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Production1
Department of Anatomy/Cell Biology, Wayne State University School of Medicine, Detroit, MI 48201
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-12, and IL-18
was tested in BALB/c mice. RT-PCR analysis detected IFN- mRNA
expression levels in cornea that were significantly increased at 1–7
days postinfection. IL-18 mRNA was detected constitutively in cornea
and, at 1–7 days postinfection, levels were elevated significantly,
while no IL-12 mRNA was similarly detected. To test whether IL-18
contributed to IFN- production, mice were treated with
anti-IL-18 mAb. Treatment decreased corneal IFN- mRNA levels,
and bacterial load and disease increased/worsened, compared with
IgG-treated mice. To stringently examine the role of IFN- in
bacterial killing, knockout (-/-) vs wild-type (wt) mice also were
tested. All corneas perforated, and bacterial load was increased
significantly in -/- vs wt mice. Because disease severity was
increased in IFN--/- vs IL-18-neutralized mice,
and since IL-18 also induces production of TNF, we tested for TNF-
in both groups. ELISA analysis demonstrated significantly elevated
corneal TNF- protein levels in IFN--/- vs wt mice
after infection. In contrast, RT-PCR analysis of IL-18-neutralized vs
IgG-treated infected mice revealed decreased corneal TNF- mRNA
expression. Next, to resolve whether TNF was required for bacterial
killing, TNF- was neutralized in BALB/c mice. No difference in
corneal bacterial load was detected in neutralized vs IgG-treated mice.
These data provide evidence that IL-18 contributes to the resistance
response by induction of IFN- and that IFN- is required for
bacterial killing. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production depends
upon both the presence of IL-12 and the ability of T cells to respond
to this cytokine (5, 6, 7). Recently, IL-12 has been
demonstrated in the cornea of susceptible B6 mice by several
techniques, including RT-PCR and protein analyses (8). In
that study, we also found that IFN- was important in bacterial
killing, but if IL-12-driven IFN- production was sustained or if
IL-12 was endogenously absent, resulting in reduced IFN-, the
susceptibility response occurred, driven either by host factors or
unchecked bacterial growth, respectively. In contrast, preliminary
studies using similar procedures failed to detect IL-12 in the infected
cornea of BALB/c mice, which control infection and restore corneal
clarity (9). Therefore, we predicted that BALB/c mice
would be better able to regulate IFN- production through
IL-12-independent mechanisms, resulting in less corneal toxicity and
destruction.
In this regard, another cytokine, IL-18, shares some of the properties
of IL-12, including inducing production of IFN- by T cells, NK
cells, and NKT cells (10, 11, 12, 13). IL-18 is produced by
macrophages and dendritic cells and, like IL-1, is released as an
inactive precursor, requiring cleavage by IL-1
-converting
enzyme/caspase-1 for its maturation (14, 15, 16). IL-18 is a
costimulus for IFN- production in the setting of microbial
stimulation of macrophage cytokines such as IL-12 and may synergize
with IL-12 to drive Th1 T cell development (7, 17, 18). In
BALB/c mice, the mechanism(s) of resistance, including control of
bacterial load in the cornea, remains incompletely defined. Because
IFN- is an important regulatory cytokine of host defense, often
required for development of innate resistance and control of other
microbial pathogens such as Toxoplasma gondii
(19) and Chlamydia pneumoniae
(20), we began the pathogenesis studies described in this
study by testing for IFN-. We also tested BALB/c mice for IL-12 and
IL-18 mRNA expression in cornea before and following P.
