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*
Immunex Corporation, Seattle WA 98101; and
Department of Molecular Microbiology and Immunology, Saint Louis University, St. Louis, MO 63104
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and NK cell cytotoxicity, making it a logical
target for viral antagonism of host defense. We demonstrate that the
ectromelia poxvirus p13 protein, bearing homology to the mammalian
IL-18 binding protein, binds IL-18, and inhibits its activity in vitro.
Binding of IL-18 to the viral p13 protein was compared with binding to
the cellular IL-18R. The dissociation constant of p13 for murine IL-18
is 5 nM, compared with 0.2 nM for the cellular receptor heterodimer.
Mice infected with a p13 deletion mutant of ectromelia virus had
elevated cytotoxicity for YAC-1 tumor cell targets compared with
control animals. Additionally, the p13 deletion mutant virus exhibited
decreased levels of infectivity. Our data suggest that inactivation of
IL-18, and subsequent impairment of NK cell cytotoxicity, may be one
mechanism by which ectromelia evades the host immune
response. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production, induces NK cell cytotoxicity, and
participates in the polarization of the T-lymphocyte Th1 phenotype
(1, 2). IL-18 was originally identified as IFN-
inducing factor, purified from the livers of mice following induction
of toxic shock (3). LPS-induced liver damage is in fact
prevented upon inhibition of IL-18 by neutralizing Abs (3)
or in IL-18-deficient mice (4). IL-18 has been shown to
increase IFN- production in Ag-stimulated T cell lines, and to act
synergistically with IL-12 to stimulate IFN- production in Th1
clones (5, 6). In NK cells, IL-18 enhances IFN-
production (7, 8) and stimulates cytotoxicity
(3). IL-18-deficient mice display reduced NK cell killing
activity (4) and treatment of wild-type
(WT)4 mice with
exogenous IL-18 augments NK cell cytotoxicity in vitro and in vivo
(4, 9, 10). IL-18 is structurally related to the IL-1 family of cytokines (11) and has been shown to be processed by the IL-1ß-converting enzyme (12, 13). Furthermore, the IL-18R is related to the IL-1 family of receptors, being composed of a ligand-binding subunit, IL-1Rrp1 (14, 15) and an accessory subunit, AcPL (16), both of which share sequence homology to the IL-1R family. It has been proposed that synergy observed between IL-12 and IL-18 may be due to the induction of IL-18R expression by IL-12 in B and T cells (17, 18).
A soluble protein has recently been described which binds and inhibits IL-18, yet bears no significant homology to either IL-18R subunit (19). The IL-18 binding protein (IL-18BP) contains a single putative Ig domain. This Ig domain bears very limited homology to the third Ig domain of the type II IL-1R. Much greater homology to IL-18BP can be found in a family of proteins (p13/p16) encoded by several poxviruses (swinepox, cowpox, variola, molluscum contagiosum, and ectromelia) (19, 20).
Poxviruses infect the host mainly through the cornified epithelium of the skin or the mucosal surface of the respiratory tract. The skin probably becomes infected through microscopic abrasions, which allows the virus access to the epidermal and dermal layers. In these layers the poxvirus replicates locally, and most species of virus rapidly spread to the draining lymph node via the lymphatics, and possibly also via infected cells (21). Some poxviruses, such as molluscum contagiosum, cause a localized, self-limited infection. Others cause a fulminant, systemic infection characterized by a generalized rash and high mortality rate, as with variola virus (smallpox), human monkeypox, or ectromelia virus (EV) (mousepox). In either case, virus transmission usually originates from infection in the epithelia and persists in the face of circulating systemic immunity (21).
Poxviruses often acquire cellular proteins critical for immune regulation, thus allowing them to escape host responses (22). The first such protein identified was encoded by the T2 open reading frame (ORF) of Shope fibroma poxvirus. This ORF produced a secreted soluble form of the type II TNF receptor (23) which, when deleted in myxoma poxvirus, reduced virulence by nearly 70% (24). Indeed, many of the viral immune regulators subsequently identified represent soluble versions of cellular cytokine receptors (i.e., type II IL-1, type II TNF, and type I and type II IFN receptors), although some, such as the secreted p35 protein from variola and cowpox viruses, bind and neutralize their target molecules (ß-chemokines) despite being structurally unrelated to the cellular receptor (25, 26, 27, 28).
