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Departments of
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Anatomy/Cell Biology and
Immunology/Microbiology, 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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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our working hypothesis is that the final outcome following P. aeruginosa corneal infection (restoration of corneal clarity vs corneal perforation) depends upon the host’s ability to rapidly remove the invading pathogen. Following bacterial clearance, appropriate modulation of the corneal inflammatory response must occur to allow wound healing. We propose that mice that are unable to efficiently regulate the inflammatory response in cornea will undergo permanent tissue damage following ocular bacterial challenge. To test this, inbred strains of mice that are either susceptible or resistant to corneal infection were examined for corneal PMN number, bacterial load, and for MIP-2 and KC mRNA and protein expression after P. aeruginosa corneal challenge. In addition, recombinant murine chemokine was administered to resistant mice after infection to determine whether this treatment exacerbated disease pathology. Lastly, a neutralizing anti-chemokine polyclonal Ab (pAb) was given to susceptible mice after infection to test whether corneal stromal damage could be reduced.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Young adult (8 wk) BALB/cByJ (BALB/c) (resistant) and C57BL/6J (B6) (susceptible) mice (The Jackson Laboratory, Bar Harbor, ME) were used for these studies. Before corneal infection, mice were lightly anesthetized with isoflurane (Aerrane; Anaquest, Madison, WI) and placed beneath a stereoscopic microscope at x40 magnification. The central cornea of the left eye was scarified with three 1-mm incisions using a sterile 25 5/8-gauge needle. Random eyes were routinely examined histologically to ensure that the wounds penetrated only the epithelial basal lamina and superficial corneal stroma. A bacterial suspension (5 µl) containing 1.0 x 106 CFU of P. aeruginosa strain 19660 (American Type Culture Collection, Manassas, VA), prepared as described previously (20), was topically applied onto the wounded cornea. Eyes were examined macroscopically 24 h postinfection (p.i.) and/or at times described below to ensure that all mice were similarly infected and to monitor the course of disease in the infected mice. 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.
Ocular response to infection
Following P. aeruginosa corneal infection, ocular disease was graded using the following established scale (21): 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. Five mice from each experimental group (BALB/c vs B6, rMIP-2 vs vehicle treated, or MIP-2 pAb vs pre-immune, normal rabbit serum (NRS) treated) were examined at each time point. A mean clinical score was calculated for each group of mice to express disease severity. This was determined by summation of the scores for each group divided by the total number of mice scored at each time point (22).
Histopathology
For histopathological analysis, whole eyes were enucleated from three mice from each experimental group (BALB/c vs B6, rMIP-2 vs vehicle, and MIP-2 pAb vs NRS treated) at various selected times after infection. For the comparison of histopathology between BALB/c vs B6 mice, eyes were collected before and at 16 h and 3, 5, and 7 days p.i. For determination of the effect of rMIP-2 treatment, eyes were collected from rMIP-2- and vehicle-treated mice at 3 and 5 days p.i. For the MIP-2 neutralization studies, eyes were collected from MIP-2 pAb- and NRS-treated mice at 6 days p.i. Eyes were enucleated and immersed immediately 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. Eyes were then dehydrated in graded ethanols and embedded in Epon-araldite as described previously (7). 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, Chesire, U.K.).
Quantitation of PMN in corneal tissues
A myeloperoxidase (MPO) assay was used to quantitate the total number of PMN infiltrating the cornea following infection (23, 24). At selected times after infection (between 1 and 7 days p.i.), three corneas from each experimental group (BALB/c vs B6, rMIP-2 vs vehicle treated, and MIP-2 pAb vs NRS treated) were collected for MPO analysis. Corneas were excised at the limbus with a sterile razor blade, and noncorneal tissue was removed by dissection. Following 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 s on ice, freeze-thawed three times, sonicated a second time, 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 absorbancy 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 the individual samples 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 (23). Results are reported as units of MPO/cornea.
Quantitation of viable bacteria in corneal tissues
At selected times after infection (between 1 and 9 days p.i.), three corneas from each experimental group (BALB/c vs B6 and MIP-2 pAb vs NRS) were collected for the determination of viable bacteria in infected corneal tissue. Individual corneas were homogenized in sterile 0.9% NaCl containing 0.25% BSA (25). A 100 µl of each sample was diluted serially 1:10 in the same solution and plated in triplicate on Pseudomonas isolation agar (Difco, Detroit, MI) plates. Plates were incubated for 16 h at 37°C. The number of viable bacteria in an individual cornea was determined by counting the individual colonies on the plates from the various dilutions and multiplying the number of colonies by the appropriate dilution. Results are reported as log10 number of CFU/cornea.
Quantitation of corneal MIP-2 and KC mRNA levels
RNase protection assays were used to quantitate corneal levels of MIP-2 and KC mRNA (26, 27). MIP-2 and KC cDNA clones were generated by RT-PCR using total RNA from P. aeruginosa-infected corneas as the template for the RT reaction. PCR primers were designed (MacVector Software; Oxford Molecular, Madison, WI) to amplify nucleotides 228–462 of murine MIP-2 (accession number X53798) (28) and nucleotides 139–351 of murine KC (accession number X12531) (29). EcoRI and XbaI restriction sites were added to the 5' ends of the primers to facilitate ligation of the PCR product into the pGEM-3Z vector. 32P-labeled MIP-2 and KC anti-sense strand riboprobes were generated from the cDNA clones by in vitro transcription. Likewise, unlabeled sense strand MIP-2 and KC RNAs were created to produce a standard curve to quantitate the amount of the respective mRNAs in corneal tissue.