aeruginosa ocular challenge. In addition, a neutralizing,
anti-IL-18 mAb was administered to BALB/c mice to determine whether
this treatment modified the resistance phenotype. The role of IFN-
and TNF- in bacterial killing also was tested in IL-18 and/or
TNF--neutralized and IFN--/- mice that
endogenously lacked the cytokine.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Eight-week-old female BALB/c and
IFN--/- mice on the BALB/c background (The
Jackson Laboratory, Bar Harbor, ME) were used in this study. For
corneal infection, mice were anesthetized (Aerrane; Anaquest, Madison,
WI) and placed beneath a stereoscopic microscope, and the cornea was
scarified, as described before (3, 21). A 5-µl bacterial
suspension containing 1 x 106 CFU/µl
P. aeruginosa strain 19660 (American Type Culture
Collection, Manassas, VA), prepared as described before
(3), was topically applied onto the scarified cornea. Eyes
were examined macroscopically at 1 day postinfection
(p.i.)3 and at times
described below to ensure that all mice were similarly infected and to
monitor the course of disease. Animals were treated humanely and in
full compliance with the Association for Research in Vision and
Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision
Research.
Ocular response to infection
After bacterial infection, corneal disease was graded using an
established scale (22): 0, clear or slight opacity
partially covering the pupil; +1, slight opacity fully covering the
entire anterior segment; +2, dense opacity partially or fully covering
the pupil; +3, dense opacity covering the entire anterior segment; and
+4, corneal perforation. A mean clinical score was calculated for each
group of mice (n = 5/group/treatment) to express
disease severity. Five mice from each group, along with a similar
number of controls, were examined at 1–5 days p.i. in the IL-18 mAb
neutralization and in the IFN--/-
studies.
RT-PCR
Infected corneas were removed from mice before infection and at
6 and 12 h and 1, 3, 5, and 7 days p.i., immediately frozen in
liquid nitrogen, and stored at -70°C. Frozen tissue samples were
homogenized in RNA STAT-60 (Tel-Test, Friendsville, TX), and total RNA
was isolated following the manufacturer’s instruction. Total RNA (100
ng) was reverse transcribed using oligo(dT) primers (Life Technologies,
Grand Island, NY) and reverse transcriptase (Life Technologies) in the
presence of 10 U of RNase inhibitor (Promega, Madison, WI).
Amplification of cDNA was conducted with Taq polymerase
(Life Technologies) and specific primers for IFN-, IL-12, IL-18,
TNF-, and -actin in a GeneMate Thermal Cycler (ISC BioExpress,
Kaysville, UT). Optimum conditions for RT-PCR were established using
routine methods (23). Conditions used were 94°C for
40 s, 60°C for 50 s, and 72°C for 1 min, for 42, 30, 28,
30 cycles for IFN-, IL-12 p40 chain, IL-18, and TNF-,
respectively, and a final extension at 72°C for 10 min. The primers
used were 5'-TGC ATC TTG GCT TTG CAG CTC TTC CTC ATG GC-3' (sense) and
5'-TGG ACC TGT GGG TTG TTG ACC TCA AAC TTG GC-3' (antisense) for
IFN-; 5'-GTG AAC CTC ACC TGT GAC ACG C-3' (sense) and 5'-TGA ATA CTT
CTC ATA GTC CCT TTG G-3' (antisense) for IL-12; 5'-ACC GAA TTC ACT GTA
CAA CCG CAG TAA TAC GGA-3' (sense) and 5'-GCC TCT AGA GTG AAC ATT ACA
GAT TTA TCC CCA-3' (antisense) for IL-18; 5'-GCA AGC TTC GCT CTT CTG
TCT ACT GAA CTT CGG-3' (sense) and 5'-GCT CTA GAA TGA GAT AGC AAA TCG
GCT GAC GG-3' (antisense) for TNF-; and 5'-GTG GGC CGC TCT AGG CAC
CAA-3' (sense) and 5'-CTC TTT GAT GTC ACG CAC GAT TTC-3' (antisense)
for -actin, respectively. Control RT-PCR without reverse
transcriptase during reverse transcription was done to confirm
the lack of DNA contamination in the total RNA samples. A total of 20
µl of final PCR products was analyzed by electrophoresis on 1.2%
agarose gels with SYBR Green I Nucleic Acid Gel Stain (Molecular
Probes, Eugene, OR, and Invitrogen, Carlsbad, CA). The bands were
visualized under UV transillumination and quantitated using an
AlphaImager 2000 Documentation and Analysis system (Alpha Innotech, San
Leandro, CA). Integrated density values (IDV) for the IFN-, IL-12,
IL-18, and TNF- PCR products were corrected for the amount of
-actin on each sample. Data are expressed as the mean IDV of samples
from five separate mice for each experimental time point.
