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Departments of
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Pathology and Kaplan Comprehensive Cancer Center,
Cell Biology, and
Pathology and Heidelberger Division of Immunology, New York University School of Medicine, New York, NY 10016;
§
Children’s Hospital Research Foundation and Ohio State University Comprehensive Cancer Center, Columbus, OH 43205; and
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Department of Microbiology, Mount Sinai School of Medicine, New York, NY 10029
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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was not required to resolve infection, loss
of type I IFN responsiveness led to exacerbated disease pathology
characterized by granulocytic pulmonary inflammatory infiltrates.
Responsiveness to type I IFN did not influence the generation of
virus-specific cytotoxic lymphocytes or the rate of viral clearance,
but induction of IL-10 and IL-15 in infected lungs through a type I
IFN-dependent pathway correlated with a protective response to virus.
Combined loss of both IFN pathways led to a severely polarized
proinflammatory immune response and exacerbated disease. These results
reveal an unexpected role for type I IFN in coordinating the host
response to viral infection and controlling inflammation in the absence
of a direct effect on virus replication. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß and type II or ) can be distinguished on the basis of
primary sequence homology and use of distinct cell surface receptors;
however, their signaling mechanisms partially overlap, leading to the
activation of a partially overlapping set of genes (reviewed in Refs.
2, 3, 4). Both types of IFN activate intracellular protein
tyrosine kinases of the Jak family, leading to the phosphorylation and
activation of Stat transcription factors. The Stat1 protein is required
for signaling from both type I and type II IFN (5). In
fact, ablation of the Stat1 gene through gene targeting produced
animals exhibiting total resistance to the action of IFN, rendering
them highly susceptible to viral and microbial pathogens (6, 7). In addition to their antiviral action, a variety of
immunomodulatory and antiproliferative activities have been ascribed to
IFNs. The biological relevance of these nonantiviral effects has
remained elusive, particularly in the case of type I IFN
(8). The direct antiviral effects of IFN are mediated by induction of a set of IFN-stimulated genes. The precise mechanisms of action of these genes in "interfering" with viral replication are not well understood, and viruses differ in their sensitivity to IFN (9). We have previously demonstrated (10) that mice lacking Stat1 consistently show increased sensitivity to infection with the influenza virus strain A/PR/8/34 (PR8). Surprisingly, the susceptibility of Stat1-/- animals to this pathogen did not reflect a defect in innate antiviral immunity. In contrast to the response to other pathogens (6, 7), Stat1-/- mice were capable of preventing uncontrolled viral replication and of clearing PR8 virus. PR8 virus titers and the kinetics of clearance in Stat1-/- mice were comparable with those seen in wild-type animals (10). Nonetheless, PR8 virus infection produced enhanced lethality in Stat1-/- mice.
It has been demonstrated that the pathology resulting from influenza
virus infection results substantially from host inflammatory processes
rather than directly from virus-mediated damage to respiratory
epithelium (11), particularly in the absence of the
Mx gene product, which is defective in most inbred strains
of mice (9). We took advantage of this model to explore
the role of IFNs in the host response and development of Ag-specific
immunity to influenza virus infection by comparing the course of a
primary PR8 virus infection in the absence of responsiveness to type I
IFN, type II IFN, or both. We found that in the absence of Stat1,
influenza virus infection produces a distinctive, proinflammatory
pathological process. This exacerbated disease correlates with a lack
of the normal, strong induction of IL-15 by influenza virus infection
and with a Th2-biased host immune response. Although a Th2-type
cytokine profile was present in infected lungs of
IFN--/- animals, this polarized response appeared to
be largely compensated by the effects of type I IFN. The converse was
not true; in the absence of the IFN- receptor, IFN- produced in
response to virus infection was not sufficient to prevent enhanced
disease.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The production of Stat1-/- mice on a CD1
background has been previously described (6). For
comparison with mice disrupted for the IFN- gene on the C57BL6
background and with IFN- receptor knockout or IFN-- receptor
double knockout mice on the 129Sv/Ev background, Stat1-/-
mice were backcrossed to C57BL6 or to 129Sv, respectively (8th
backcross generation). Similar results to those described here were
also observed with Stat1-/- mice carrying a distinct
mutation (7) that were maintained on a 129Sv/Ev background
(data not shown). IFN--/- mice (12) were
purchased from The Jackson Laboratory, Bar Harbor, ME. Mice with
targeted mutations at the IFN- receptor locus
(IFNAR-/-) and at both the IFN- receptor and IFN-
receptor loci (IFNAR/GR-/-) (13, 14) were
kindly provided by Michel Aguet (Lausanne Switzerland) and Robert
Schreiber (Washington University, St. Louis, MO). Wild-type C57BL6,
129SvEv, and CD1 mice were purchased from Taconic Laboratories
(Germantown, NY). Animals were bred and maintained under specific
pathogen-free conditions. All work with animals conformed to guidelines
approved by the Institutional Animal Care and Use Committee of New York
University School of Medicine.
