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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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and ß) production after
Pseudomonas aeruginosa corneal infection was examined in
susceptible (cornea perforates) C57BL/6J (B6) and resistant (cornea
heals) BALB/cByJ (BALB/c) mice. IL-1 and -1ß (mRNA and protein)
were elevated in both mouse strains, and levels peaked at 1 day
postinfection (p.i.). Significantly greater amounts of IL-1 protein
were detected in B6 vs BALB/c mice at 1 and 3 days p.i. At 5 days p.i.,
IL-1 and -1ß (mRNA and protein) remained elevated in B6, but began
to decline in BALB/c mice. To test the significance of elevated IL-1 in
B6 mice, a polyclonal neutralizing Ab against IL-1ß was used to treat
infected B6 mice. A combination of subconjunctival and i.p.
administration of IL-1ß polyclonal Ab significantly reduced corneal
disease. The reduction in disease severity in infected B6 mice was
accompanied by a reduction in corneal polymorphonuclear neutrophil
number, bacterial load, and macrophage inflammatory protein-2 mRNA and
protein levels. These data provide evidence that IL-1 is an important
contributor to P. aeruginosa corneal infection. At least
one mechanism by which prolonged and/or elevated IL-1 expression
contributes to irreversible corneal tissue destruction appears to be by
increasing macrophage inflammatory protein-2 production, resulting in a
prolonged stimulation of polymorphonuclear neutrophil influx into
cornea. In contrast, a timely down-regulation of IL-1 appears
consistent with an inflammatory response that is sufficient to clear
the bacterial infection with less corneal
damage. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Many cytokines and chemokines influence PMN influx into tissues,
including the potent proinflammatory cytokine IL-1 (10).
IL-1 is primarily produced by macrophages and monocytes but also by
resident corneal cells (11, 12, 13). It has a wide range of
activities, including mediation of the acute-phase response,
chemotaxis, activation of inflammatory cells and APC (such as
Langerhans cells and macrophages), and stimulation of
neovascularization (10, 13, 14, 15). Prior studies in our
laboratory have examined the role of IL-1 early after P.
aeruginosa infection. Message for IL-1 and -1ß was elevated
as early as 12 h postinfection (p.i.) in the P.
aeruginosa-infected cornea in outbred, resistant (cornea heals)
Swiss ICR mice (16). Protein levels were not tested in
this study. In addition, lack of up-regulation of ICAM-1 was associated
with the dysregulation of IL-1ß protein expression in the corneas of
infected aged Swiss ICR mice (susceptible, cornea perforates)
(17). However, further immune mechanistic studies with
Swiss ICR mice would be limited due to the outbred nature of the
animals. Therefore, to vigorously test the role of IL-1 in P.
aeruginosa ocular infection, the genetic resistance vs
susceptibility model was selected. The current broad hypothesis tested
using this model predicts that balance between the induction of a
protective vs a destructive response to corneal infection is critical.
Furthermore, it is predicted more specifically that prolonged elevation
of IL-1 expression contributes to corneal destruction by continued
stimulation of PMN influx into cornea. To test this, susceptible
C57BL/6J (B6) and resistant BALB/cByJ (BALB/c) mice were tested for
IL-1 and -1ß mRNA and protein expression after P.
aeruginosa infection. In addition, IL-1ß Ab (polyclonal Ab
(pAb)) was administered to susceptible B6 mice to determine whether
this treatment prevented or lessened stromal destruction.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Eight-week-old female BALB/c (resistant, cornea heals) and B6 (susceptible, cornea perforates) mice (The Jackson Laboratory, Bar Harbor, ME) were used in this study. Before corneal infection, mice were anesthetized with isoflurane (Aerrane; Anaquest, Madison, WI) and placed beneath a stereoscopic microscope at x40 magnification. Using a sterile 25 5/8-gauge needle, the central cornea of the left eye was scarified with three 1-mm incisions. Random eyes were routinely examined histologically to ensure that the wounds were shallow and penetrated no deeper than the superficial corneal stroma. A 5-µl bacterial suspension containing 1.0 x 106 CFU of P. aeruginosa (American Type Culture Collection strain 19660) prepared as described previously (18) was topically applied onto the scarified cornea. Eyes were examined macroscopically at 24 h p.i. and at times described below to ensure that all mice were similarly infected and to monitor the course of disease, respectively. All animals were treated humanely and in full compliance with the Association for Research in Vision and Ophthalmology resolution on usage and treatment of animals in research.
Quantitation of corneal IL-1 and -1ß mRNA
RNase protection assays were used to quantitate corneal levels
of IL-1 and -1ß mRNA. IL-1 and -1ß cDNA clones were generated
by RT-PCR using total RNA from P. aeruginosa-infected
corneas as the template for the reverse transcription reaction. PCR
primers were designed (MacVector Software; Oxford Molecular, Madison,
WI) to amplify nt 304–548 of murine IL-1 (accession number X01450)
and nt 294–474 of murine IL-1ß (accession number M15131).