We have examined the capacity of the EV homologue within the p13/p16 family (subsequently referred to as p13) to bind IL-18 and inhibit its function both in vitro and in vivo. We show that p13 binds IL-18 with nanomolar avidity, that p13 inhibits the binding of IL-18 to its cellular receptor, and that it inhibits the biological activity of IL-18 in vitro. Intraperitoneal infection of mice with a WT vs a p13 knockout form of EV indicates that p13 down-regulates NK cell cytotoxic activation in vivo. Furthermore, deletion of p13 results in decreased viral infectivity in the liver.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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EV p13 ORF has been described (20). It was
amplified by PCR and subcloned into pDC409 (29) as a
fusion with the CH2 and CH3 domains of human IgG1 (30).
Expression vectors encoding murine (m)IL-1Rrp1, mAcPL, soluble
(s)IL-1Rrp1-Fc, and sAcPL-Fc have been described previously
(16). Plasmids encoding fusions with FlagpolyHis contain
the same gene-specific regions as the above described plasmids, but the
mutein Fc has been replaced with the peptide RSDYKDDDDKPGHHHHHHPG. The
luciferase reporter plasmid containing three multimerized NF-
B sites
and a minimal c-Fos promoter has been described previously
(31). The murine IL-18 sequence was cloned into pDC206
(3, 32).
Following transient transfection of CV-1/EBNA cells by the DEAE-dextran method (33), fusion proteins were purified from culture supernatants by chromatography on protein A-Poros or Nickel columns (PerSeptive Biosystems, Framingham, MA). Purity and relative amounts of each Fc protein were assessed by PAGE, and purity was consistently found to be >98%. Furthermore, the N-terminal sequence was determined after each independent purification, to verify the identity of the purified protein. Mixed heterodimeric proteins yielded sIL-1Rrp1 sequence, presumably due to the observation that sAcPL-Fc was expressed at significantly lower levels than sIL-1Rrp1-Fc. Protein was quantitated by amino acid analysis and by the BCA method (Pierce, Rockford, IL).
Cell culture and transfection
COS-7 and BS-C-1 cells were maintained in DMEM/5% and 10% FBS,
respectively. To assess inhibition of NF-B activation, COS-7 cells
were transiently transfected by the DEAE for consistency dextran method
with 10 ng of each receptor plasmid (mIL-1Rrp1 and mAcPL) and 50 ng of
the reporter plasmid per 4.5 x 104 cells.
The total amount of DNA in the transfection was increased to 1
µg/well by addition of empty pDC304 vector. Two days
posttransfection, mIL-18 (PeproTech, Rocky Hill, NJ) (20 ng/ml final
concentration) was preincubated with varying amounts of Fc fusion
protein (3–12 µg/ml final concentration) for 15 min at room
temperature. Transfected cells were stimulated with the pretreated
mIL-18 for 4 h, at which time cells were lysed and luciferase
activity assessed using Reporter Lysis Buffer and Luciferase Assay
Reagent (Promega, Madison, WI).
Immunoprecipitation of [35S]IL-18
COS-7 cells (4.5 x 104) were transiently transfected by the DEAE-dextran method with an empty expression plasmid, or the same plasmid encoding mIL-18 (1 µg each). Two days posttransfection, cells were starved for 1 h then labeled in [35S]Cys-[35S]Met containing medium for 4 h. The supernatants were removed, subjected to centrifugation, and adjusted to 0.4 M NaCl/1.0% Triton X-100 in the presence of protease inhibitors. Fc fusion protein (1 µg) was added to the supernatants along with 30 µl of a 50% slurry of protein G-Sepharose (Boehringer Mannheim, Indianapolis, IN). As indicated, various amounts of FlagpolyHis fusion proteins were also included in the immunoprecipitations. Following precipitation overnight at 4°C, immunocomplexes were washed extensively in buffer (0.4 M NaCl, 0.05% SDS, and 1.0% Nonidet P-40) and then separated by electrophoresis in a 4–20% Tris-Glycine gel (Novex, San Diego, CA). The gel was fixed, incubated in Amplify (Amersham Pharmacia Biotech, Piscataway, NJ), dried, and exposed to X-OMAT AR film (Eastman Kodak, Rochester, NY).
BIAcore binding
Binding of mIL-18 to the Fc fusion proteins was evaluated using the BIAcore 3000 (Pharmacia Biacore, Uppsula, Sweden). All binding experiments were performed at 25°C. Goat anti-human IgG (Jackson ImmunoResearch, West Grove, PA) was immobilized to a CM5 BIAcore sensor chip via amine coupling at pH 4.8 as previously described (34). The Fc proteins were then loaded onto the surface at equimolar levels of protein. Various levels of mIL-18 (0.15 nM to 1 µM) in 3-fold serial dilutions were then run over the flow cells for 6 min at 10 µl/min and then allowed to dissociate for 20 min. Between binding cycles the flow cell was regenerated using 100 mM glycine (pH 1.65) plus 0.15 M NaCl, then reloaded with Fc protein before addition of the next higher concentration of mIL-18. Before evaluation, a background curve of the same concentration of mIL-18 run over a surface coated with capture Ab only was subtracted. Data was evaluated using the BIAcore BIA Evaluation 3.0.2 software (Pharmacia Biacore). All data obtained were utilized in the data evaluation except in the case of p13-Fc, where we only used the data from 0.15 to 111 nM to determine the binding constant.
