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From the Department of Anatomy and Cell Biology, Wayne State University School of Medicine, Detroit, Michigan.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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METHODS. Total PMN number, macrophage inflammatory protein (MIP)-2 mRNA and protein expression, and ocular bacterial load were determined in 8-week- and 12-month-old inbred BALB/c mice at various times after infection with P. aeruginosa. In addition, 12-month-old mice were treated systemically with the MIP-2 polyclonal antibody (pAb) to determine the effects of MIP-2 neutralization on ocular disease and PMN recruitment.
RESULTS. Histologically, PMN infiltration into the cornea of 12-month-old mice was delayed initially and was associated with an inability to reduce bacterial load at later postinfection (PI) times. In addition, a significantly greater number of PMNs were found in the cornea of 12-month-old mice at later PI times. The increase in PMN number in 12-month-old mice correlated with a persistence of MIP-2 expression in cornea at these later times. Systemic treatment of 12-month-old mice with neutralizing MIP-2 pAb versus normal rabbit serum (NRS) resulted in reduced corneal PMN number and ocular disease.
CONCLUSIONS. These data provide evidence that persistence of PMN in the cornea of 12-month-old mice contributes to corneal tissue destruction after P. aeruginosa challenge. Further evidence also is provided that the chemoattractant MIP-2 contributes to the altered PMN response in these animals.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous experimental data from this laboratory using a young (8-week-old) versus aged (12-month-old) outbred mouse model suggest that susceptibility to P. aeruginosa challenge involves defects in inflammatory mediator expression and/or PMN function.9 10 11 12 13 The current studies were performed to elucidate which specific host factors (e.g., cytokines and/or chemokines) contribute to the extensive stromal destruction and corneal perforation in 12-month-old animals.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Ocular Response to Infection
After P. aeruginosa corneal infection, ocular disease
was graded using the following established scale15
: 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. Five mice from
each experimental group were examined from postinfection days (DPI) 1
to 7. The experiment was repeated once similarly, and representative
data are shown.
Histopathology
For histopathologic analysis, whole eyes were enucleated from
three mice from each experimental group (8-week- versus 12-month-old
BALB/c mice) at 1 and 5 DPI. Eyes were enucleated and immersed
immediately in phosphate-buffered saline (PBS), rinsed, and placed in a
fixative containing 1% osmium tetroxide, 2.5% glutaraldehyde, and 0.2
M Sörensen’s phosphate buffer (pH 7.4; 1:1:1) at 4°C for a
total of 3 hours. Eyes were then dehydrated in graded ethanols and
embedded in Epon-Araldite, as described previously.14
Thick sections (1.5 µm) of the eye were cut from each group of three
mice, stained with a modified Richardson’s stain, and observed.
Representative sections were photographed with a photomicroscope
(Axiophot; Carl Zeiss, Thornwood, NY) equipped with bright field optics
and film (pan F film; Ilford, Cheshire, UK).
Quantitation of PMN in Corneal Tissues
A myeloperoxidase (MPO) assay was used to quantitate the total
number of PMN in cornea after infection.16
17
At selected
times after infection, three corneas from each experimental group were
collected for analysis. After collection, individual corneas were
immersed in 1.0 ml of 50 mM phosphate buffer (pH 6.0) containing 0.5%
hexadecyltrimethylammonium bromide. Samples were sonicated for 10
seconds on ice and subsequently freeze thawed three times. After
centrifugation, 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 absorbancy at 460 nm was monitored for 5 minutes using a
spectrophotometer (Genesis 2; Spectronics, Rochester, NY). The slope of
the line was determined for each sample and used to calculate units of
MPO in the tissue.17
Results are reported as units of
MPO/cornea. The experiment was repeated once similarly, and
representative data from a single experiment are shown.
Quantitation of Viable Bacteria in Corneal Tissues
Three corneas from each experimental group were collected at
select times after infection for the quantitation of viable bacteria.
Individual corneas were homogenized in sterile 0.9% NaCl containing
0.25% BSA.18
Serial 10-fold dilutions of the samples were
plated on Pseudomonas isolation agar, and plates were
incubated overnight at 37oC. Results from one
of two separate similar experiments are reported as
log10 number of CFU/cornea.
Quantitation of MIP-2 mRNA
RNase protection assays (RPAs) were used to quantitate the level
of macrophage inflammatory protein (MIP)-2 mRNA in cornea, as described
previously.19
Briefly, corneal tissue was collected from
8-week- and 12-month-old mice before and at various times after
infection. Samples were flash frozen in liquid nitrogen and stored at
-70°C until extraction of RNA. Four corneas were pooled for an
individual sample. Total RNA was extracted from corneal tissue using
RNazol B (Tel-Test; Friendsville, TX). Three micrograms of total RNA
from each sample were hybridized overnight at 56°C to 300 pg of
32P-labeled, antisense strand MIP-2 riboprobe.
Similarly, various concentrations of unlabeled sense strand MIP-2
standard (7.8–250 pg) were hybridized to the same amount of riboprobe.