EcoRI and XbaI restriction sites were added to 5'
ends of primers to facilitate ligation of PCR products to the pGEM-3Z
vector. 32P-labeled IL-1 and -1ß antisense
RNA probes and unlabeled IL-1 and -1ß sense-strand RNAs were
generated from the cDNA clones by in vitro transcription.
Sense-strand RNAs were used to produce standard curves to
quantitate amounts of respective mRNA in cornea.
Corneas were collected from BALB/c and B6 mice before and at 6 and
12 h and 1, 3, and 5 days p.i. Immediately after collection,
corneas were flash-frozen in liquid nitrogen and stored at -70°C
until RNA extraction. Eight corneas were pooled, and total RNA was
extracted using RNA STAT-60 (Tel-Test, Friendswood, TX) according to
the manufacturer’s instructions. Five micrograms of total RNA from
each sample was hybridized overnight at 56°C to 300 pg of the IL-1
and -1ß riboprobes. Similarly, various concentrations of the
unlabeled sense-strand standards (5–250 pg) were hybridized to the
same amount of riboprobe. After hybridization, samples were digested
with 1000 U of T1 nuclease (Life Technologies, Gaithersburg,
MD). Nuclease-protected fragments were resolved on a 4.5%
urea-containing sequencing gel. Protected bands were observed by
exposing the dried gel to x-ray film and were quantitated using an
AlphaImager 2000 documentation and analysis
system (Inotech, San Leandro, CA). This experiment was performed at
least three times to ensure reproducibility of the data. Results from
two separate experiments are shown and reported as pg cytokine mRNA/5
µg total RNA.
Quantitation of corneal IL-1 and -1ß protein
Protein for IL-1 and -1ß was determined using ELISA kits
(R&D Systems, Minneapolis, MN). For these studies, individual corneas
(n = 3 at each time point) were collected from mice
before and at 12 h and 1, 3, and 5 days p.i. as described above.
Before storage at -70°C, the total weight of each cornea was
determined. Immediately before analysis, samples were thawed and
homogenized in 0.5 ml of 0.1% Tween 20-PBS with a glass Kontes pestle
(Fischer, Itasca, IL). Samples were centrifuged at 5000 x
g for 10 min, and an aliquot of each supernatant was assayed
for IL-1 and -1ß protein. The sensitivity of the ELISA was 2.5
pg/ml for IL-1 and 3.0 pg/ml for IL-1ß. ELISA experiments were
performed in duplicate to ensure reproducibility of the data. Results
are reported as pg cytokine/mg cornea.
IL-1ß neutralization
Rabbit anti-murine IL-1ß pAb was purchased from PeproTech (Rocky Hill, NJ). The lyophilized powder (500 µg) was reconstituted in 0.5 ml of water as recommended by the manufacturer. B6 mice (n = 5) were anesthetized with Aerrane and were injected with IL-1ß pAb subconjunctivally (10 µg; 1 day before infection) and i.p. (150 µg; 1 day before and 1 and 3 days after infection). Control mice (n = 5) received an equal volume of PBS subconjunctivally and i.p. at the same times. The neutralization experiments were repeated similarly three times to ensure reproducibility of the data.
Ocular response to infection
After P. aeruginosa corneal infection in IL-1ß pAb- vs PBS-treated mice, ocular disease was graded using the following established scale (19): 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. To observe eyes whose lids were sealed, mice were anesthetized with isoflurane, and sterile PBS was applied to the lids to permit their careful partial opening, without inducing corneal perforation. A mean clinical score was calculated for each group of mice (n = 5 for each group) to express disease severity. This was done by summation of the ocular disease scores for each group divided by the total number of mice scored at each time point (18).
Histopathology
For histopathological analysis, eyes were enucleated at 5 days p.i. from three mice from each group (IL-1ß pAb- vs PBS-treated). Eyes were immediately immersed in PBS, rinsed, and placed in a fixative containing 1% osmium tetroxide, 2.5% glutaraldehyde, and 0.2 M Sorenson’s phosphate buffer (pH 7.4) (1:1:1) at 4°C for a total of 3 h. Eyes were transferred into fresh fixative after 1.5 h and then dehydrated in graded ethanols and embedded in Epon-araldite as described previously (18, 20). Thick sections (1.5 µm) were cut, stained with a modified Richardson’s stain, and observed. Representative sections were photographed with a Zeiss Axiophot photomicroscope (Carl Zeiss, Morgan Instruments, Cincinnati, OH) equipped with bright field optics using Ilford pan F film (Mobberley, Cheshire, U.K.).
Quantitation of PMN in cornea
A myeloperoxidase (MPO) assay (20, 21) was used to quantitate the total number of PMN infiltrating the cornea after infection. At 3, 5, and 7 days p.i., three corneas from each group were collected for MPO analysis. Corneas were excised at the limbus with a sterile razor blade, and noncorneal tissue was removed by dissection. After collection, individual corneas were homogenized with a glass Kontes pestle in 1.0 ml of 50 mM phosphate buffer (pH 6.0) containing 0.5% hexadecyltrimethyl-ammonium bromide, freeze-thawed three times, and centrifuged at 14,000 x g for 10 min to remove cellular debris. An aliquot of the supernatant (0.1 ml) was added to 2.9 ml of the 50 mM phosphate buffer containing o-dianisidine dihydrochloride (16.7 mg/100 ml) and hydrogen peroxide (0.0005%). The change in absorbance at 460 nm was continuously monitored for 5 min using a Genesis 2 spectrophotometer (Spectronics, Rochester, NY). The slope of the line was determined for each individual sample and used to calculate the number of units of MPO in the tissue. One unit of MPO activity is defined as that degrading 1 µmol of peroxide per minute (21). To ensure reproducibility of the data, the MPO assay was repeated once similarly. Results are reported as units of MPO/cornea ± SEM.