Viruses
Influenza A/WSN (H1N1) and NA/B-NS viruses were grown in Madin-Darby bovine kidney cells and titered by plaque assay on Madin-Darby bovine kidney cells, as previously described (10). Influenza A/PR/8/34 (H1N1) (PR8) was grown in the allantoic cavities of 10-day-old embryonated eggs (SPAFAS, Preston, CT) and titered by plaque formation on Madin-Darby canine kidney cells in the presence of trypsin or by hemagglutination (HA)3 assay. Virus production assays to determine tissue culture infectious dose (TCID) were performed on confluent monolayers of Madin-Darby canine kidney cells in 96-well plates infected with dilutions of viral suspensions. Following 2–3 days of incubation, supernatants were titrated by HA, and the lowest concentration of virus to produce an infection that scored positive by HA was defined as 1 TCID. The HA assay was done in V-bottom 96-well plates. Serial 2-fold dilutions of each sample were incubated for 1 h on ice with an equal volume of a 0.5% suspension of chicken erythrocytes (Truslow Farms, Chestertown, MD). Wells containing an adherent, homogeneous layer of erythrocytes were scored as positive.
Infections
Mice 6–10 wk old were anesthetized and inoculated intranasally (i.n.) with 50 µl virus diluted in PBS. For determination of LD50 in mice of different genotypes, various doses of PR8, ranging from 10 to 105 TCID per animal, were administered and animals were monitored daily for signs of illness. Survival was scored after 10 days. For rechallenge experiments, mice were initially inoculated with 103 TCID of the attenuated NA/B-NS viral strain, and serum samples were obtained after 3 wk. Animals with positive influenza virus-specific Ab titers and control animals that had been inoculated with PBS alone were rechallenged with 105 PFU WSN, and animals were monitored as described above.
For comparison of virus replication, clearance, and pathogenesis, animals were infected with 102 or 103 TCID of PR8. Groups of 3–5 mice of each genotype were sacrificed at 3- to 4-day intervals. One lung from each animal was homogenized in 2 ml PBS, and the presence of influenza virus was quantified by virus production/HA assay. The second lung from each animal was divided. One lung lobe was inflated and fixed in 10% buffered formalin for histological examination. The remainder was homogenized in TRIZOL (Life Technologies, Gaithersburg, MD) for RNA isolation and evaluation of cytokine transcripts. Samples for microscopic examination were paraffin embedded after fixation and 5-µm sections were stained with hematoxylin and eosin.
Cytokine determinations
Spleen cells after lysis of RBC were cultured at 2 x
106 cells/ml for 24 h in the presence of
soluble anti-CD3 Ab 2C11. Subsequently, cells were washed,
incubated with 50 U/ml IL-2 in the absence of Ab for 7 days, replated
at 2 x 106 cells/ml, and then left
untreated or restimulated with anti-CD3 Ab. Supernatants were
harvested from triplicate cultures after 48 h. Production of
IFN-, IL-4, IL-5, and IL-10 was measured by cytokine-specfic ELISA
(PharMingen, San Diego, CA).
For measurement of cytokine transcripts from lymphocytes, splenocytes
were cultured for 4 days with plate-bound anti-CD3 (2C11) plus
anti-mouse CD28 (37.51) Abs and IL-2. Cells were washed and
cultured for an additional 48 h with IL-2. After restimulation
with plate-bound Ab for 6 h, cells were lysed with TRIZOL (Life
Technologies). RNA was isolated from the splenocyte lysates and from
lung tissue-TRIZOL homogenates after phenol and phenol-chloroform
extractions. Cytokine transcripts were assayed by RNase protection with
the use of RiboQuant multiprobes according to the manufacturer’s
protocols (PharMingen). Splenocyte RNA (5–8 µg/sample) and lung RNA
(20–30 µg/sample) were used. For intracellular staining of cytokine
protein, cells were pretreated with brefeldin A (1 µg/ml) for 3
h to enhance accumulation of secreted proteins followed by surface
staining with anti-CD4-Tricolor or anti-CD8-Tricolor Abs
(Caltag, South San Francisco, CA). Cells were subsequently fixed and
permeabilized with Fix & Perm reagent (Caltag), followed by staining
with anti-IFN--FITC and anti-IL-5-PE Abs (Caltag).