mAb treatment
Neutralizing rat anti-mouse IL-18 (endotoxin <10 ng/ml Ab)
and TNF- (endotoxin not measured) mAbs, both of IgG1 isotype, were
purchased from Medical & Biological Laboratories (Naka-Ku Nagoya,
Japan) and BioSource International (Camarillo, CA), respectively.
BALB/c mice (n = 5/group) were injected
subconjunctivally 1 day before infection with 10 µl (10 µg/cornea)
of anti-IL-18 or TNF- mAb. At 4 h and again at 2 days p.i.,
each mouse was injected i.p. with 150 µl (150 µg) of anti-IL-18
or TNF- mAb diluted in 0.05% PBS. Control mice similarly received
an equal volume of rat IgG (Sigma-Aldrich, St. Louis, MO) diluted in
0.05% PBS.
Quantitation of viable bacteria in cornea
At 3 and 5 days p.i., three corneas from each experimental group
(anti-IL-18 and TNF- mAb- vs rat IgG-treated mice and at 1, 3,
and 5 days p.i. from IFN--/- vs wild-type
(wt) BALB/c mice) were collected, and the number of viable bacteria was
quantitated. For this, individual corneas were homogenized in sterile
0.9% NaCl containing 0.25% BSA (4). A total of 100 µl
of each sample was diluted serially 1/10 in the same solution, plated
in triplicate on Pseudomonas isolation agar (Difco, Detroit,
MI) plates, and incubated overnight at 37°C. The number of viable
bacteria in an individual cornea was determined by counting individual
colonies on plates from the various dilutions and multiplying the
number of colonies by the appropriate dilution. Results are reported as
log10 number of CFU/cornea ± SEM.
Histopathology of IFN--/- vs wt BALB/c mice
For histopathology, eyes from three mice of each group were enucleated at 5 days p.i., immersed in PBS, rinsed, and fixed in 1% osmium tetroxide, 2.5% glutaraldehyde, and 0.2 M Sorenson’s phosphate buffer (pH 7.4; 1:1:1) at 4°C for 3 h. Eyes were dehydrated in ethanols and embedded in Epon-araldite, and thick sections (1.5 µm) were cut, stained, observed, and photographed, as described before (3, 4, 21).
Quantitation of TNF- protein
TNF- protein levels were determined using an ELISA kit (R&D
Systems, Minneapolis, MN), as described previously (21).
Individual corneas (n = 5/time/group) were collected
from -/- and wt as well as TNF--neutralized and rat IgG-treated
control mice at 3 and 5 days p.i., the total weight of each cornea was
determined, and samples were immediately analyzed. Samples were
homogenized with a glass pestle (Kontes; Fischer, Itasca, IL) and
centrifuged, and an aliquot of each supernatant was assayed for TNF-
protein levels. The sensitivity of the assay was 5.1 pg/ml.
Statistical analysis
An unpaired, two-tailed Student’s t test was used to determine statistical significance for data from RT-PCR, mean clinical scores, bacterial counts, and ELISA analyses. Mean differences were considered significant at the confidence level of p = 0.05. All experiments were repeated at least twice to ensure reproducibility, and representative data from a single experiment are shown.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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mRNA expression in the cornea
To determine whether IFN- was produced in the cornea of BALB/c
mice before and after infection with P. aeruginosa, we
tested for mRNA expression levels in uninfected and infected corneas
using RT-PCR. Data from a representative experiment are shown in Fig. 1
. IFN- mRNA transcripts were not
detected in the uninfected (0 h) cornea nor at 6 or 12 h p.i.