Quantitation of viable bacteria in cornea
At 5 and 7 days after infection, three corneas from each experimental group were collected for the determination of viable bacteria in infected cornea. Individual corneas were homogenized in sterile 0.9% NaCl containing 0.25% BSA (22). A total of 100 µl of each sample was diluted serially 1:10 in the same solution and plated in triplicate on peptone-tryptic soy agar plates (Difco, Detroit, MI). Plates were 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. This experiment was repeated once to ensure reproducibility of the data. Results are reported as log10 number of CFU/cornea ± SEM.
Quantitation of corneal macrophage-inflammatory protein-2 (MIP-2) level in IL-1ß pAb-treated B6 mice
A RNase protection assay and ELISA were used to quantitate MIP-2 in IL-1ß pAb- vs PBS-treated B6 mice at 5 and 7 days p.i. MIP-2 probe was prepared from cDNA obtained by RT-PCR using total RNA from P. aeruginosa-infected mouse corneas as the template. Five micrograms of total RNA from each group was hybridized to 32P-labeled MIP-2 probe as described above and in previous work (20). The level of MIP-2 protein in each cornea was determined using an ELISA kit (R&D Systems). This was done according to the manufacturer’s instructions as described above and as reported previously (20).
Statistical analysis
An unpaired, two-tailed Student t test was used to
determine statistical significance for ELISA, mean clinical scores, MPO
assays, and bacterial plate counts. Mean differences were considered
significant at the confidence level of p 
0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Corneal IL-1 and -1ß mRNA levels were quantitated in BALB/c
and B6 mice before and after P. aeruginosa challenge. This
was done to determine whether there were differences in regulation
of the proinflammatory cytokines that could contribute to the disparate
response to infection observed between the two mouse strains
(20). IL-1 and -1ß mRNA levels were measured before
and at 6 and 12 h and 1, 3, and 5 days p.i. using RNase protection
assays. Data from two separate but similar experiments are shown in
Table I
. A low level of IL-1 mRNA was
detected in uninfected corneas (time 0) as well as in corneas at 6
h p.i. in both mouse strains. In contrast, IL-1ß mRNA was not
detected at any of these times in either group of mice under the assay
conditions tested. Both IL-1 and -1ß mRNA levels began to rise by
12 h p.i., and peak mRNA expression of both cytokines was detected
in each mouse strain at 1 day p.i. By 5 days p.i., both IL-1 and
-1ß mRNA began to decline in resistant BALB/c mice, but the level of
each cytokine remained elevated in B6 mice.
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The amount of IL-1 and -1ß protein also was determined in
uninfected and infected corneas of resistant vs susceptible mice by
ELISA. Data from a representative experiment are shown in Figs. 1 and 2. A
low level of IL-1 but no -1ß protein was detected in uninfected
corneal tissue in both mouse strains (data not shown). In BALB/c mice,
IL-1 and -1ß was detected in the cornea as early as 12 h p.i.
The level of each cytokine remained relatively constant in BALB/c mice
from 12 h to 5 days p.i. In B6 mice, the level of both IL-1 and
-1ß protein began to rise by 12 h p.i., peaked at 1 day, and
then gradually declined thereafter. The amount of IL-1 was
significantly greater in B6 vs BALB/c mice at 1 (IL-1,
p = 0.0124; IL-1ß, p = 0.0002) and 3
days p.i. (IL-1, p = 0.0059; IL-1ß,
p = 0.0404). Although differences between levels of the
two cytokines in B6 and BALB/c were not statistically significant at 5
days p.i., the concentrations of IL-1 and -1ß in B6 mice were 1.5-
and 1.2-fold greater, respectively, than in BALB/c mice.
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Because both IL-1 and -1ß were produced in significantly
greater amounts from 1 to 3 days p.i. in B6 when compared with BALB/c
mice, we hypothesized that IL-1 promoted the destructive inflammation
observed in the cornea of susceptible B6 mice. Therefore, we next
tested whether the susceptibility phenotype of B6 mice could be altered
by administration of a neutralizing IL-1ß pAb. B6 mice were injected
subconjunctivally with 10 µg of IL-1ß pAb 1 day before and i.p.
with 150 µg 1 day before and 1 and 3 days after ocular challenge. The
ocular response to infection was examined from 1 to 7 days p.i. Mean
clinical scores for the IL-1ß pAb- and PBS-treated mice were
calculated and are shown in Fig. 3. Both
groups initially (1 day p.i.) displayed similar disease grades.