Preparation of bone marrow-derived macrophages
Marrow harvested from four femurs of each mouse strain were plated in a six-well tissue culture dish in DMEM supplemented with 10% mouse L cell-conditioned medium and 20% FCS (Life Technologies). The medium was changed after 48 h, and adherent cells were allowed to grow to near confluence. For FACS analysis, cells were dislodged by scraping and then stained with FITC-conjugated hamster anti-mouse CD11b (clone 500-A2, Caltag). Individual wells were left untreated, treated with LPS at 5 µg/ml (from Escherichia coli strain 0127:B8, Sigma, St. Louis, MO), or infected with 106 PFU influenza virus A/WSN/33. After 24 h incubation, cells were lysed in TRIZOL and RNA extracted with phenol-chloroform.
Cellular cytotoxicity assay
CTL activity from mice that had survived infection with influenza virus was measured on autologous cells. Spleen cells from virus-infected mice were treated to lyse RBC and divided into three samples. One sample was irradiated for use as stimulator cells by infecting with influenza A/PR8/34 virus at a multiplicity of infection of 4. Stimulators were incubated together with a second sample of spleen cells (responders) at a ratio of 4 x 106 stimulators plus 4 x 106 responders/ml for 5 days at 37°C. A third sample of splenocytes was stimulated with 25 µg/ml Salmonella typhosa LPS for 5 days at 2 x 106 cells/ml and used as targets in cytotoxicity assays. Virus-specific cytotoxicity was measured by mixing spleen cells at a ratio of 40:1 with autologous target cells labeled with sodium [51Cr]chromate, either uninfected or infected with influenza PR8 virus at a multiplicity of infection of 10. Cells were incubated for 4 h at 37°C and pelleted, and radioactivity released into supernatants was measured. Maximal release was measured by incubating target cells with 0.5% Nonidet P-40 and spontaneous release by incubating target cells with medium alone (routinely <10% of maximal release). Percent cytotoxicity was calculated as [(test release - spontaneous release)/(maximal release - spontaneous release)] x 100. Ab-redirected lysis, a measure of activated CD8 T lymphocytes, was determined by similar assays but using 51Cr-labeled P815 tumor cells coated with anti-CD3 Ab 2C11 as targets as previously described (15).
CTL activity from immunized mice was measured against heterologous targets pulsed with influenza virus antigenic peptide. Immunity to influenza virus was generated in C57BL6/J mice (wild-type and Stat1-/-) by i.p. injection of 250 hemagglutinating units of influenza virus A/PR/8/34 per animal 10 d before harvest of spleens. Spleen cells were isolated as described above and stimulated in vitro for 5 days by cocultivation with irradiated (2000 rad) influenza virus-infected spleen cells (1 x 108 spleen cells were infected with 1 x 103 hemagglutinating units A/PR8/34). In vitro-stimulated CTL activity was measured against 51Cr-labeled EL4 (H-2b) target cells incubated with a 100 nM concentration of a synthetic peptide representing aa 366–374 of A/PR8/34 nucleoprotein (16) in a standard 4-h chromium release assay, as described above.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have previously demonstrated an increased sensitivity of Stat1-/- mice relative to wild-type animals to infection by human influenza virus strain A/PR/8/34 (10). Stat1-/- mice infected i.n. with this virus displayed an LD50 10-fold lower than their wild-type counterparts. This increased sensitivity could not be explained by increased virus replication in the absence of IFN responsiveness, because peak virus titers were comparable in lungs harvested from infected wild-type or mutant animals. When viral titers were determined throughout the course of the infection, the kinetics of viral clearance were comparable in wild-type, Stat1-/-, and IFNAR-/- mice (10).
To characterize the cellular immune response to influenza virus, CTL
activity was measured with the use of splenocytes derived from C57BL6
mice immunized in vivo by i.p. injection of a sublethal dose of PR8
virus (Fig. 1
). Both wild-type and
Stat1-/- mice generated cytotoxic lymphocytes capable of
killing target cells pulsed with the immunodominant influenza virus
peptide presented by H-2Db (16).
Cytotoxicity measurements over a range of peptide concentrations or
with virus-infected target cells showed no significant differences
between wild-type and Stat1-/- effector cells, indicating
no impairment of either the specificity or the efficiency of the
cytotoxic response in the absence of Stat1 (Fig. 1 and data not shown).