(data not shown) in BALB/c mice. Readily detectable low levels of
IFN- mRNA were seen at 1 day p.i. in the cornea, and these levels
continued to rise at 3 and peaked at 5 days p.i. By 7 days p.i.,
cytokine expression levels had decreased, but still remained
significantly greater when compared with 3-day values. Statistically,
IFN- mRNA levels in cornea were significantly increased
(p = 0.02, 0.0006, 0.0001, and 0.001 at 1, 3,
5, and 7 days p.i., respectively) when compared with levels in the
uninfected cornea.
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Since mRNA for IFN- was detected in the infected cornea of
BALB/c mice, we next tested the infected cornea for the presence of
IL-12, a major IFN--inducing cytokine. In preliminary
(9) and the current experiments (Fig. 2), IL-12 p40 was not detectable at any
time p.i. in the cornea of BALB/c mice. We then tested for the presence
of IL-18, a cytokine, with functional similarities to IL-12
(10, 11, 12, 13). Low levels of mRNA transcripts for IL-18 were
constitutively expressed in the uninfected BALB/c mouse cornea (Fig. 3). At 6 and 12 h p.i., mRNA levels
began to rise, but neither time point was significantly different from
expression levels detected in the uninfected cornea. However, by 1 day
p.i., IL-18 mRNA levels were significantly elevated, peaked at 3 days
p.i., and remained significantly elevated at 5 and 7 days p.i. when
compared with constitutive levels of expression
(p = 0.664, 0.36, 0.001, 0.0001, 0.0001, 0.0001
at 6 and 12 h and 1, 3, 5, and 7 days p.i., respectively).
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Because our data showed that both IL-18 and IFN- mRNA levels
were significantly increased in the BALB/c mouse cornea after bacterial
infection, the next series of in vivo studies tested the significance
of these data. mAb neutralization of IL-18 was used to determine
whether IL-18 induced IFN- production in the cornea after bacterial
infection. Corneal IFN- mRNA expression was analyzed by RT-PCR at 3
and 5 days p.i., and the data are shown in Fig. 4. Treatment with anti-IL-18 mAb led
to a significant decrease in IFN- (p = 0.007
and 0.02 at 3 and 5 days p.i., respectively) when compared with mRNA
levels of cytokine in the cornea of rat IgG-treated control mice. Mean
clinical scores in IL-18 mAb- vs rat IgG-treated mice showed a trend
for disease worsening, but corneal perforation did not occur in the
IL-18-neutralized mice (data not shown).
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We next tested whether mAb neutralization of IL-18, resulting in
decreased mRNA expression for IFN-, contributed to increased
bacterial growth in the cornea. Direct plate count was used to
quantitate bacterial load in the cornea of IL-18 mAb vs rat IgG-treated
mice at 3 and 5 days p.i. The mean log10 number
of viable bacteria per cornea (±SEM) is shown in Fig. 5. A significant increase in bacterial
load (1- to 2-log increase, p = 0.0014 and 0.002 at 3 and 5
days p.i., respectively) was observed in the cornea of anti-IL-18
mAb- vs rat IgG-treated mice.
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-/- mice
To assure that IFN- expression was required for bacterial
killing in resistant BALB/c mice, corneas of
IFN--/- vs wt BALB/c mice were infected with
P. aeruginosa, and ocular disease was graded. Mean clinical
scores were significantly different at 3 and 5 days p.i.
(p = 0.0003 and 0.0001 at 3 and 5 days p.i.,
respectively) in IFN--/- vs wt mice (Fig. 6). In addition, all of the corneas
perforated in the -/- animals at 5 days p.i., in contrast with a +2
ocular disease grade observed in wt mice. Viable bacteria in the cornea
of IFN--/- vs wt mice also were quantitated
at 1, 3, and 5 days p.i. (Fig. 7). A
significantly increased number of bacteria was detected in the -/- vs
wt mouse cornea at all times tested (p = 0.011,
0.006, and 0.001 at 1, 3, and 5 days p.i., respectively).