Construction of EV-p13-
The p13 gene in pL28-5 (20) was inactivated by the insertion of a 2-kbp cassette containing the Escherichia coli guanine phosphoribosyltransferase (gpt) gene under the control of the vaccinia virus 7.5 K promoter (20). The mutated gene was transfected into CV-1 cells coinfected with WT EV, and a recombinant mutant virus (EV-gpt+, p13-; hereafter EV-p13-) was selected by three plaque purifications in the presence of 25 µg/ml of mycophenolic acid, 250 µg/ml xanthine, and 15 µg/ml hypoxanthine. The genotype of EV-p13- was verified by PCR using combinations of primers 5' and 3' to the p13 gene and the selection cassette. The mutant virus was expanded in BS-C-1 cells (35) and stored as a cell lysate at -70°C.
Animals
Specific-pathogen-free, female, C57BL/6 mice (Charles River Laboratories, Wilmington, MA) were procured through the National Cancer Institute (Frederick, MD) and used at 6–8 wk of age. Mice were treated in accordance with institutional policies and the Guide for Care and Use of Laboratory Animals.
Virus infection
Groups of mice were injected i.p with 1 x 104 PFU of EV-WT, EV-p13-, or saline. At 1, 2, or 3 days postinfection (p.i.) mice were euthanized, the peritoneal cavities were washed with a total of 8 ml of HBSS, and the spleens and livers were removed. Peritoneal exudate cells (PECs) were pooled, washed, and enumerated. Splenocytes from two mice per group were pooled, and the mononuclear fraction was isolated using Lymphocyte Separation Medium (ICN Biomedicals, Costa Mesa, CA). Virus infectivity was determined as described previously (36).
Cytotoxicity assays
Standard 51Cr-release assays to measure NK cytotoxic activity in splenocyte and PEC populations were performed as described in detail elsewhere (37), except that target cells were labeled for 2 h and 1 x 104 cells were added per well. In vitro depletion of CD8+ or NK cells from PECs before use in the 51Cr-release assay was conducted by incubation for 30 min at 37°C with a mAb specific for CD8 (clone 2.43; rat IgG2b), or by sequential incubation of cells with anti-CD8 and anti-asialo-GM1 (Wako Pure Chemicals, Richmond, VA) and human complement (adsorbed on mouse splenocytes before use). An aliquot was analyzed by flow cytometry to assess efficacy of depletion. Cells were added to target without adjustment for reduction in cell number.
Flow cytometry
PECs were incubated with Fc block (PharMingen, San Diego, CA) for 15 min at 4°C, then aliquots were stained with FITC-conjugated anti-NK1.1 (clone PK136, mouse IgG2a; PharMingen), FITC-anti-CD4 (clone RM2501, rat IgG2a; Caltag Laboratories, Burlingame, CA), or FITC-anti-CD8 (clone CT-CD8a, rat IgG2a; Caltag Laboratories) at 0.5 µg/106 cells in FACS buffer for 30 min at 4°C. A total of 7,000–10,000 cells were analyzed by flow cytometry using a FACSCalibur flow cytometer and CellQuest analysis software (Becton Dickinson, San Jose, CA).
Measurement of IFN- and IL-10 cytokines
Cultures of 1 x 106 splenocytes or
PECs were incubated in 96-well plates for 5.5 h in RPMI plus 2%
FCS with 50 µg/ml PMA and 500 ng/ml calcium ionophore. Culture
supernatants were harvested and frozen until cytokines were assayed.
IFN- and IL-10 were detected by a sandwich ELISA protocol using an
anti-IFN- mAb (clone 37801.11; R&D Systems, Minneapolis, MN) and
an anti-IL-10 mAb (JES5-2A5; PharMingen) as capture reagents. Bound
IFN- and IL-10 were detected by addition of biotinylated goat
anti-mouse IFN- polyclonal Ab (R&D Systems) and biotinylated rat
anti-mouse IL-10 mAb (SXC-1, PharMingen), respectively, followed by
streptavidin-HRP (Zymed Laboratories, South San Francisco, CA) and the
substrate o-Phenylenediamine. The optical density was
measured at 490 nm and was converted to ng/ml using sets of IFN- and
IL-10 standards. Each value is a geometric mean of cultures from three
individual mice. Statistical significance was determined using the
unpaired t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The recently reported IL-18BP displays extremely limited similarity to the third putative Ig domain of the ligand binding subunit of the IL-18R, IL-1Rrp1 (19). IL-18BP shows higher homology to the EV p13 protein: 23.7% identity over 118 aa compared with 16.2% identity over 105 aa with IL-1Rrp1 (T. L. Born, unpublished observation). Therefore, we examined whether EV p13 was able to bind IL-18, and compared this binding with the binding of IL-18 to its cellular receptor.
