved survival
by the partial depletion of macrophages suggested that the toxicity of
this model is the result of interactions between these two cell
populations. NK cells constitutively express the IL-2/15R, and
activation of this receptor complex results in further up-regulation of
NK cell IL-12R expression (55). Thus, we suspect that the toxicity of
this model first involves stimulation of NK cells by IL-2/15 plus
IL-12, followed by an NK cell-dependent activation of macrophages. The
inability of IL-2 plus IL-12 administration to induce TNF- in
NK-depleted SCID mice also supports our proposed sequence of events, as
NK cells and macrophages are both sources of this cytokine. Thus,
mobilization of macrophage effector function may occur as a result of
continuous and uncontrolled production of NK cell factors in response
to IL-2 plus IL-12 (2), although the precise cellular source of these
proinflammatory cytokines cannot be determined based upon measurements
of serum cytokines. Our postulate that chronic stimulation of the NK
cell compartment via daily injection of IL-2 plus IL-12 results in
macrophage activation is not an unprecedented one (1, 2, 56, 57).
However, the inability of IFN- and TNF- neutralization strategies
to prevent death was unexpected and suggests that NK cells may be able
to activate macrophage effector functions via alternate
pathways.
To the best of our knowledge, the present model represents the
first example of a cytokine-induced shock syndrome that is mediated by
components of the innate immune system. Our current understanding of
the role of the NK cell in other models of shock is quite limited, but
there is evidence that NK cells can be activated during septic events
and may contribute to the pathogenesis of this condition via the
secretion of IFN-, which acts primarily to augment macrophage
function. One well-studied example of this phenomena is the Shwartzman
reaction in which an intradermal priming dose of LPS is followed
24 h later by an i.v. LPS challenge (58). The first LPS dose
induces the production of IL-12, which stimulates the release of
IFN- (presumably by NK cells) and permits the priming of
macrophages. Upon subsequent LPS challenge, sensitized macrophages
release massive amounts of TNF- and IL-1ß, which mediate the
lethal effects of this treatment (58). Heremans et al. have
demonstrated that depletion of NK cells before the induction of the
generalized Shwartzman reaction leads to a 70% reduction in mortality
and significantly lower levels of IFN- and TNF- following the
systemic injection of LPS (59). In contrast, mice depleted of
CD4+ or CD8+ T cells were still highly
susceptible to the lethal effects of the Shwartzman reaction. Ozmen et
al. analyzed the Shwartzman reaction in detail and found that simple
coinjection of TNF- plus IL-1ß or TNF- plus IFN- was
sufficient to induce lethality following priming of mice with IL-12 or
IFN- alone (60). However, regardless of the priming event, no
mortality was observed if IL-1ß and IFN- were administered in the
absence of TNF-. Thus, in distinct contrast to our model, TNF- is
crucial for the lethality of the Shwartzman reaction. Furthermore, our
experiments with IFN--/- and STAT1-/-
mice would suggest that IFN- priming (following IL-12
administration) is not a critical event in our model.
The induction of NK cell apoptosis in the present model was dependent
upon the coadministration of IL-2 with IL-12. Nonadherent splenocytes
from cytokine-treated SCID mice did not exhibit endonucleosomal DNA
degradation or the morphologic features of apoptosis unless mice had
received both cytokines, results that were confirmed in vitro (not
shown). Apoptosis of NK cells has also been observed following
coactivation with IL-2 and engagement of the low affinity FcR (or
cross-linking of CD94), and after cell-mediated lysis of leukemic
targets cells in the presence of IL-2 (61, 62, 63, 64). We have demonstrated
previously that in vitro stimulation of resting human NK cells with
IL-2/15 and IL-12 leads to the production of large amounts of IFN-
and TNF-, followed by the induction of programmed cell death at
approximately 48–72 h (12). Activation-induced apoptosis appears to be
a common strategy for the removal of activated effector cells and
attenuation of the immune response to specific pathogens (65). The
ability of IL-2 plus IL-12 to induce NK cell apoptosis in vivo is
further evidence for the central role of the NK cell compartment in the
toxicity of this treatment. The existence of this suicide pathway
following NK cell activation further suggests that unbridled
amplification of NK cell effector functions may be deleterious to the
organism. This apoptotic regulatory mechanism was apparently
insufficient to limit toxicity in the present model, possibly due to
the intensity and rapidity of the NK cell response to administration of
IL-2/15 plus IL-12. Activation-induced apoptosis associated with the
induction of a shocklike state has also been described following
experimental activation of T cells with staphylococcal enterotoxins
(superantigens) or anti-CD3 Ab (66, 67). However, in contrast to
our model, the toxicity of these T cell-mediated shock syndromes is
mediated primarily by TNF-.