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At 5 days p.i., eyes from IFN--/- and
wt BALB/c mice were enucleated and prepared for histopathology, and
these data are shown in Fig. 8. The
cornea of the -/- mice lacked epithelium; stromal cytoarchitecture
was destroyed; and perforation, observed visually by mean clinical
score grading, was confirmed. In contrast, the cornea of the wt mice
had begun to reepithelialize, less stromal damage was apparent, and no
perforation was observed. Inflammatory infiltrate, however, remained
and was concentrated in the deep stroma and anterior chamber.
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Because the cornea of anti-IL-18 mAb-treated infected mice did
not perforate and corneal perforation was routinely observed at 5 days
p.i. in IFN--/- mice, we postulated that
other proinflammatory cytokines must be affected in the -/- mice that
contributed to corneal perforation. ELISA analysis (Fig. 9) revealed that the cornea of the -/-
vs wt BALB/c mouse had significantly elevated TNF- protein levels at
3 days p.i. (p = 0.016) that remained elevated,
but not significantly, at 5 days p.i. (p =
0.197). TNF- mRNA levels in cornea were then tested in
IL-18-neutralized vs IgG-treated mice at similar times p.i. TNF-
levels were significantly (p = 0.001 and 0.02,
at 3 and 5 days p.i., respectively) decreased in IL-18- vs IgG-treated
mouse cornea (Fig. 10).
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Data from the IFN--/- experiment
suggested that despite elevated levels of TNF-, without IFN-,
bacterial killing was impaired. In addition, data from the IL-18
neutralization study showed that after neutralization of IL-18, both
IFN- and TNF- levels decreased significantly. To resolve the role
of TNF- in bacterial killing, TNF- neutralization studies were
done using BALB/c mice. ELISA analysis confirmed that TNF- protein
levels were decreased (p = 0.04 at both 3 and 5
days p.i.) in neutralized vs IgG control-treated mice (Fig. 11). At 3 and 5 days p.i., no
difference (p = 0.256 and 0.854) in viable
bacterial counts was seen in TNF-- vs rat IgG-treated mouse cornea,
indicating that TNF- is not required for bacterial killing (Fig. 12).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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is an important regulatory cytokine of host defense in
both innate and acquired immunity (24, 25, 26). It plays a
critical role in inflammation and regulates the
antimicrobial/tumoricidal potential of macrophages by up-regulation of
MHC class I and class II protein expression; enhanced production of
macrophage-derived mediators such as TNF-, IL-1, IL-6, IL-12, IL-18,
and NO; and down-regulation of the synthesis of anti-inflammatory
mediators such as IL-10 (27, 28, 29, 30). Scharton-Kersten et al.
(19) demonstrated that in the absence of endogenous
IFN-, -/- mice failed to control acute infection with T.
gondii and no evidence for an IFN--independent protective
function was apparent. Similar results were reported recently showing
that IFN- was necessary for development of innate resistance against
infection with C. pneumoniae. In contrast, a
previous study using mAb neutralization of IFN- in susceptible B6
mice suggested that prolonged and elevated expression of IFN- was
associated with the susceptibility response and corneal perforation
(3), and further work showed that significantly reduced
levels of IFN- were equally deleterious (8). Since no
information existed on the role of IFN- in the BALB/c mouse and
development of the resistance phenotype, we tested for this cytokine.
Our data showed that IFN- was detectable at 1, peaked at 5, and
declined at 7 days p.i. in the infected cornea of these mice,
suggesting that tighter regulation of IFN- might be important in
resistance to P. aeruginosa infection. Direct comparative
measurement of IFN- levels in infected cornea of B6 vs BALB/c mice
would directly test this hypothesis, but has not yet been done.
Nevertheless, together, these data support the tenet that controlled
up- and then down-regulation vs persistence of IFN- in the cornea
leads to resolution of disease in BALB/c vs tissue destruction and
perforation, in B6 mice.