It has been shown that the extracellular domain of IL-1Rrp1
(sIL-1Rrp1-Fc) is able to bind IL-18, whereas the extracellular
region of the accessory subunit of the IL-18R (sAcPL-Fc) is
unable to bind IL-18 under the same conditions (14, 15, 16).
Dimerization of the extracellular regions of IL-1Rrp1 and AcPL, as
demonstrated by coexpression of the soluble Fc fusion proteins in the
same cell, creates a higher avidity IL-18R protein (T. L. Born and
J. E. Sims, unpublished observation). Protein purified from the
supernatant of cells cotransfected with sIL-1Rrp1-Fc and sAcPL-Fc
contains, theoretically, 25% sIL-1Rrp1-Fc homodimers, 25% sAcPL-Fc
homodimers, and 50% Fc heterodimers. To investigate the ability of
IL-18 to bind these receptor proteins or the EV p13 protein, we
incubated radiolabeled mIL-18 with equal amounts of sIL-1Rrp1-Fc,
heterogeneous sIL-1Rrp1-Fc + sAcPL-Fc, or p13-Fc. Precipitation of the
Fc proteins using protein G-Sepharose revealed that the mixed
heterodimeric receptor and the p13 protein bound similar amounts of
IL-18, whereas a lesser amount of IL-18 was bound by the IL-1Rrp1
homodimer (Fig. 1
). We observed binding
of p13-Fc to both murine and human IL-18, whereas receptor homodimers
and heterodimers both failed to display cross-species binding (data not
shown). The mammalian IL-18BPs are similarly able to bind either human
or murine IL-18 (19). In summary, under the conditions
tested, the avidity of IL-18 for p13 and for the receptor heterodimer
appears to be higher than its avidity for IL-1Rrp1 alone. Specificity
was demonstrated in that type I sIL-1R-Fc did not bind IL-18 under the
same conditions (Fig. 1).
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Analysis of the data over the entire range of IL-18 concentrations
tested demonstrates that sIL-1R-rp1-Fc binds to mIL-18 with a
dissociation constant of 39 nM (
2 = 0.897)
(Table I). This avidity is in agreement
with the affinity reported previously for both soluble and full-length
IL-1Rrp1 (14, 15).
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20% above baseline following removal
of IL-18 (Fig. 2
), suggesting a slower dissociation rate of the
cytokine from the receptor heterodimer. This result is consistent with
the immunoprecipitation results reported above. The data were fit with
a heterogeneous ligand/parallel reaction model
(2 = 1.16) because the Fc proteins represent a
mixture of receptor heterodimers and homodimers. A portion of the
binding was of an avidity similar to that observed for sIL-1Rrp1-Fc (51
nM), whereas the other binding component displayed a dissociation
constant of 0.2 nM (Table I). We interpret this latter result to
represent binding to receptor heterodimers in the heterogeneous
preparation. Thus, heterodimerization of the receptor results in an
100-fold increase in avidity for mIL-18.
The flow cell coated with p13-Fc generally displayed a greater overall
RRU shift in response to IL-18 as compared with either of the
receptor-Fc proteins. Moreover, both the association and dissociation
of IL-18 from p13-Fc were much slower than was observed with the
receptor homodimer or heterodimer (Fig. 2). Analysis of the data over
the range of 0.15–111 nM mIL-18 indicates that p13-Fc has a
dissociation constant of 5 nM (2 = 1.73)(Table I). This avidity is intermediate between the avidities of IL-18 for
sIL-1Rrp1-Fc and for the putative heterodimeric portion of sIL-1Rrp1-Fc
+ sAcPL-Fc.
As a control, IL-1ß was run over flow cells coated with the same proteins and no shift was observed (data not shown). Taken together, these results suggest that mIL-18 has a higher avidity for the heterodimeric IL-1Rrp1 + AcPL than it does for IL-1Rrp1 or EV p13.