Antiviral immunity was also measured in mice that had survived
respiratory infection with an attenuated strain of influenza virus,
NA/B-NS (17). Splenocytes from animals of both genotypes
were harvested, stimulated in vitro with influenza-virus infected
spleen cells, and assayed for Ag-specific cytotoxicity against
autologous infected blasts. As a second measure of the abundance of CTL
generated during influenza virus infection, Ab-redirected cellular
cytotoxicity was also measured against P815 tumor cells. In all cases,
significant influenza virus-specific cellular cytotoxicity was detected
in recovered mice, but no significant differences were observed to
indicate a deficiency in the absence of Stat1 (data not shown). This
antiviral cytotoxic response likely contributed to the equal virus
elimination observed in infected mice of both genotypes. Increased
sensitivity to influenza virus infection in vivo in the absence of
Stat1 was therefore not due to loss of any direct antiviral effect of
IFNs on either virus replication or virus elimination by the
host.
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To compare disease progression in mice lacking type I and/or type
II IFN responsiveness, we monitored pulmonary histopathology in
wild-type (C57BL6, 129SvEv, and CD1 backgrounds),
IFNAR-/- (129SvEv background), IFN--/-
(C57BL6 background), and Stat1-/- mice (C57BL6, 129SvJ,
and CD1 backgrounds). Five animals of each genotype were infected i.n.
with 102 TCID of PR8 virus, and lung tissues were
harvested and formalin fixed at 6 days postinoculation. Dramatic
differences in disease pathology were revealed by comparative lung
histology, and very different inflammatory responses to influenza virus
infection were observed dependent on the genotype of the infected
animals (Fig. 2). Although some variation
was noted among the three different strain backgrounds tested,
presumably reflecting differences in virus susceptibility not related
to the IFN system, significant differences in disease pathology were
detected only in mutant strains. Moreover, the major differences in
host response dependent on targeted loss of IFN responsiveness were
replicated with the Stat1 mutation bred onto each of the
different strain backgrounds 129, C57BL6, and outbred CD1.
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, A and E). Inflammation in
IFN--/- mice was similar to wild-type, although
somewhat more cellular (Fig. 2, C and F),
consistent with previous reports showing little differential
sensitivity to influenza virus infection in animals lacking IFN-
(18). In contrast, lungs from infected
Stat1-/- mice (Fig. 2, B and H)
showed a markedly exacerbated inflammatory process. Infiltrates were
much more cellular and diffuse. In addition to the increased number of
inflammatory cells, the character of the infiltrates was changed in the
Stat1-/- mice. Although small numbers of lymphocytes were
identified in the infected tissue, the majority of responding cells
were granulocytes, and infiltrating macrophages, neutrophils, and
eosinophils were abundant (Fig. 2H). Interestingly, whereas
the inflammatory process detected in lungs harvested from
IFNAR-/- animals (Fig. 2D) was significantly
worse than that in wild-type or IFN--/- mice, the
severity was consistently intermediate between that observed in
wild-type relative to Stat1-/- animals. Similar to the
Stat1-/- mice, the responding inflammatory cells in PR8
virus-infected IFNAR-/- mice were primarily (>50%)
granulocytes (Fig. 2G). However, fewer infiltrating cells
and less tissue destruction were observed relative to infected
Stat1-/- tissue (compare Fig. 2B and Fig. 2D). The histopathology of PR8 virus-infected lungs from
IFNAR/GR-/- mice was comparable with that seen in the
Stat1-/- animals (data not shown), suggesting that both
IFN- and IFN- contributed to regulation of the inflammatory
response to influenza virus infection. Humoral immune response to influenza virus infection in Stat1-deficient and wild-type mice
Serum samples were taken from wild-type and Stat1-/-
mice before and after i.n. inoculation with the attenuated influenza
virus strain, NA/B-NS (17), and were assayed for the
presence of virus-specific Abs. Influenza virus-specific Ig was
detected at equivalent levels in animals of both genotypes (Fig. 3A). Interestingly, whereas
wild-type animals produced Abs against influenza virus of both IgG1 and
IgG2a subclasses, no IgG2a virus-specific Abs were detected in serum
from infected Stat1-/- mice. Despite these differences in
Ab isotype, both wild-type and Stat1-/- mice displaying
detectable anti-influenza virus Abs were fully protected from
rechallenge with a lethal dose of WSN virus (data not shown).