Significant differences between IL-1ß pAb- and PBS-treated mice were
observed at 3, 5, and 7 days p.i. (p = 0.0001
at all three time points, respectively). Representative eyes from both
groups were photographed using a slit lamp at 5 days p.i., and these
data are shown in Fig. 4. IL-1ß
pAb-treated mice showed only slight opacity covering the anterior
segment of the eye (Fig. 4A), whereas PBS-treated mice
exhibited centrally thinned and/or a perforated corneas (Fig. 4B).
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Specifically, in IL-1ß pAb-treated mice, the corneal epithelium was
thinned with wide intercellular spaces between the cells. Nevertheless,
the epithelium remained intact from limbus to limbus and contained few
inflammatory cells. In the stroma, inflammatory cells were seen spread
throughout as well as clustered into densely packed foci of cells.
Also, few infiltrating cells were commonly observed in the peripheral
portion of the anterior chamber between the iris and the corneal
endothelium. The corneal epithelial basal lamina, Descement’s
membrane, and the endothelium also appeared intact. In PBS-treated
control mice, total dissolution of the corneal stroma was observed,
consistent with corneal perforation that was routinely observed in B6
mice within 7 days p.i. The corneal epithelium was denuded centrally,
the stroma was thinned with extensive destruction of collagen
fibers/extracellular matrix, and numerous free bacteria were commonly
observed. A heavy infiltration of inflammatory cells was noted in the
peripheral stroma and anterior chamber. Necrotic and/or apoptotic
inflammatory cells also were observed superficially in the stroma near
large foci of free bacteria.
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Treatment with IL-1ß pAb significantly reduced ocular disease
grades at 3–7 days p.i. as determined by mean clinical score data. To
ascertain whether these data correlated with reduced PMN number in the
cornea of IL-1ß pAb- vs PBS-treated B6 mice, MPO assays were used.
Data from a representative experiment are shown in Fig. 6. At 3 and 7 days p.i., MPO activity was
significantly decreased in IL-1ß pAb- vs PBS-treated mice
(p = 0.0182 and 0.0227, respectively). At 5
days p.i., MPO activity also was reduced in pAb-treated mice, but these
data were not significant (p = 0.4392).
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. Although a slight decrease in
bacterial load was observed in IL-1ß pAb- vs PBS-treated mice at 5
days p.i., these data were not statistically significant. However, at 7
days p.i., there were significantly fewer CFU bacteria
(p = 0.0006) in IL-1ß pAb- vs PBS-treated
mice. These data indicate that treatment with IL-1ß pAb resulted in
reduction in the number of corneal PMN number at 7 days p.i. without a
concomitant increase in bacterial load.
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Because PMN number was reduced after IL-1ß pAb treatment, we
next tested whether this effect was mediated by IL-1 regulation of
other cytokines/chemokines. MIP-2, a potent PMN chemoattractant and the
mouse homologue of IL-8 (23), was shown recently to be
associated with the recruitment and persistence of PMN in the infected
cornea (20). Therefore, we used RNase protection assay and
ELISA to test whether the reduction in PMN number observed in IL-1ß
pAb-treated mice was associated with a concomitant decrease in MIP-2
chemokine production. Although the amount of MIP-2 mRNA transcript
(data not shown) and protein (Fig. 8) was
decreased by 31.7% and 30.0%, respectively, in IL-1ß pAb- vs
PBS-treated mice at 5 days p.i., these data were not significant. At 7
days p.i., a significant decrease in the amount of MIP-2 protein (60%
reduction, p = 0.0245) was observed in IL-1ß
pAb-treated cornea (Fig. 8). These data confirmed that reduction in PMN
number in the cornea of IL-1ß pAb-treated mice was directly
associated with down-regulation of MIP-2 expression.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-1
initiates the host immune response (12). IL-1 also directs
the secretion of other cytokines, including TNF-, IL-6, and IFN-
(12). In the eye, increased IL-1 activity has been shown
previously to correlate with the severity of inflammation in several
experimental models, including the injured vitamin A-deficient rat
cornea (24), the herpes-infected cornea (25),
and corneal allograft rejection (26). In addition,
previous studies from this laboratory showed that differences in
IL-1ß protein expression early after infection (6–48 h p.i.) were
observed in the outbred aged (susceptible, cornea perforates) vs young
(resistant, cornea heals) Swiss ICR mice (17). Based upon
these studies, we postulated that increased expression of IL-1 in
cornea may play a significant regulatory role in the destructive host
response to P. aeruginosa corneal infection. As a corollary
to this, we hypothesized that an appropriate balance of IL-1 expression
in the cornea is consistent with an inflammatory response, which would
be sufficient to reduce the bacterial load yet did not produce
irreversible corneal destruction.
Using RNase protection assays and ELISA, we document that message for
both IL-1 and -1ß was elevated in susceptible B6 and resistant
BALB/c mice within 12 h after infection and reached peak levels of
expression (mRNA and protein) at 1 day p.i. Although the mRNA level of
each cytokine remained elevated in B6 mice, the message for these
cytokines began to decline in BALB/c mice by 5 days p.i. In addition,
although IL-1 and -1ß protein concentrations were significantly
higher in B6 vs BALB/c mice at both 1 and 3 days p.i., protein levels
were relatively constant in resistant BALB/c mice through 5 days p.i.