EV p13 inhibits the binding of IL-18 to its receptor
Although the mammalian IL-18BP was shown to inhibit the activity
of IL-18 (19), it was never formally shown that binding of
IL-18 to IL-18BP prevents binding to the cellular receptor. It is
possible that p13 binds IL-18 at a site distinct from that of cellular
receptor binding. To address this question, radiolabeled IL-18 was
bound to sIL-1Rrp1-Fc or to sIL-1Rrp1-Fc + sAcPL-Fc in the presence of
increasing amounts of p13-FlagpolyHis protein. The amount of
35S-labeled IL-18 bound to the receptors was
analyzed by precipitation with protein G-Sepharose. As shown in Fig. 3, an 6-fold molar excess of
sAcPL-FlagpolyHis had no effect on the binding of IL-18 to
sIL-1Rrp1-Fc, whereas the same molar ratio of p13-FlagpolyHis
completely ablated the observed binding. Similarly, addition of a
6-fold molar excess of sAcPL-FlagpolyHis had no effect on the binding
of IL-18 to sIL-1Rrp1-Fc + sAcPL-Fc, yet 4-, 16-, and 38-fold excesses
of p13-FlagpolyHis reduced, in a dose-dependent manner, the amount of
IL-18 binding to the mixed heterodimeric receptor-Fc protein. We show
in this experiment that p13-Fc efficiently bound and precipitated
IL-18, whereas no IL-18 was precipitated with p13-FlagpolyHis,
indicating that the precipitation with protein G was specific for the
Fc region (Fig. 3). Thus, p13 competes with the IL-18 receptor for
cytokine binding.
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Because p13 inhibits binding of IL-18 to its receptor, one would
predict that it also inhibits the biological activity of IL-18. To test
this, we preincubated rIL-18 with the indicated purified proteins, then
used these mixtures to stimulate COS-7 cells transfected with an
NF-B-responsive luciferase reporter gene and both IL-18R subunits.
Consistent with its inability to bind IL-18 with high avidity,
sIL-1Rrp1-Fc displayed no significant inhibition of IL-18 activity in
this assay (Fig. 4); however,
sIL-1Rrp1-Fc + sAcPL-Fc was an efficient inhibitor, displaying 78%
inhibition at an 160-fold molar excess over IL-18. Preincubation of
mIL-18 with p13-Fc resulted in a 51% inhibition at an 240-fold
molar excess (Fig. 4). p13-FlagpolyHis showed similar inhibition in
separate experiments (data not shown).
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B activity (data not shown). The potency of inhibition
displayed by sIL-1Rrp1-Fc + sAcPL-Fc relative to p13-Fc is in agreement
with the relative avidities we determined by BIAcore measurements
(Table I). The lack of inhibition by sIL-1Rrp1-Fc is likely due to the
combination of a low avidity of this protein for IL-18, and the fact
that the full-length receptors are overexpressed on the COS cell
surface in this experiment. We have tested the ability of sIL-1Rrp1-Fc
and sIL-1Rrp1-FlagpolyHis to inhibit IL-18-mediated proliferation of
the murine T cell line AE7, and do see a modest level of inhibition
under these conditions (data not shown). An in vivo role for p13 in NK cell activation
NK cells are an important component of the innate immune response that contributes to recovery from infection. Because IL-18 is known to induce NK cell cytotoxicity, and our results demonstrate that p13 inhibits the activity of IL-18, it is possible that p13 secretion from EV-infected cells may attenuate the activation of NK cells proximal and/or distal to the site of EV replication. To test this hypothesis, we constructed an EV deletion mutant in which nt 60–149 of the coding region of the p13 gene (416 nt in length) were deleted and replaced with an E. coli gpt selection cassette. The genotype of EV-p13- was confirmed by PCR and DNA sequence analysis. Moreover, Western blot analysis failed to detect p13 protein in the EV-p13--infected cells or culture supernatant (data not shown). This EV deletion mutant was used to investigate the role of p13 in viral pathogenesis using the peritoneal route of infection. This route of inoculation permitted us to measure the kinetics of appearance of NK cytotoxic activity and cytokine production at the site of infection.
EV-p13-, EV-WT, or saline was injected into the
peritoneum of C57BL/6 mice, and spleens and PECs were collected 1, 2,
or 3 days p.i. NK cytotoxic activity was measured using a standard
51Cr-release assay with YAC-1 tumor cell targets.