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(19), suggesting that the lack of this Ig isotype was the
result of a direct loss of the ability of cells to respond
transcriptionally to IFN- in the absence of Stat1. Preferential
production of the IgG1 subclass of Abs is characteristic of B
lymphocytes maturing under the influence of Th2 cells
(20). A hallmark of Th2-influenced responses, which
normally characterize allergic, antiparasitic, or autoimmune
responses rather than antiviral ones, is the production of IgE. We
detected abnormally high levels of total IgE in the serum of influenza
virus-infected Stat1-/- mice (Fig. 3B), and
even naive animals showed significant levels of circulating IgE. IgE
levels were below the limit of detection (<0.04 ng/ml) in control
animals, whether or not they were infected with virus. Thus,
Stat1-/- mice displayed a default Th2 bias that was
greatly augmented by viral infection. Neither the enhanced production
of IgE nor the complete suppression of IgG2a synthesis were noted
in influenza virus-infected IFN--/- mice
(18), although a heavy bias toward IgG1 was noted
previously in IFNAR/GR-/- mice (13).
These results strongly suggest that type I IFN in addition to type II
IFN controls heavy chain class switch recombination. Th2 cytokine production in Stat1-/- mice
T cell responses in the absence of IFN signaling were further
characterized by comparison of wild-type and Stat1-/-
splenic lymphocyte differentiation. Th1 and Th2 T cell subsets are
defined functionally in terms of the cytokines that they produce
(21). The high levels of IgE found even in serum from
naive Stat1-/- mice suggested an intrinsic bias toward
Th2-influenced responses. Therefore, we measured cytokines produced by
spleen cell cultures isolated from uninfected wild-type and
Stat1-/- mice (Table I).
Splenocytes were stimulated in vitro through the TCR and allowed to
differentiate for 1 wk in the presence of IL-2. Restimulation of
Stat1+/+ splenocytes led to exclusive production of
IFN-. In contrast, significant amounts of IL-5 were secreted by
rested Stat1-/- splenocytes even in the absence of
restimulation. Moreover, restimulation of Stat1-/- cells
produced significant production of the Th2-type cytokines IL-4, IL-5,
and IL-10 not seen in wild-type cultures, in addition to IFN-.
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-producing response was also observed when cytokine
transcripts from in vitro differentiated splenocytes were assayed by
RNase protection. By this method, wild-type splenocytes were found to
make mRNA corresponding to the expected IFN- and IL-2, as well as
IL-10 (Fig. 4A, lane 11).
Stat1-/- splenocytes, differentiated and assayed in
parallel, produced transcripts corresponding to IL-5, IL-9, IL-10, and
IL-13 as well as comparable amounts of IL-2 and IFN- (Fig. 4A,
lane 12). Cytokine mRNA profiles varied somewhat with strain
background in these experiments. Lymphocytes harvested from wild-type
129 animals were much more Th0 in character than those from C57BL6 or
CD1 mice after in vitro differentiation and restimulation with
anti-CD3 and anti-CD28. Nonetheless, both
Stat1-/- (Fig. 4A) and IFNAR-/-
(data not shown) splenocytes showed a marked Th2 bias compared with
wild-type, strain matched controls.
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suggested a mixed
response, possibly due to the absence of IFN--mediated inhibition.
Because unfractionated splenocytes were used for the in vitro
differentiation and restimulation, the production of IFN- could
reflect contributions from cell types other than CD4 helper cells, such
as CD8 or NK cells. To dissect the contribution of CD4 and CD8 cells to
these cytokine profiles, we measured cytokine protein levels on a per
cell basis by flow cytometry (Fig. 4E). We scored
IFN--producing cells as a marker for Th1-type and IL-5-producing
cells as a marker of Th2-type. Approximately one-half the
differentiated wild-type CD4 cells produced IFN- and very few
produced IL-5, indicative of a Th1 response. In contrast, few
Stat1-/- CD4 cells produced IFN-, whereas nearly 10%
produced IL-5. The majority of CD8 cells of both genotypes produced
IFN-. Therefore, the majority of Stat1-/- CD4 cells
would appear to be biased toward a Th2 response or at least against a
Th1 response, and the vast majority of T lymphocyte-derived IFN-
produced by Stat1-/- cultures was not from helper T cells
but rather from CD8 cells. Cytokine production in infected lung tissue
We examined whether this Th2 bias observed with in vitro
differentiated Stat1-/- splenocytes was also present at
the site of infection. Fig. 4, A–D, shows cytokine mRNA
profiles from lungs of wild-type, Stat1-/-,
IFN--/-, and IFNAR-/- mice, harvested at
various times after i.n. inoculation with PR8 virus. On day 3 of
infection, lungs from wild-type animals (Fig. 4A, lanes
1–6) showed strong induction of IL-6 and IL-15, and a clear but
less robust induction of IFN-. By day 6 (Fig. 4B), the
IL-15 signal had begun to wane, and a delayed IL-10 induction was
evident.