After hybridization, samples were digested with T1 nuclease (Gibco,
Gaithersburg, MD), and 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 quantitated using a
densitometer with image analysis software (MDX Persen Densitometer S II
and Image Quant Densitometric software; Molecular Dynamics; Sunnyvale,
CA). Representative results from two similar experiments are reported,
and the data expressed as atomoles chemokine mRNA per microgram
total RNA.
Quantitation of MIP-2 Protein Levels
Corneal MIP-2 protein levels were determined using an
enzyme-linked immunosorbent assay (ELISA) kit (R&D; Minneapolis, MN),
as described previously.19
For these studies, individual
corneas (n = 3/time point) were collected from mice
before and at various times after infection. Before storage at
-70°C, the total weight of each cornea was determined. Corneas were
then stored in 1.0 ml of PBS containing 0.5% Tween 20. Immediately
before analysis, samples were thawed and homogenized with a glass
pestle (Kontes; Fischer, Itasca, IL). After centrifugation, an aliquot
of each supernatant was assayed for MIP-2 protein. Representative
results from one of two similar experiments are reported, and the data
expressed as picograms MIP-2 per milliliter corneal tissue.
MIP-2 Neutralization
Neutralizing polyclonal antibody (pAb) to murine
MIP-220
was kindly provided by Nicholas Lukacs (Department
of Pathology, University of Michigan, Ann Arbor). This pAb (1:1000
dilution) has been shown previously to completely neutralize PMN
chemotaxis to 30 ng of murine MIP-2 per milliliter.20
For
the MIP-2 neutralization studies, 12-month-old BALB/c mice
(n = 5) were anesthetized with isoflurane and injected
intraperitoneally with 0.2 ml of neutralizing MIP-2 pAb at 1 day before
and at 1 and 3 days after infection. Control mice (n =
5) were similarly treated with 0.2 ml of preimmune, normal rabbit serum
(NRS). This experiment was repeated similarly three times, and
representative data from one experiment are presented.
Statistical Analysis
An unpaired, two-tailed Student’s t-test was used to
determine statistical significance for the ocular disease grades, MPO
assays, bacterial plate counts and ELISAs. 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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Histopathology
Corneas of 8-week- and 12-month-old BALB/c mice also were examined
histopathologically at 1 and 5 DPI and marked differences in the
progression of corneal disease were observed (Fig. 1)
. At 1 DPI, the epithelium was irregular (patchy or denuded centrally
while intact peripherally) in the cornea of 8-week-old mice (Figs. 1A
1C)
. Numerous PMN were observed in the central (Fig. 1A)
and peripheral
(Fig. 1C) cornea, and corneal swelling was modest. In contrast, in
12-month-old mice, PMN were not observed in the central cornea at 1 DPI
(Figs. 1B 1D)
. Instead, PMN were distributed throughout the entire
thickness of the peripheral cornea (Fig. 1D)
, but centripetally
migrating cells were also evident. Similar to 8-week-old mice, the
epithelium was patchy or denuded in the central cornea of 12-month-old
mice, but remained intact at the periphery. Figure 1E
shows the cornea
of an 8-week-old mouse assigned a +1 ocular disease grade at 5 DPI. In
this cornea, the epithelium had resurfaced almost completely, and
collagen fibrils in the stroma appeared intact and regularly spaced.
Only a few PMN remained in the cornea of 8-week-old mice at this time.
At a similar time in 12-month-old mice, the epithelium was completely
stripped in the central cornea (Fig. 1F) . Numerous PMN were present
throughout the entire thickness of the cornea and in the anterior
chamber. Extensive degradation of stromal collagen fibrils was observed
and was accompanied by pronounced corneal edema. These findings are
consistent with an ocular disease grade of +4 (corneal perforation) in
aged animals.
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Because disease activity was reduced in MIP-2 pAb–treated 12-month-old mice, corneal MPO activity was measured in the two experimental groups to determine whether an effect of MIP-2 pAb treatment was the reduction of corneal PMN number. These data are shown in Figure 5 . A similar amount of MPO activity was detected in the cornea of MIP-2 pAb– and NRS-treated mice at 3 DPI. In contrast, mice treated with MIP-2 pAb versus NRS had significantly less corneal MPO activity at 5 DPI (P = 0.0347).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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When progression of ocular disease was monitored macroscopically in BALB/c mice, these data showed that 8-week- and 12-month-old inbred mice responded to P. aeruginosa challenge similar to outbred animals.12 That is, corneal perforation was observed in all 12-month-old mice by 7 DPI, whereas corneal healing had begun in 8-week-old mice. Corneal histopathology confirmed the macroscopic observations that PMN were reduced or absent from the central cornea initially (up to 1 DPI) in 12-month-old mice. Whereas the histopathology data showed marked differences in the distribution of PMN in the cornea of 8-week- versus 12-month-old mice at 1 DPI, the overall total number of PMN, as measured by MPO activity, was unexpectedly similar in both groups. In contrast, by 5 DPI, histopathology and MPO assays confirmed similarly that PMN were distributed throughout the entire cornea and were more numerous, respectively, in the cornea of 12-month- versus 8-week-old mice.