We did not observe any significant difference between cytotoxic
activity associated with splenocytes from
EV-p13-, EV-WT, or saline-treated animals up to
3 days p.i. (data not shown). In contrast, PECs from
EV-p13--infected mice showed progressive and
dramatic increases in cytototoxic activity compared with WT
virus-infected mice 2 and 3 days p.i. (Fig. 5). The cytotoxic activity was likely due
to NK cells in the PECs rather than CTL because the YAC-1 target cells
were uninfected and MHC mismatched with the C57BL/6 effectors. To
verify that lysis of the targets was attributable to NK cells, PECs
collected from mice infected 3 days earlier with
EV-p13- were depleted of
CD8+ cells or CD8+ and
asialo-GM1+ cells by incubation
with Ab and complement. The remaining PECs, without adjustment for
reduction of cell numbers due to depletion of
CD8+ and/or
asialo-GM1+ cells, were tested
for cytotoxic activity on YAC-1 target cells (Fig. 5D). This
assay method ensures that remaining PEC populations would be present at
their original concentrations and thus would not bias the
anti-target activity. The results indicate that the majority of the
cytotoxic activity exhibited by PECs from
EV-p13--infected mice is attributable to NK
cells. Taken together, these data suggest that NK cell cytotoxic
activity at the site of infection was enhanced in mice infected with
the EV-p13- relative to EV-WT.
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). The total number of cells collected
in the peritoneal washes of EV-p13-- and
EV-WT-infected mice varied by 53% of the saline control group. A
slight increase in the number of CD4+ and
CD8+ cells in the peritoneal cavities of mice
infected with EV-p13- was observed, relative to
saline-treated animals, 3 days p.i. (Fig. 6). These data suggest that
the increased cytotoxic activity observed following infection with
p13- mutant virus is due primarily to elevated
activation of NK cells in PECs, and not an increased number of NK
cells.
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and IL-10 (data not shown). However, IFN- production in
PEC cultures from EV-WT-infected mice was 20-fold lower than comparable
cultures from saline and EV-p13--infected mice
(Table II). This is in agreement with our
observations above indicating a difference in NK cell activation in PEC
but not splenocytes from EV-p13--infected mice
relative to EV-WT infection. Interestingly, IL-10 production
reproducibly tended to be lower in PECs of mice infected with
EV-p13- compared with EV-WT-infected or control
mice. These data suggest that the loss of p13 may result in an enhanced
type 1 pattern of cytokine production by PECs of EV-infected
mice.
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Given the observed increase in NK cell activation, we would
predict an effect on the pathogenesis of infection in
EV-p13--infected mice. To test this hypothesis,
groups of mice were injected i.p. with EV-p13-
or EV-WT, and virus titers in spleens, livers and peritoneal cavities
were determined 3 days p.i. Virus titers in peritoneal washes were
similar between the two infections (data not shown). The viral titer in
liver and spleen from mice infected with EV-p13-
was significantly lower compared with that from EV-WT-infected animals
in one experiment (Table III). When the
experiment was repeated with a smaller sample size, we again observed a
decrease in viral titer in the liver of mice infected with
EV-p13- compared with EV-WT. Splenic titers of
EV, however, were similar between EV-p13-- and
EV-WT-infected mice in the second experiment (Table III). The
p13- mutant virus replicates with similar
efficiency to WT virus in tissue culture (data not shown), suggesting
that at least the difference in infectivity levels in liver may be
attributed to the action of host antiviral mechanisms. The
correspondence of the p13- deletion with poorer
growth rate in liver suggests that binding of IL-18 by p13 may
contribute to the capacity of EV to evade host defenses.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The maximum RRU shift observed by BIAcore for p13-Fc was consistently
higher than that observed for either of the receptor preparations
tested. Decreasing the amount of p13-Fc loaded onto the chip decreased
the magnitude of the difference. However, we did not observe similar
maximal RRU shifts for p13 and the cellular receptors even when
equimolar amounts of each protein were loaded (Fig. 2). Furthermore,
the binding of IL-18 to p13-Fc did not reach a plateau by 6 min, as did
binding to the cellular receptor Fc fusion proteins. These differences
observed in the BIAcore binding profiles could be due to a difference
in conformation between the smaller p13-Fc (45 kDa) and larger
IL-18R-Fc proteins (85 kDa by SDS-PAGE), resulting in differences in
accessibility to cytokine when immobilized on the BIAcore flow cell.
Alternatively, it could represent differences in the percentage of
active (IL-18 binding) protein in equimolar amounts of total protein,
or errors in determination of protein concentration between the
samples. Protein concentrations were determined in at least two
independent assays and verified by SDS-PAGE and Coomassie stain.
Regardless, BIAcore affinity measurements are dependent on the
concentration of the soluble analyte (IL-18), but independent of the
concentration of the immobilized protein. Each affinity constant was
determined for at least two different concentrations of loaded Fc
fusion protein, and in all cases the results were consistent.