This pattern of cytokine gene induction, consistently present in each
animal assayed, was distinct from that seen in lungs from PR8
virus-infected Stat1-/- mice. In Stat1 mutant animals,
IL-6 and IFN- were also induced strongly by day 3 (Fig. 4A,
lanes 6–10), similar to wild-type animals. However, missing from
the Stat1-/- profile at day 3 was the strong induction of
IL-15 transcripts, but present was weak induction of the Th2 cytokines
IL-5 and occasionally IL-13. Missing from the 6-day infected
Stat1-/- lungs was the induction of IL-10 seen in
wild-type samples (Fig. 4B). This pattern of cytokine
expression within the infected tissue was quite different from that
seen in differentiated splenocytes and could not be classified in terms
of a simple Th1-Th2 dichotomy. Therefore, although the presence of Th2
cytokines may have contributed to disease exacerbation in
Stat1-/- animals, it is likely that Th2 cytokines alone
were not sufficient and additional factors influenced the altered
pathogenesis observed.
This conclusion was reinforced by examination of the cytokine response
in virus-infected IFN--/- mice (Fig. 4C).
Animals were infected i.n. with PR8 virus, and groups of four were
sacrificed at 3-day intervals and analyzed individually. Lung samples
harvested at day 3 showed strong induction of IL-6 and IL-15 (Fig. 4C, lanes 1–4). Similar to wild-type animals, IL-10 was
induced by day 6. Unlike wild-type animals, however, IL-5 and IL-13
were also occasionally detected in lungs of animals harvested on day 6
(lanes 5 and 8) similar to the pattern
observed in Stat1-/- samples and indicative of the Th2
bias observed in the absence of IFN-. This result suggests that the
presence of IL-5 and IL-13 were not sufficient to cause significant
disease exacerbation, at least in the presence of an intact type I IFN
response, and reinforces the conclusion that the mere presence of Th2
cytokines is insufficient to explain the altered pathology of
virus-infected Stat1-/- mice.
Fig. 4D shows results of influenza virus infection in
IFNAR-/- mice. In PR8 virus-infected lungs harvested on
day 3, IL-6 and IFN- transcripts were detected in all animals (Fig. 4D, lanes 1–5). Juxtaposition of samples from wild-type,
background-matched controls (Fig. 4D, lanes 1–5)
underscores the reduction in IL-15 induction in the absence of IFN-
responsiveness (Fig. 4D, lanes 6–10). No increase in IL-5
or IL-13 was detected by RNase protection assay in these animals. IL-10
induction was present in the day 6 IFNAR-/- lung samples,
consistent with kinetics seen in wild-type animals. The increased
IFN- mRNA synthesis seen in the IFNAR-/- mice from 3
to 6 days (Fig. 4D, lanes 6–10) was consistent between
experiments and not seen with other mutant strains.
Macrophage cytokine production in vitro
We examined potential sources of IL-15 mRNA detected in influenza
virus-infected lungs of wild-type and IFN--/- mice.
Macrophages were derived from each mouse strain by culturing bone
marrow for 7 days in L cell-conditioned media. Homogeneity of cultured
cells was estimated to be between 80 and 90% by FACS analysis after
staining for CD11b (data not shown). Monolayers of adherent macrophages
were treated with LPS (5 µg/ml) or infected at high multiplicity with
influenza virus A/WSN/33. RNA was isolated from each culture after
24 h and cytokine transcripts were analyzed by RNase protection.
As expected (22, 23), IL-15 was strongly induced by LPS in
wild-type macrophages derived from 129SvEv or C57BL6 mice (Fig. 5, left). Cytokine profiles
from IFN--/- cells resembled wild-type controls (Fig. 5A, right). However, no IL-15 induction occurred in
LPS-treated IFNAR-/- or Stat1-/-
macrophages. IL-15 was also induced in wild-type and
IFN--/- cultures in response to virus infection,
although to a lesser extent than by LPS. It is likely that type I IFN
induction by either stimulus mediates the increased transcription of
IL-15. IL-10 induction paralleled that of IL-15 in response to LPS or
virus, whereas IL-6 was induced in all strains regardless of genotype
only in response to LPS. Thus, the prominent induction of IL-15
observed in infected lungs likely originated from macrophages, either
resident or infiltrating. IL-10, found in infected lungs of all but
Stat1-/- mice, may have been elaborated by other cell
types in addition to macrophages (24, 25) since
IFNAR-/- macrophages appear deficient in IL-10
production, and yet this cytokine was clearly induced in infected lungs
(see Fig. 4D).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have observed a markedly different disease process in influenza
virus-infected Stat1-/- mice that is characterized by a
proinflammatory response mediated by diffuse, largely granulocytic
pulmonary infiltrates. Stat1-/- mice were 
10-fold more
susceptible to lethal infection than congenic wild-type,
IFN--/-, or IFNAR-/- strains, although
the virulence of the PR8 virus strain makes subtle alterations in
sensitivity difficult to measure by LD50.