The next series of experiments tested whether this histologically observed absence of PMN in the cornea was associated with impaired clearance of P. aeruginosa from the cornea of 12-month-old animals. A similar bacterial load was found in the cornea of 8-week- and 12-month-old mice at 1 and 3 DPI, but as early as 4 DPI, 8-week-old mice exhibited a significant reduction in bacterial number. In contrast, bacterial number did not begin to decline in the cornea of 12-month-old mice until 8 DPI, after corneal perforation had already occurred. From these data, we hypothesize that the early absence of PMNs from the central cornea of 12-month-old mice may allow for continued replication of bacteria in the cornea. This increase in bacterial number theoretically would require a greater number of PMNs to ultimately clear the infection. Both the histopathology and MPO data support this theory. In addition to age-related defects in PMN localization in cornea, other functional defects such as in phagocytosis of P. aeruginosa also may contribute to the impaired clearance of bacteria in 12-month-old mice. In this regard, in vitro studies have shown impaired uptake and killing of various pathogens, including P. aeruginosa, by PMN isolated from aged individuals or rodents.3 21 22
It is also possible that aberrations in the early synthesis and
secretion of inflammatory mediators may account for the initial
defective PMN response in 12-month-old animals.4
5
13
Accordingly, we showed previously that a reduction in interleukin
(IL)-1ß and interferon (IFN)-
levels during the first 12 hours
after infection in outbred 12-month-old mice correlated with the
absence of corneal intercellular adhesion molecule (ICAM)-1
upregulation and PMN infiltration. Alternatively, previous studies
using 8-week-old inbred mice classified as either susceptible or
resistant to corneal P. aeruginosa challenge suggested that
the early PMN response to infection was not mediated by the PMN
chemoattractant MIP-2.19
We have provided evidence that
this is also the case with 8-week-versus 12-month-old mice, because
similar amounts of MIP-2 mRNA and protein were found in the cornea of
both experimental groups at 12 hours and 1 DPI.
In contrast to the early response to infection, our previous studies using the 8-week-old genetically susceptible–resistant mouse model system showed that continued increased expression of MIP-2 and interleukin (IL)-1 in the cornea of a susceptible strain of mice at later times after infection was associated with the persistence of PMN and ultimate corneal perforation.19 23 The current studies also show a similar persistence of MIP-2 expression in the cornea of 12-month-old susceptible animals. Increased later expression of MIP-2 in the cornea of 12-month-old mice could be due to the continued presence of an inflammatory stimulus (e.g., P. aeruginosa) in the cornea of these mice. Alternatively, studies by Himi et al.5 and Bruunsgaard et al.4 suggest that the observed prolonged inflammatory response in the aged also could be due to an altered regulation in the expression and/or secretion of proinflammatory and antiinflammatory cytokines.
Because the corneal expression of MIP-2 in 12-month-old mice remained significantly elevated at 5 to 7 DPI, we next tested whether this chemokine was biologically relevant to this model of infection. For these studies 12-month-old BALB/c mice were systemically administered either neutralizing MIP-2 pAb or NRS (control) before and after P. aeruginosa challenge. Whereas the cornea of all mice treated with NRS perforated at 7 DPI, mice treated with MIP-2 pAb displayed reduced ocular disease (+2 to +3 ocular disease grades) at this time. In addition, significantly fewer PMNs were detected in the cornea of MIP-2 pAb– versus NRS-treated mice at both 3 and 5 DPI. These studies indicated that an increase in and/or persistence of local MIP-2 expression in the cornea of 12-month-old mice is one mechanism by which continued PMN recruitment to the inflamed cornea occurred. The increased number or extended presence of this cell type in cornea has been implicated previously as a major contributor to irreversible stromal damage.16 19 Although perforation was not observed in MIP-2 pAb–treated mice, the cornea of these animals was not normal. Thus, it is likely that other inflammatory mediators participate in the recruitment of PMN to the cornea. In this regard, we tested and found significantly higher amounts of the pleiotropic proinflammatory cytokine IL-125 in the infected cornea of 12-month- versus 8-week-old inbred mice at later times after infection (Hazlett et al., unpublished data, 2000).
In summary, the present study provides evidence that, in 12-month-old mice, aberrant regulation of the host response to corneal challenge with P. aeruginosa contributes to corneal stromal destruction and perforation. The histopathology studies together with bacterial load data suggest that early absence of PMNs from the central cornea of 12-month-old mice after infection may occur because in these animals P. aeruginosa is not efficiently removed from the cornea. In addition, these studies show that the chemokine MIP-2 (and possibly the cytokine IL-1) contribute to the susceptible phenotype by enhancing recruitment of PMN to the cornea. Because activated PMN secrete various proteolytic enzymes and oxidative metabolites,26 the increased number and persistence of this cell type in 12-month-old animals leads to degradation of various matrix components of the cornea and eventually to corneal perforation.
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Footnotes |
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Submitted for publication February 28, 2000; revised April 18, 2000; accepted April 27, 2000.
Commercial relationships policy: N.
Corresponding author: Linda D. Hazlett, Department of Anatomy/Cell Biology, Wayne State University School of Medicine, 540 E. Canfield Avenue, Detroit, MI 48201. lhazlett{at}med.wayne.edu
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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