It is well established that recruitment of AcP to the active IL-1/IL-1R
complex increases its stability. In stably transfected CHO cells, an
4-fold increase in affinity has been reported in the presence of AcP
(38). In EL4 cells, the
KD of IL-1
has been reported
to be the same for cells expressing or lacking AcP, yet the stability
of binding was increased in the presence of AcP, suggesting that AcP
most likely alters the off rate of IL-1 from the receptor complex
(39). A similar alteration in the binding of IL-18 would
be expected in the presence of AcPL, yet the results reported above
offer the first direct examination of this issue. Our in vitro assays,
such as immunoprecipitation and BIAcore binding, suggest an increased
avidity of IL-18 for the receptor heterodimer (sIL-1Rrp1-Fc/sAcPL-Fc)
as compared with its avidity toward sIL-1Rrp1-Fc alone. It should be
noted, however, that due to inadequacies of current Abs, we have not
formally demonstrated the presence of receptor heterodimers in our
sIL-1Rrp1-Fc/sAcPL-Fc cotransfected cell supernatants. We have assumed
that the differences we observe in this protein preparation as compared
with IL-1Rrp1-Fc protein are due to the presence of heterodimeric
soluble receptor molecules. An independent approach to verify the
altered avidity of the receptor heterodimer is needed. We have
attempted these experiments in standard cell-surface binding
experiments, and have had difficulties with background IL-18 binding
which makes quantitative results inconclusive. In qualitative assays,
however, we have observed a higher level of iodinated IL-18 binding to
cells overexpressing both receptor subunits relative to cells
overexpressing IL-1Rrp1 alone (data not shown).
The demonstration that EV p13 binds IL-18 with high avidity in vitro
suggested that it has the capacity to sequester IL-18 in vivo,
affecting the innate immune response to EV infection. To test this
possibility, we constructed an EV mutant in which the p13 gene was
deleted and then monitored NK cell activation and cytokine production
in infected C57BL/6 mice. We wished to examine the activation of NK
cells both proximal and distal to the initial site of infection. EV
infects mice in the wild through minor skin abrasions, yet it is
technically difficult to quantitate, and to measure the activation
state of, NK cells entering epidermal/dermal lesions. Therefore, we
chose to infect mice by the i.p. route and measure NK cell numbers and
activation state in spleen and peritoneal cavity. The most dramatic
differences between WT-EV and EV-p13- infections
were observed in NK cells proximal to the site of infection (from the
peritoneal cavity). First, PECs from p13-
virus-infected mice showed a progressive increase in NK-associated
cytotoxic activity against YAC-1 targets as compared with samples from
EV-WT or saline controls. The elevated NK cell cytotoxicity was not due
to enhanced trafficking of NK1.1+ cells into the
peritoneal cavity, as mice injected with EV-WT,
EV-p13-, or saline had similar numbers of
NK1.1+ PECs over a 3-day period. Rather, the
elevated cytotoxicity is likely due to an increased frequency of
activated NK cells in PECs of EV-p13--infected
mice. This is consistent with the lack of an IL-18BP in the
p13- virus, because IL-18 is known to activate
NK cells, at least in part, through stimulation of Fas ligand-mediated
cytotoxicity (40, 41). Second, higher levels of IFN-
were observed in PECs of mice infected with
EV-p13- compared with those infected with EV-WT.
This is consistent with the observed increase in NK cytotoxic activity
in PECs of EV-p13--infected mice, and with the
fact that IL-18 is a potent inducer of IFN-. The enhanced IFN-
production was observed proximal to the initial site of infection but
not distally in the spleen. While this correlated with our data
indicating a lack of splenic NK cytotoxic activation, the reasons for
differential NK cell activation between PECs and splenocytes remain
unresolved.
Titer of virus in the peritoneal cavity, to which abdominal organs as
well as resident PECs contribute, was not significantly affected by
increased NK cytotoxic activity or IFN- secretion by PECs in
p13- virus-infected mice. However, liver titers
were significantly influenced by loss of p13. A number of factors could
affect the phenotype of p13- mutant in various
cells and tissues. It has been shown that IL-18-dependent responses can
differ according to the tissue source of the treated cell. For example,
studies have shown that liver lymphocytes respond to IL-18 by
production of IFN-, and that this production can occur independently
of IL-12 (42). In contrast, splenic T cells from C57BL/6
mice apparently do not produce IFN- in response to IL-18, alone or
in combination with IL-12 (43), and indeed, we did not
find elevated IFN- production or NK activity in the spleen of
EV-p13--infected mice. Also, in a recent study
the effectiveness of IFN- in controlling EV replication was shown to
vary among tested tissues (44). And finally, EV-infected
splenocytes, PECs, or liver cells may differ in effectiveness as
targets of NK-mediated cytotoxicity. These observations may help to
explain the differential IFN- production, NK activation, and viral
titer we detected in cells from the liver, spleen and peritoneal cavity
of infected mice. Further work will be required to determine whether NK
activity is elevated in the liver of
EV-p13--infected mice, and the suitability of
cell types in the various organs as NK targets.