We also tested and confirmed preliminary data that BALB/c, unlike B6,
mice (8) do not express detectable levels of IL-12 after
P. aeruginosa infection. The absence of IL-12 in infected
cornea of BALB/c vs B6 mice implies that IL-12-driven production of
IFN- may in turn positively regulate IL-12, establishing a dangerous
loop that leads to excessive proinflammatory production, toxicity, and
corneal perforation, as seen in B6, but not BALB/c, mice. Next, because
IL-18 is known to induce the synthesis of IFN-, often in
collaboration with IL-12 (10, 24, 25, 26, 31), we tested for
IL-18 expression in the uninfected and infected BALB/c mouse cornea.
Rationale for these studies was provided by the experiments of Muller
et al. (12), who demonstrated that IL-12-independent
IFN- production in experimental Chagas’ disease is mediated by
IL-18. Kawakami et al. (11) also recently reported that
IL-18 contributes to host resistance against infection with
Cryptococcus neoformans in mice with defective IL-12
synthesis through induction of IFN- synthesis. Furthermore, in IL-12
p40-/--infected mice, low serum levels of
IFN- (20–30% of that in wt mice) were detected after infection
with C. neoformans, which further indicated the existence of
IL-12-independent mechanisms for IFN- production and eradicating
this pathogen.
After establishing elevated levels of IL-18 mRNA expression in the
infected BALB/c mouse cornea, we used Ab neutralization to determine
whether IL-18 induced production of IFN-. Neutralization of IL-18
significantly reduced corneal levels of IFN- mRNA compared with
control mice (Fig. 4
), implicating the importance of IL-18 in induction
of IFN- in the infected cornea. Whether IL-18 neutralization induced
systemic effects in draining cervical lymph nodes, for example, was not
tested, due to the lack of T cell participation in corneal pathogenesis
in these mice, but such effects cannot be ruled out by this study.
We next determined whether IFN- was required for bacterial killing,
by quantitating bacteria in the cornea of anti-IL-18 mAb-treated
mice. Viable bacterial number was increased 
2 logs (Fig. 5) in
mAb-neutralized vs control-treated mice, implying that IL-18 protects
mice against P. aeruginosa infection by inducing IFN-
production and bacterial killing in the cornea. To confirm the
importance of IFN- in this model, IFN--/-
BALB/c mice also were tested. Mean clinical score data and
histopathology showed that IFN--/- vs wt
mice were susceptible to P. aeruginosa infection, and by 5
days p.i. all of the infected corneas of the -/- mice had perforated,
whereas the corneas of wt BALB/c had begun to recover. Furthermore,
viable bacterial load increased significantly (1–2 logs) in the cornea
of -/- vs wt BALB/c mice (Fig. 7). Together, these data further
suggested that IFN- is critical in bacterial killing in P.
aeruginosa-induced keratitis and development of the resistance
response. However, the precise mechanism(s) whereby IFN- contributes
to bacterial killing in this model remains untested. In this vein,
other studies have provided direct evidence of IFN- killing of
Legionella pneumophila after administering an adenovirus
vector containing murine IFN- cDNA concomitant with the bacterial
inoculum and showed a 10-fold decrease in lung bacterial CFU compared
with controls (32). Also in studies with C.
pneumoniae, IFN- was found necessary for control of
bacterial load by increasing NO release and superoxide peroduction,
both of which the authors concluded related to bacterial killing
(20).
Nonetheless, a difference was noted between IL-18 mAb-neutralized vs
-/- mice, namely, that although disease worsened in the
Ab-neutralized mouse cornea, none of the corneas perforated, whereas
all infected corneas in the IFN--/- animals
perforated after infection. Thus, we next predicted that although
IFN- is important in clearance of P. aeruginosa in the
cornea, other cytokines may also be required for killing. Others using
IFN--/- mice in a viral infection model
(33) similarly concluded that although IFN- played an
important role in the clearance of HSV from the eye, the pathogenesis
of herpetic stromal keratitis lesions involved additional cytokines.