In summary, we have demonstrated that the administration of IL-2 or IL-15 in combination with IL-12 results in a fatal systemic inflammatory response that is critically dependent upon the NK cell compartment. This lethal shocklike reaction does not appear to be mediated by any of the known cytokine products of the NK cell compartment, nor any of the effector molecules associated with NK cell cytotoxic activity. This is the first time that the NK cell (or any cellular compartment) has been identified as an important component of cytokine-induced systemic inflammation. Elucidation of the factors involved in this novel inflammatory pathway may therefore have relevance for understanding the complications of high dose cytokine therapy as well as other forms of shock.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. William E. Carson III, Arthur G. James Comprehensive Cancer Center, Ohio State University, N924 Doan Hall, 410 W. 10th Street, Columbus, OH 43210. E-mail address:
3 Abbreviations used in this paper: MIP, macrophage-inflammatory protein; hu, human; I-B, inhibitor of NF-B; ICE, IL-1ß-converting enzyme; iNOS, inducible nitric oxide synthase; KC, ??; mu, murine; PE, phycoerythrin; rAd, recombinant adenovirus.
Received for publication October 13, 1998. Accepted for publication January 13, 1999.
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References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
in defense against murine cytomegalovirus infection and enhancement of this defense pathway by interleukin 12 administration. J. Exp. Med. 182:1045.-inducing factor in enhanced production of IFN-. J. Immunol. 159:2125. production by NK cell stimulatory factor (NKSF): characterization of the responder cells and synergy with other inducers. J. Exp. Med. 173:869. by natural killer cells in vitro. J. Clin. Invest. 96:2578.
in response to monocyte-derived cytokines. J. Clin. Invest. 97:2722.[Medline]
genes. Science 259:1739. for an inflammatory response to viral infection. Science 269:1583.B prevents apoptosis and liver dysfunction during liver regeneration. J. Clin. Invest. 101:802.[Medline]
chain of the IL-2 receptor. EMBO J. 14:3654.[Medline]
-induced apoptosis in STAT1-null cells due to low constitutive levels of caspases. Science 278:1630. interferon modulation determines the outcome of infection. Infect. Immun. 65:4761.[Abstract]
B in the mortality of sepsis. J. Clin. Oncol. 100:972.