NK cells, and their capacity to secrete IFN-, are critical in the
early defense against infection by viral pathogens, and are targets of
poxvirus host response modifier genes. EV-infected cells secrete a
binding protein with high affinity for IFN-
(KD 1 nM) (45, 46). Also, EV-infected cells synthesize a double-stranded RNA
binding protein (E3L) which blocks IFN- action by inhibiting
dsRNA-activated protein kinase and 2-'-5' oligonucleotide adenylate
synthase (47). IL-18 is an important inducer of IFN-
production and cytotoxicity, and it appears that EV can inhibit IL-18
action through multiple mechanisms. It has been shown that the
orthopoxvirus crm a gene, which is expressed by EV, blocks
the action of IL-1ß-converting enzyme, a protease that converts both
the IL-1ß and IL-18 precursors into mature forms (48).
Because crmA is a cytoplasmic protein, it can only affect IL-18
maturation in the infected cell, conceivably allowing IL-18 secretion
by uninfected neighboring cells triggered by inflammatory mediators.
The discovery that EV p13 encodes a secreted protein that binds and
inactivates IL-18 suggests that EV may also have a mechanism to block
IL-18 action at a distance from the infected cell. Together crmA and
p13 would provide the virus with a means to inhibit IL-18-mediated NK
cell activation proximal to a focus of infection. It is probable that
the effectiveness of IL-18 inhibition will vary from tissue to tissue,
but likely would be greatest in the skin, the primary site of infection
and transmission.
Although IL-18 has been shown to affect differentiation and expansion of Th1 cells, we believe the importance of poxvirus IL-18BP homologues to the virus life cycle will reside in the attenuation of NK, or perhaps memory T cell, effector function. This hypothesis is based on several observations. Molluscum contagiosum virus encodes at least three IL-18BP homologues (49), yet replicates only in keratinocytes of the human epidermis, at a distance from any T cell-rich lymphoid organ. The epidermal keratinocytes are major producers of IL-18 (50), and mouse skin is a source of potentially IL-18-responsive NK, NK-like, or memory T cells (51, 52). Finally, NK and memory T cells have been shown to be important in controlling poxvirus infections (21, 53).
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. John E. Sims, Department of Molecular Biology, Immunex Corporation, 51 University Street, Seattle, WA 98101. E-mail address:
3 Address correspondence and reprint requests to Dr. R. Mark L. Buller, Department of Molecular Microbiology and Immunology, 1402 South Grand Boulevard, Saint Louis University, St. Louis, MO 63104. E-mail address:
4 Abbreviations used in this paper: WT, wild type; AcPL, accessary protein-like; IL-18BP, IL-18 binding protein; ORF, open reading frame; EV, ectromelia virus; m, murine; s, soluble; RRU, relative response units; PEC, peritoneal exudate cell; p.i., postinfection.
Received for publication August 23, 1999. Accepted for publication January 5, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
production by T-cells. Nature 378:88.[Medline]
-inducing factor enhances T helper 1 cytokine production by stimulated human T cells: synergism with interleukin-12 for interferon- production. Eur. J. Immunol. 26:1647.[Medline]
, interleukin-1ß and interferon--inducing factor on the production of interferon- by natural killer. Eur. J. Immunol. 27:2787.[Medline]
- and Fas ligand-mediated hepatotoxic pathways in endotoxin-induced liver injury in mice. J. Immunol. 159:3961.[Abstract]
inducing factor mediated by ICE. Science 275:206. inducing factor and regulates LPS induced IFN induction. Nature 386:619.[Medline]
-inducing factor in enhanced production of IFN-. J. Immunol. 159:2125. production. J. Immunol. 161:3400. chains of the IL-2 receptor by the novel cytokine IL-15. EMBO J. 13:2822.[Medline]
B-mediated transcription by mutant derivatives of the latent membrane protein of Epstein-Barr virus. J. Virol. 69:2968.[Abstract]
-inducing factor up-regulates Fas ligand-mediated cytotoxic activity of murine natural killer cell clones. J. Immunol. 157:3967.[Abstract]
-inducing factor, a novel cytokine, enhances Fas ligand-mediated cytotoxicity of murine T helper 1 cells. Cell. Immunol. 173:230.[Medline]
production and activates IRAK and NF-B. Immunity 7:571.[Medline]
receptor by myxoma virus. Science 258:1369. binding proteins. Virology 208:762.[Medline]
-inducing factor) messenger RNA and functional protein by murine keratinocytes. J. Immunol. 159:298.[Abstract]
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