In B6 mice, the prolonged and elevated expression of IL-1 was
consistent with an increased severity of inflammation, extensive
stromal damage, and corneal perforation. Thus, it appears that the
susceptibility of B6 mice to P. aeruginosa ocular infection
is associated with an aberrant regulation of the proinflammatory
cytokine IL-1. In contrast, the data from resistant BALB/c mice suggest
that 1) an appropriate up- and down-regulation of IL-1 contributes to
wound healing and the reestablishment of corneal integrity, or 2)
BALB/c mice express more effective negative regulators of IL-1 activity
(e.g., IL-1 receptor antagonist) than B6 mice do.
IL-1 and -1ß bind to the same receptor and have very similar if
not identical biological properties. To examine the biological
relevance of elevated IL-1 in corneal infection, we reduced total IL-1
activity in B6 mice using an IL-1-specific neutralizing pAb against
IL-1ß. We hypothesized that reduction in the level of either IL-1
or -1ß in B6 mice would reduce total IL-1 activity sufficiently to
promote an inflammatory response that would allow clearance of bacteria
from cornea without severe corneal disruption. Alternately, because
IL-1 receptor antagonist is structurally related to IL-1ß and also
binds to the same receptor sites as the two molecular forms of IL-1
(27), it is possible that the level of bound antagonist,
coupled with reduction in IL-1ß, together may have resulted in
ameliorated disease. The latter hypothesis remains to be tested. To
directly test the first hypothesis, IL-1ß pAb was injected
subconjunctivally and systemically. The progress of ocular disease was
monitored from 1 to 7 days p.i., and mean clinical scores were
calculated. By 5–7 days p.i., it was apparent that the corneas of
IL-1ß pAb-treated B6 mice exhibited significantly less severe corneal
disease than did those of PBS-injected control animals. These
macroscopic data (both mean clinical scores and slit lamp) were
confirmed by histopathology studies, which provided morphological
evidence that less stromal damage was induced in the cornea of IL-1ß
pAb- vs PBS-treated mice. As a final measure to confirm that pAb
neutralization of IL-1ß resulted in a decrease in the concentration
of IL-1ß in the cornea of pAb-treated mice, ELISA analysis was
performed. These studies (data not shown) confirmed that there was a
significant decrease (p = 0.0168) in IL-1ß
protein in the cornea of pAb- vs PBS-treated control mice at 7
days p.i.
Because administration of IL-1ß pAb significantly reduced ocular disease, the next series of studies were performed to elucidate the possible mechanism(s) by which this occurred. Among the multifaceted roles of IL-1 is its ability to mediate PMN influx to inflamed sites (13). In this regard, infiltration of PMN is a central feature in the ocular pathogenesis of P. aeruginosa (8, 9). Furthermore, recent work from this laboratory has shown that the persistence of an increased number of PMN in cornea at later times p.i. (5 and 7 days) correlated with the development of corneal perforation in susceptible B6 mice (20). Therefore, we next examined whether reduction in ocular disease after IL-1ß pAb treatment coincided with down-regulation in the number of PMN in the cornea. Mice treated with IL-1ß pAb vs PBS demonstrated fewer PMN in the cornea from 3 to 7 days p.i. with significant decreases at both 3 and 7 days p.i. These data demonstrate that IL-1ß can directly or indirectly regulate a PMN response during P. aeruginosa infection and that a decrease in the number of PMN after IL-1ß pAb treatment correlates with less stromal damage.
We next explored the mechanism by which IL-1ß pAb mediated PMN
down-regulation in IL-1ß pAb-treated B6 mice. Recent work from this
laboratory has shown that MIP-2 is a mediator of corneal PMN
infiltration and that persistence of this chemokine correlated with the
susceptible phenotype of B6 mice (20). Furthermore, other
investigators have shown that IL-1 induced MIP-2 production in
different inflammatory models, such as the HSV-1-infected cornea
(25), and in the injured lung (28).
Therefore, we hypothesized that in our susceptible model IL-1ß pAb
treatment down-regulated expression of MIP-2, which in turn led to
fewer PMN in the cornea. To test this hypothesis, MIP-2 mRNA and
protein levels were determined in the cornea of IL-1ß pAb- vs
PBS-treated B6 mice. By 7 days p.i., a significant reduction of MIP-2
protein expression was found in IL-1ß pAb-treated cornea. These data
suggest that at least one function of IL-1ß is to up-regulate MIP-2
production in the cornea of susceptible B6 mice. The decrease in PMN
number in the cornea of IL-1ß pAb-treated mice appears due, at least
in part, to a down-regulation of this chemokine. Thus, from data
reported herein, it appears that IL-1, released early after P.
aeruginosa infection, provides an initial warning signal to induce
the expression of MIP-2, which then augments infiltration of PMN to the
infection site. Confirming reports from another model also support this
conclusion. In the HSV-1-infected cornea, neutralization of IL-1 or
-1ß resulted in substantial inhibition of MIP-2 production
(25).