B. Science 274:784. production. Blood 90:2541. by an intracellular parasite and induces resistance in T-cell-deficient hosts. Proc. Natl. Acad. Sci. USA 90:6115., a mediator of lethal lipopolysaccharide-induced Shwartzman-like shock reactions in mice. J. Exp. Med. 171:1853., and tumor necrosis factor are the key cytokines of the generalized Shwartzman reaction. J. Exp. Med. 180:907.This article has been cited by other articles:
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  M. C. Rodriguez-Galan, D. Reynolds, S. G. Correa, P. Iribarren, M. Watanabe, and H. A. Young Coexpression of IL-18 Strongly Attenuates IL-12-Induced Systemic Toxicity through a Rapid Induction of IL-10 without Affecting its Antitumor Capacity J. Immunol., July 1, 2009; 183(1): 740 - 748. [Abstract] [Full Text] [PDF]
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A. L. Coelho, M. A. Schaller, C. F. Benjamim, A. Z. Orlofsky, C. M. Hogaboam, and S. L. Kunkel The Chemokine CCL6 Promotes Innate Immunity via Immune Cell Activation and Recruitment J. Immunol., October 15, 2007; 179(8): 5474 - 5482. [Abstract] [Full Text] [PDF]
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 M. Gendelman, N. Halligan, R. Komorowski, B. Logan, W. J. Murphy, B. R. Blazar, K. A. Pritchard Jr, and W. R. Drobyski Alpha phenyl-tert-butyl nitrone (PBN) protects syngeneic marrow transplant recipients from the lethal cytokine syndrome occurring after agonistic CD40 antibody administration Blood, January 1, 2005; 105(1): 428 - 431. [Abstract] [Full Text] [PDF]
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G. B. Lesinski, B. Badgwell, J. Zimmerer, T. Crespin, Y. Hu, G. Abood, and W. E. Carson III IL-12 Pretreatments Enhance IFN-{alpha}-Induced Janus Kinase-STAT Signaling and Potentiate the Antitumor Effects of IFN-{alpha} in a Murine Model of Malignant Melanoma J. Immunol., June 15, 2004; 172(12): 7368 - 7376. [Abstract] [Full Text] [PDF]
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 U. Deiters, M. Gumenscheimer, C. Galanos, and P. F. Muhlradt Toll-Like Receptor 2- and 6-Mediated Stimulation by Macrophage-Activating Lipopeptide 2 Induces Lipopolysaccharide (LPS) Cross Tolerance in Mice, Which Results in Protection from Tumor Necrosis Factor Alpha but in Only Partial Protection from Lethal LPS Doses Infect. Immun., August 1, 2003; 71(8): 4456 - 4462. [Abstract] [Full Text] [PDF]
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M. Okamoto, S. Kato, K. Oizumi, M. Kinoshita, Y. Inoue, K. Hoshino, S. Akira, A. N. J. Mckenzie, H. A. Young, and T. Hoshino Interleukin 18 (IL-18) in synergy with IL-2 induces lethal lung injury in mice: a potential role for cytokines, chemokines, and natural killer cells in the pathogenesis of interstitial pneumonia Blood, February 15, 2002; 99(4): 1289 - 1298. [Abstract] [Full Text] [PDF]
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 C. J. Godshall, A. B. Lentsch, J. C. Peyton, M. J. Scott, and W. G. Cheadle STAT4 Is Required for Antibacterial Defense but Enhances Mortality during Polymicrobial Sepsis Clin. Vaccine Immunol., November 1, 2001; 8(6): 1044 - 1048. [Abstract] [Full Text] [PDF]
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S. Zeerleder, C. E. Hack, C. Caliezi, R. Hebeisen, and W. A. Wuillemin Bacillus Calmette-Guerin sepsis: shift of an intended local toward a detrimental systemic cytotoxic immune response Blood, August 1, 2001; 98(3): 890 - 891. [Full Text] [PDF]
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T. A. Fehniger and M. A. Caligiuri Interleukin 15: biology and relevance to human disease Blood, January 1, 2001; 97(1): 14 - 32. [Full Text] [PDF]
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T. Hussell and P. J. M. Openshaw IL-12-Activated NK Cells Reduce Lung Eosinophilia to the Attachment Protein of Respiratory Syncytial Virus But Do Not Enhance the Severity of Illness in CD8 T Cell-Immunodeficient Conditions J. Immunol., December 15, 2000; 165(12): 7109 - 7115. [Abstract] [Full Text] [PDF]
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W. E. Carson, J. E. Dierksheide, S. Jabbour, M. Anghelina, P. Bouchard, G. Ku, H. Yu, H. Baumann, M. H. Shah, M. A. Cooper, et al. Coadministration of interleukin-18 and interleukin-12 induces a fatal inflammatory response in mice: critical role of natural killer cell interferon-gamma production and STAT-mediated signal transduction Blood, August 15, 2000; 96(4): 1465 - 1473. [Abstract] [Full Text] [PDF]
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K. S. Wang, D. A. Frank, and J. Ritz Interleukin-2 enhances the response of natural killer cells to interleukin-12 through up-regulation of the interleukin-12 receptor and STAT4 Blood, May 15, 2000; 95(10): 3183 - 3190. [Abstract] [Full Text] [PDF]
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