However, comparative histology of lungs harvested from infected animals
reinforced this conclusion, consistently showing a gradation of
pathology, from primarily lymphocytic bronchitis in wild-type and
IFN--/- animals, to patchy bronchopneumonia with mixed
inflammatory infiltrates in IFNAR-/- mice, to diffuse,
severe bronchopneumonia in Stat1-/- animals. The
inflammation differed not only in terms of amount but also by its
composition. Although lymphocytes were clearly recruited to the site of
infection in all infected animals, there was a predominance of
macrophages and acute inflammatory cells in lungs of type I
IFN-nonresponsive animals not seen in wild-type mice. Inflammation in
the IFN--/- mice was largely lymphocytic, although it
was somewhat more mixed than in wild-type mice. In contrast, the
proportion of granulocytes increased in the absence of the type I IFN
receptor (50–60%) and became overwhelming in the absence of
Stat1.
It is likely that some component of the distinct histopathology and
survival between influenza virus-infected wild-type and Stat1 mice can
be explained by differences in helper T cell subsets responding to
infection. An inappropriate Th2 bias was observed in
Stat1-/- mice both in vitro and in vivo. The resulting
production of IL-5 and IL-13 at the site of infection may explain some
part of the deviation from a typical antiviral Th1 response as well as
the proinflammatory nature of the response. Graham et al.
(26) have demonstrated the requirement for Th1 cells in
adoptive transfer experiments using influenza virus-specific Th1 or Th2
CD4+ cell clones. Th1 clones provided protection
against lethal challenge whereas transfer of Th2 clones was
nonprotective and led instead to disease exacerbation. However, the
Th2-biased cytokine profile in infected IFN--/- mice
was accompanied by a very different disease pathology and outcome than
observed in the absence of type I IFN responsiveness, suggesting that
Th2 cytokines alone cannot be the full explanation for the
Stat1-/- phenotype.
IFN--/- animals responded to influenza virus in a
manner very similar to that of wild-type animals, as previously
observed by others (18). Although IFN--/-
mice produced more virus-specific IgG1 than wild-type animals, they
nonetheless displayed significant levels of heavy chain class switching
to IgG2a and did not display increased concentrations of IgE
(18). In contrast, Stat1-/- animals produced
no antiviral IgG2a Abs and displayed high levels of circulating IgE. It
is therefore likely that type I IFN- and Stat1-mediated events were of
major importance in shaping the normal, protective, antiviral response
that consisted of both IgG1 and IgG2a anti-influenza Abs as well as
the absence of IgE. The absence of IFN- signaling alone, despite the
presence of Th2 type cytokines in lungs of influenza virus-infected
IFN--/- mice, was not sufficient to increase disease
severity or to bias the primary immune response to the degree observed
in the Stat1-/- animals.
We have observed a strong induction of IL-15 mRNA in infected lungs of
the relatively protected wild-type and IFN--/- animals
and reduction or absence of its induction in IFNAR-/- and
Stat1-/- animals which showed exacerbated disease. IL-15
transcription has been shown to be up-regulated by IFN and by
inducers of IFN- (23). Whereas IL-15 mRNA is expressed
abundantly by many tissues (27), protein production has
been identified only in culture supernatants of bone marrow stromal
cells and activated monocytes (22, 28). It is likely that
the IL-15 produced in the lung after infection was made by resident or
infiltrating macrophages in response to infection and/or to type I IFN.
This normal IL-15 induction was absent in both Stat1-/-
and IFNAR-/- macrophages in vitro and in infected
lungs. IL-15 has been shown to play an essential role in NK cell
development and activation as well as in T cell proliferation and
homing (29, 30). Although cytotoxicity by NK cells does
not appear to be essential in protection from influenza virus
(31), a role as cytokine producers may be important for
resolution of disease. In addition, other IL-15 effects may be
important for T cell differentiation, migration, and function in the
setting of acute viral infection. Recent studies showing enhanced
IFN- synthesis by IL-15-treated CD4+
lymphocytes lend support to its role as a T cell modulator
(32).