Because PMN play an important role in bacterial clearance, one would predict that the decrease in PMN number in IL-1ß pAb-treated mice would be associated with enhanced bacterial growth in cornea. To determine whether IL-1ß pAb-treated mice were capable of effectively clearing bacteria from infected tissue, viable bacteria were quantitated in the cornea of IL-1ß pAb- vs PBS-treated mice. Slightly fewer viable bacteria were detected in the cornea of pAb- vs PBS-treated mice at 5 days p.i. but, unexpectedly, by 7 days p.i. the difference was significant between the two groups. These data imply either that IL-1ß pAb-treated B6 mice are capable of more efficient clearance of viable bacteria from the cornea or that bacterial growth and spreading is reduced in the absence of extensive breakdown of stromal proteins by PMN.
In summary, we have investigated the role of IL-1 in P.
aeruginosa corneal infection in two inbred mouse strains, one
susceptible, the other resistant. Our data have shown that the levels
of both IL-1 and -1ß are elevated after infection in both mouse
strains. However, significantly higher concentrations of IL-1 and
-1ß were seen at 1 and 3 days p.i., and the level of expression
remained elevated at 5 days p.i. in susceptible B6 vs resistant BALB/c
mice. We also have shown that administration of IL-1ß pAb
subconjunctivally and i.p. offered an effective means of reducing
host-mediated stromal destruction in susceptible mice. Such treatment
decreased MIP-2 expression and resulted in a reduction in PMN number at
later times p.i. Based on the evidence provided herein, we suggest that
prolonged and elevated IL-1 expression leads to an increase in MIP-2
production, which induces persistence of PMN influx and ultimately
corneal perforation in susceptible mice. In contrast, balanced
regulation of IL-1 facilitates sufficient MIP-2 production to attract a
cellular infiltrate that is sufficient to eliminate the bacteria with
less corneal damage.
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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.
3 Abbreviations used in this paper: PMN, polymorphonuclear neutrophil; pAb, polyclonal Ab; B6, C57BL/6J; BALB/c, BALB/cByJ; p.i., postinfection; MIP-2, macrophage-inflammatory protein-2; MPO, myeloperoxidase.
Received for publication December 13, 1999. Accepted for publication March 28, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 P. Karicherla and J. A. Hobden Nona-D-Arginine Therapy for Pseudomonas aeruginosa Keratitis Invest. Ophthalmol. Vis. Sci., January 1, 2009; 50(1): 256 - 262. [Abstract] [Full Text] [PDF]
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 A. B. Tarabishy, B. Aldabagh, Y. Sun, Y. Imamura, P. K. Mukherjee, J. H. Lass, M. A. Ghannoum, and E. Pearlman MyD88 Regulation of Fusarium Keratitis Is Dependent on TLR4 and IL-1R1 but Not TLR2 J. Immunol., July 1, 2008; 181(1): 593 - 600. [Abstract] [Full Text] [PDF]
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S. A. McClellan, Y. Zhang, R. P. Barrett, and L. D. Hazlett Substance P Promotes Susceptibility to Pseudomonas aeruginosa Keratitis in Resistant Mice: Anti-inflammatory Mediators Downregulated Invest. Ophthalmol. Vis. Sci., April 1, 2008; 49(4): 1502 - 1511. [Abstract] [Full Text] [PDF]
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L. C. Huang, R. Y. Reins, R. L. Gallo, and A. M. McDermott Cathelicidin-Deficient (Cnlp / ) Mice Show Increased Susceptibility to Pseudomonas aeruginosa Keratitis Invest. Ophthalmol. Vis. Sci., October 1, 2007; 48(10): 4498 - 4508. [Abstract] [Full Text] [PDF]
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L. D. Hazlett, Q. Li, J. Liu, S. McClellan, W. Du, and R. P. Barrett NKT Cells Are Critical to Initiate an Inflammatory Response after Pseudomonas aeruginosa Ocular Infection in Susceptible Mice J. Immunol., July 15, 2007; 179(2): 1138 - 1146. [Abstract] [Full Text] [PDF]
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 M. Lin, E. Carlson, E. Diaconu, and E. Pearlman CXCL1/KC and CXCL5/LIX are selectively produced by corneal fibroblasts and mediate neutrophil infiltration to the corneal stroma in LPS keratitis J. Leukoc. Biol., March 1, 2007; 81(3): 786 - 792. [Abstract] [Full Text] [PDF]
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L. D. Hazlett, S. A. McClellan, R. P. Barrett, J. Liu, Y. Zhang, and S. Lighvani Spantide I Decreases Type I Cytokines, Enhances IL-10, and Reduces Corneal Perforation in Susceptible Mice after Pseudomonas aeruginosa Infection Invest. Ophthalmol. Vis. Sci., February 1, 2007; 48(2): 797 - 807. [Abstract] [Full Text] [PDF]