However, to the best of our knowledge, no other cytokines were tested
in that study.
In this regard, although IL-18 exerts some of its proinflammatory
effects through induction of IFN- (11, 34), recent data
suggest that IL-18 also induces TNF- production through stimulating
activation of NF-
B (17), inducing production of not
only TNF-, but IL-1 and chemokines such as IL-8 and
macrophage-inflammatory protein-1 (35). All of the
latter cytokines, except TNF-, have been shown previously to play
critical roles in T cell chemotaxis and polymorphonuclear neutrophil
(PMN) persistence following P. aeruginosa-induced corneal
infection (21, 36, 37).
TNF- has been shown to play a crucial role in response to tissue
injury, infection, and inflammation (38). Siegmund et al.
(39) demonstrated that neutralization of IL-18 reduced
disease severity in murine colitis and reduced intestinal IFN- and
TNF- production. Moreover, similar results were reported by Netea et
al. (17), who demonstrated that neutralization of IL-18
during lethal endotoxemia protected mice against the lethal effects of
LPS, by reduction of TNF- and PMN infiltration. IL-18 also
up-regulates expression of adhesion molecules such as ICAM-1
(40), also shown to be of importance in PMN infiltration
into cornea in ocular models of P. aeruginosa infection
(41). Therefore, we next tested for TNF- levels in
IFN--/- and IL-18-neutralized mice. ELISA
analysis confirmed significantly elevated levels of TNF- protein in
cornea in IFN--/- vs wt BALB/c mice at 3
days p.i. with levels remaining elevated, but not significant, at 5
days p.i. (Fig. 9). These data support the importance of IFN- in
bacterial killing and imply that in the absence of endogenous IFN-,
TNF- alone, even at elevated levels, does not contribute to
bacterial killing, but rather, may contribute to increased pathology
and corneal perforation. In contrast, in IL-18-neutralized mice, RT-PCR
revealed decreased levels of TNF- at similar time points. Thus,
neutralization of IL-18 significantly decreased TNF- as well as
IFN- levels when compared with levels in controls. To further test
the role of TNF- in bacterial killing, we neutralized TNF in BALB/c
mice. No difference was detected in corneal bacterial load in
mAb-neutralized vs IgG-treated mice, confirming that TNF- is not
critical for bacterial killing. In P. aeruginosa-induced
models of pneumonia, TNF- is regarded as somewhat a double-edged
sword, and the role of the cytokine remains controversial. It has been
reported as necessary for PMN recruitment and bacterial clearance in
mice (42), but in a rat model, levels of IL-1 and
TNF- increased consistently following infection until death,
implicating these cytokines in the pathogenesis of acute P.
aeruginosa-induced pneumonia (43). We hypothesize,
but have not tested, that tissue-specific mechanisms of bacterial
killing (cornea vs lung) are a contributing factor to these disparate
data in the mouse.
In summary, the data presented demonstrate that IFN- is produced in
BALB/c mice following ocular bacterial challenge and that IL-18 plays
an important role in inducing production of the cytokine. We also
provide evidence that neutralization of IL-18 decreased both IFN-
and TNF- production in cornea and that neutralization of TNF-
does not significantly change bacterial load in the cornea when
compared with IgG-treated mice. Data from the
IFN--/- studies also imply that elevated
levels of TNF-, in the absence of IFN-, fail to control bacterial
load, and suggest that TNF- contributes to corneal pathogenesis and
perforation.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Linda D. Hazlett, Department of Anatomy/Cell Biology, Wayne State University School of Medicine, 540 East Canfield Avenue, Detroit, MI 48201. E-mail address: lhazlett{at}med.wayne.edu
3 Abbreviations used in this paper: p.i., postinfection; IDV, integrated density value; PMN, polymorphonuclear neutrophil; wt, wild type.
Received for publication December 20, 2001. Accepted for publication March 19, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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