Another cytokine lacking in infected lungs from Stat1-/-
mice was IL-10 which appeared late during infection of wild-type
animals. Although IL-10 production is usually associated with a Th2
bias, studies of cytokine production profiles in mediastinal lymph
nodes of wild-type mice infected with influenza virus showed a
concurrent synthesis of IL-2, IFN-, and IL-10 (24). We
do not know which population of responding cells within the lung
produced IL-10; IL-10 can be produced by CD4 cells, CD8 cells, B cells,
and macrophages (24, 25). Nor do we know why there is a
lag in its appearance in the lung. We observed induction of IL-10 mRNA
synthesis in infected wild-type and IFN--/- macrophage
cultures, but not in cells derived from IFNAR-/- or
Stat1-/- mice (Fig. 4). The similar lung IL-10 mRNA
profiles in all but the Stat1-/- animals suggests either
a nonmacrophage source at the site of infection, at least in the case
of IFNAR-/- animals, or that tissue macrophages in the
lung respond differently than the bone marrow-derived cells tested in
vitro. It is possible that the presence of IL-10, which can act as a
down-regulatory cytokine, serves to blunt somewhat the proinflammatory
response to influenza virus in IFNAR-/-
animals, resulting in milder inflammation than observed in
Stat1-/- mice. Although the lung pathology in
IFNAR-/- animals was exacerbated compared with wild-type
or IFN--/- mice, it was consistently less severe than
that seen in Stat1-/- or IFNAR/GR-/-
knockouts. The preservation of type II IFN signaling may also act to
modulate inflammation in the lungs of IFNAR-/- mice
relative to Stat1-/- animals.
Therefore, although the Th2-predominant T cell response observed in
Stat1-/- mice after influenza virus infection may
partially account for the altered course of their disease, the absence
of IFN- signaling alone is not sufficient to perturb the primary
immune response to virus infection. Additionally, although Th2
predominance was observed in the progression of disease in
Stat1-/- animals, a substantial population of
IFN--secreting CD8 cells was also present in the
Stat1-/- mice, as were virus-specific cytotoxic T cells
competent for virus elimination. This differs from the Th2 bias that
can be produced by exogenous IL-4 administration during influenza virus
infection which resulted in a marked delay in viral clearance
(33). Thus, the physiological defects resulting from the
absence of Stat1 appear to be at least 2-fold. The first is an
inappropriate production of Th2-type cells, cytokines, and Ig,
apparently due to an inability to prevent Th2 cell differentiation
and/or proliferation normally mediated by both type I and type II IFN.
The second defect is the marked recruitment of proinflammatory
macrophages, neutrophils, and eosinophils to the site of infection, a
defect due substantially to the loss of type I IFN responsiveness and
not due to the Th2 bias alone. This disease pattern is remarkably
reminiscent of vaccine-enhanced respiratory syncytial virus disease and
vaccine-induced atypical measles illness of children where an
inappropriate Th2-like response to some vaccine preparations produces a
debilitating inflammatory response rather than cell-mediated viral
immunity (34, 35). Disregulated IFN production and/or
responsiveness could be a factor in these syndromes.
IL-12- and Stat4-deficient mice also exhibit impaired Th1 and enhanced
Th2 responses (36, 37, 38, 39). Coupled with the results reported
here, these polarized responses suggest that IL-12 is required for Th1
differentiation whereas IL-12-, type I IFN-, and type II IFN-mediated
responses work in concert for suppression of Th2 cells. The finding
that loss of the suppression of Th2 cells mediated by IFN-
(40) does not result in exacerbated influenza
virus-induced pathology implicates IFN- as an important coordinator
of both innate and specific responses to virus infection. Recent
observations that either IL-12 or type I IFN, but not type II IFN, are
capable of inducing the IL-12 receptor ß2-chain and therefore IL-12
responsiveness on human T lymphocytes (41) lend support to
this hypothesis as does the requirement of type I IFN for induction of
IL-15 and IL-10, additional cytokines potentially important for disease
resolution. There may also be an essential role for Stat1-mediated
responses in immunoregulation by cytokines other than the IFNs, for
example, in some responses to IL-12 (42). Along with
recent studies of mice deleted for other Stat genes (38, 39, 43, 44), the results reported here show the intimate relationship
among Stat gene function, the functional diversity of lymphocytes, and
the development of innate and acquired immunity to pathogens.
|
Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. David E. Levy, Department of Pathology, New York University School of Medicine, 550 First Avenue, New York, NY 10016.
3 Abbreviations used in this paper: HA, hemagglutination; i.n., intranasal(ly); TCID, tissue culture infectious dose.
Received for publication August 31, 1999. Accepted for publication February 8, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, 
) and
Stat1-/- C57BL6/J mice (
, •) were immunized with a
sublethal dose of influenza virus A/PR/8, and splenocytes were assayed
for cytotoxic activity against EL4 target cells either without (