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E. A. Szliter, S. Lighvani, R. P. Barrett, and L. D. Hazlett Vasoactive Intestinal Peptide Balances Pro- and Anti-Inflammatory Cytokines in the Pseudomonas aeruginosa-Infected Cornea and Protects against Corneal Perforation J. Immunol., January 15, 2007; 178(2): 1105 - 1114. [Abstract] [Full Text] [PDF]
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I. Zolfaghar, D. J. Evans, R. Ronaghi, and S. M. J. Fleiszig Type III Secretion-Dependent Modulation of Innate Immunity as One of Multiple Factors Regulated by Pseudomonas aeruginosa RetS Infect. Immun., July 1, 2006; 74(7): 3880 - 3889. [Abstract] [Full Text] [PDF]
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 M Oka, K Norose, K Matsushima, C Nishigori, and M Herlyn Overexpression of IL-8 in the cornea induces ulcer formation in the SCID mouse Br. J. Ophthalmol., May 1, 2006; 90(5): 612 - 615. [Abstract] [Full Text] [PDF]
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E. C. Carlson, J. Drazba, X. Yang, and V. L. Perez Visualization and Characterization of Inflammatory Cell Recruitment and Migration through the Corneal Stroma in Endotoxin-Induced Keratitis Invest. Ophthalmol. Vis. Sci., January 1, 2006; 47(1): 241 - 248. [Abstract] [Full Text] [PDF]
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S. A. McClellan, X. Huang, R. P. Barrett, S. Lighvani, Y. Zhang, D. Richiert, and L. D. Hazlett Matrix Metalloproteinase-9 Amplifies the Immune Response to Pseudomonas aeruginosa Corneal Infection Invest. Ophthalmol. Vis. Sci., January 1, 2006; 47(1): 256 - 264. [Abstract] [Full Text] [PDF]
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X. Huang, R. P. Barrett, S. A. McClellan, and L. D. Hazlett Silencing Toll-like Receptor-9 in Pseudomonas aeruginosa Keratitis Invest. Ophthalmol. Vis. Sci., November 1, 2005; 46(11): 4209 - 4216. [Abstract] [Full Text] [PDF]
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G. Sosne, P. L. Christopherson, R. P. Barrett, and R. Fridman Thymosin-{beta}4 Modulates Corneal Matrix Metalloproteinase Levels and Polymorphonuclear Cell Infiltration after Alkali Injury Invest. Ophthalmol. Vis. Sci., July 1, 2005; 46(7): 2388 - 2395. [Abstract] [Full Text] [PDF]
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 C. Winstanley, S. B Kaye, T. J Neal, H. J Chilton, S. Miksch, C A. Hart, and and the Microbiology Ophthalmic Group Genotypic and phenotypic characteristics of Pseudomonas aeruginosa isolates associated with ulcerative keratitis J. Med. Microbiol., June 1, 2005; 54(6): 519 - 526. [Abstract] [Full Text] [PDF]
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M. R. Power, Y. Peng, E. Maydanski, J. S. Marshall, and T.-J. Lin The Development of Early Host Response to Pseudomonas aeruginosa Lung Infection Is Critically Dependent on Myeloid Differentiation Factor 88 in Mice J. Biol. Chem., November 19, 2004; 279(47): 49315 - 49322. [Abstract] [Full Text] [PDF]
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A. Thakur, R. P. Barrett, J. A. Hobden, and L. D. Hazlett Caspase-1 Inhibitor Reduces Severity of Pseudomonas aeruginosa Keratitis in Mice Invest. Ophthalmol. Vis. Sci., September 1, 2004; 45(9): 3177 - 3184. [Abstract] [Full Text] [PDF]
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Y. Lu, K. Fukuda, K. Seki, Y. Nakamura, N. Kumagai, and T. Nishida Inhibition by Triptolide of IL-1-Induced Collagen Degradation by Corneal Fibroblasts Invest. Ophthalmol. Vis. Sci., December 1, 2003; 44(12): 5082 - 5088. [Abstract] [Full Text] [PDF]
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X. Huang and L. D. Hazlett Analysis of Pseudomonas aeruginosa Corneal Infection Using an Oligonucleotide Microarray Invest. Ophthalmol. Vis. Sci., August 1, 2003; 44(8): 3409 - 3416. [Abstract] [Full Text] [PDF]
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S. A. McClellan, X. Huang, R. P. Barrett, N. van Rooijen, and L. D. Hazlett Macrophages Restrict Pseudomonas aeruginosa Growth, Regulate Polymorphonuclear Neutrophil Influx, and Balance Pro- and Anti-Inflammatory Cytokines in BALB/c Mice J. Immunol., May 15, 2003; 170(10): 5219 - 5227. [Abstract] [Full Text] [PDF]
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M. L. Xue, D. Wakefield, M. D. P. Willcox, A. R. Lloyd, N. Di Girolamo, N. Cole, and A. Thakur Regulation of MMPs and TIMPs by IL-1{beta} during Corneal Ulceration and Infection Invest. Ophthalmol. Vis. Sci., May 1, 2003; 44(5): 2020 - 2025. [Abstract] [Full Text] [PDF]
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N. Cole, M. Krockenberger, F. Stapleton, S. Khan, E. Hume, A. J. Husband, and M. Willcox Experimental Pseudomonas aeruginosa Keratitis in Interleukin-10 Gene Knockout Mice Infect. Immun., March 1, 2003; 71(3): 1328 - 1336. [Abstract] [Full Text] [PDF]
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T.-J. Lin, R. Garduno, R. T. M. Boudreau, and A. C. Issekutz Pseudomonas aeruginosa Activates Human Mast Cells to Induce Neutrophil Transendothelial Migration Via Mast Cell-Derived IL-1{alpha} and {beta} J. Immunol., October 15, 2002; 169(8): 4522 - 4530. [Abstract] [Full Text] [PDF]
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M. S. Gregory, A. C. Repp, A. M. Holhbaum, R. R. Saff, A. Marshak-Rothstein, and B. R. Ksander Membrane Fas Ligand Activates Innate Immunity and Terminates Ocular Immune Privilege J. Immunol., September 1, 2002; 169(5): 2727 - 2735. [Abstract] [Full Text] [PDF]
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B. D. Jett and M. S. Gilmore Internalization of Staphylococcus aureus by Human Corneal Epithelial Cells: Role of Bacterial Fibronectin-Binding Protein and Host Cell Factors Infect. Immun., August 1, 2002; 70(8): 4697 - 4700. [Abstract] [Full Text] [PDF]
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S. Khatri, J. H. Lass, F. P. Heinzel, W. M. Petroll, J. Gomez, E. Diaconu, C. M. Kalsow, and E. Pearlman Regulation of Endotoxin-Induced Keratitis by PECAM-1, MIP-2, and Toll-like Receptor 4 Invest. Ophthalmol. Vis. Sci., July 1, 2002; 43(7): 2278 - 2284. [Abstract] [Full Text] [PDF]
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X. Huang, S. A. McClellan, R. P. Barrett, and L. D. Hazlett IL-18 Contributes to Host Resistance Against Infection with Pseudomonas aeruginosa Through Induction of IFN-{gamma} Production J. Immunol., June 1, 2002; 168(11): 5756 - 5763. [Abstract] [Full Text] [PDF]
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C. M. Pillar and J. A. Hobden Pseudomonas aeruginosa Exotoxin A and Keratitis in Mice Invest. Ophthalmol. Vis. Sci., May 1, 2002; 43(5): 1437 - 1444. [Abstract] [Full Text] [PDF]
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A. Thakur, M. Xue, F. Stapleton, A. R. Lloyd, D. Wakefield, and M. D. P. Willcox Balance of Pro- and Anti-Inflammatory Cytokines Correlates with Outcome of Acute Experimental Pseudomonas aeruginosa Keratitis Infect. Immun., April 1, 2002; 70(4): 2187 - 2197. [Abstract] [Full Text] [PDF]
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L. D. Hazlett, X. L. Rudner, S. A. McClellan, R. P. Barrett, and S. Lighvani Role of IL-12 and IFN-{gamma} in Pseudomonas aeruginosa Corneal Infection Invest. Ophthalmol. Vis. Sci., February 1, 2002; 43(2): 419 - 424. [Abstract] [Full Text] [PDF]
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L. D. Hazlett, S. A. McClellan, X. L. Rudner, and R. P. Barrett The Role of Langerhans Cells in Pseudomonas aeruginosa Infection Invest. Ophthalmol. Vis. Sci., January 1, 2002; 43(1): 189 - 197. [Abstract] [Full Text] [PDF]
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K. A. Kernacki, R. P Barrett, S. McClellan, and L. D. Hazlett MIP-1{alpha} regulates CD4+ T cell chemotaxis and indirectly enhances PMN persistence in Pseudomonas aeruginosa corneal infection J. Leukoc. Biol., December 1, 2001; 70(6): 911 - 919. [Abstract] [Full Text] [PDF]
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S. P. Matzer, T. Baumann, N. W. Lukacs, M. Rollinghoff, and H. U. Beuscher Constitutive Expression of Macrophage-Inflammatory Protein 2 (MIP-2) mRNA in Bone Marrow Gives Rise to Peripheral Neutrophils with Preformed MIP-2 Protein J. Immunol., October 15, 2001; 167(8): 4635 - 4643. [Abstract] [Full Text] [PDF]
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M. Hurt, S. Apte, H. Leher, K. Howard, J. Niederkorn, and H. Alizadeh Exacerbation of Acanthamoeba Keratitis in Animals Treated with Anti-Macrophage Inflammatory Protein 2 or Antineutrophil Antibodies Infect. Immun., May 1, 2001; 69(5): 2988 - 2995. [Abstract] [Full Text] [PDF]
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A. Thakur, J. Kyd, M. Xue, M. D. P. Willcox, and A. Cripps Effector Mechanisms of Protection against Pseudomonas aeruginosa Keratitis in Immunized Rats Infect. Immun., May 1, 2001; 69(5): 3295 - 3304. [Abstract] [Full Text] [PDF]
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K. A. Kernacki, R. P. Barrett, S. A. McClellan, and L. D. Hazlett Aging and PMN Response to P. aeruginosa Infection Invest. Ophthalmol. Vis. Sci., September 1, 2000; 41(10): 3019 - 3025. [Abstract] [Full Text]
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