Corneal tissue was collected from BALB/c and B6 mice before and at 6 and 12 h and 1, 3, and 5 days after infection with P. aeruginosa. Following collection, samples were flash-frozen in liquid nitrogen and stored at -70°C until extraction of RNA. Six corneas were pooled for an individual sample. Total RNA was extracted from corneal tissue using RNazol B (Tel-Test, Friendsville, TX) according to the manufacturers instructions. Next, 5 µg of total RNA from each sample was hybridized overnight at 56°C to 300 pg of the MIP-2 and KC riboprobes. Similarly, various concentrations of the unlabeled sense-strand standards (6.2–150 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 quantitated using a MDX Persen Densitometer S II and Image Quant Densitometric software (Molecular Dynamics, Sunnyvale, CA). This experiment was performed at least three times to ensure reproducibility of the data. Results are reported as amol chemokine mRNA/µg total RNA.
Quantitation of corneal MIP-2 and KC protein levels
MIP-2 and KC protein levels were determined using ELISA kits (R&D Systems, Minneapolis, MN). For these studies, individual corneas were collected from mice before and at 12 h and 1, 3, and 5 days p.i., as described above. Three corneas were collected separately at each time point. Before storage at -70°C, the total weight of each individual cornea was determined. Corneas were then stored in 0.5 ml of serum-free DMEM containing 0.5% Tween 20. Immediately before analysis, samples were thawed and homogenized with a glass Kontes pestle (Fischer, Itasca, IL). Samples were centrifuged at 5,000 x g for 10 min, and an aliquot of each supernatant was assayed for MIP-2 and KC protein. Based upon a preliminary ELISA experiment using P. aeruginosa-infected corneal tissues, supernatants were diluted 1:10 for MIP-2 and 1:5 for KC in the kit’s assay diluent to permit detection of signal within the linearity of the standard curves for the respective chemokines. The sensitivity of the ELISA was 1.5 pg/ml for MIP-2 and 2.0 pg/ml for KC. Both chemokine ELISA experiments were performed in duplicate to ensure reproducibility of the data. Results are reported as pg chemokine/mg corneal tissue.
rMIP-2 administration
Murine rMIP-2 was purchased from R&D Systems (Minneapolis, MN). The lyophilized powder (10 µg) was reconstituted in 2.0 ml of PBS containing 0.1% BSA as suggested by the manufacturer. BALB/c mice (n = 5) were anesthetized with Aerrane and then injected i.p. with 0.2 ml (1.0 µg) of rMIP-2 on days 1 and 3 p.i. Control (vehicle treated) mice (n = 5) were similarly injected with 0.2 ml of PBS/0.1% BSA. Time points chosen for administration of rMIP-2 or vehicle were based on the mRNA and protein data presented herein. The rMIP-2 injection studies were performed in duplicate to ensure reproducibility of the data.
MIP-2 neutralization
Neutralizing pAb to murine MIP-2 (13) was kindly provided by Dr. Nicholas Lukacs (Department of Pathology, University of Michigan, Ann Arbor, MI). For the MIP-2 neutralization studies, B6 mice (n = 5) were anesthetized with Aerrane and subsequently injected i.p. with 0.2 ml of neutralizing MIP-2 pAb at 8 h and at 1 and 3 days after P. aeruginosa infection. Control mice (n = 5) were similarly treated with 0.2 ml of NRS. The neutralization experiments were performed in duplicate to ensure reproducibility of the data.
Statistical analysis
An unpaired, two-tailed Student’s t test was used to
determine statistical significance for the mean clinical scores, 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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Ocular disease was graded in BALB/c and B6 mice after corneal
challenge to evaluate the progression and outcome of P.
aeruginosa infection. Fig. 1
shows
the mean clinical scores for the two experimental groups from 1 to 7
days p.i. Similar ocular disease grades (+1 to +2) were observed in
corneas of BALB/c and B6 mice at 1 day p.i. Over the 7 days p.i.
examined, BALB/c mice did not progress significantly past a +2 (dense
opacity either partially or fully covering the pupil) ocular disease
grade. By 7 days p.i., two of five BALB/c mice demonstrated a +1
(slight opacity covering the anterior segment) ocular disease grade.
Alternatively, ocular disease progressively worsened in B6 mice. By 5
days p.i., one of five corneas from the B6 mice had perforated (+4),
whereas the other four mice from this group demonstrated a +3 ocular
disease grade (dense opacity covering the entire anterior segment). By
7 days p.i., perforation was evident in all of the corneas of B6 mice.
Significant differences between the BALB/c and B6 mice were found at 3,
5, and 7 days p.i. (p = 0.0353, 0.0003, and
0.0001 at 3, 5, and 7 days p.i., respectively).
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Corneal tissue from BALB/c and B6 mice were compared
histopathologically for the first week p.i., and these data are
presented in Fig. 2. At 16 h p.i.,
the epithelium was essentially intact in the cornea of resistant BALB/c
mice (Fig. 2A). PMN were observed in the central and
peripheral corneal stroma and within the anterior chamber. A few PMN
were seen in the corneal epithelium, and free bacteria were
infrequently seen in the corneal stroma. In susceptible B6 mice, the
corneal epithelium was denuded centrally (Fig. 2B), and free
bacteria were more frequently observed within the central corneal
stroma. Compared with the BALB/c mouse cornea, PMN were rarely seen in
the superior one-third of the central corneal stroma of B6 mice. The
infiltrating cells in B6 mice were particularly dense in the lower
two-thirds of the stroma and in the peripheral cornea. In the latter
location, the cellular pattern suggested centripetal migration. (The
arrows in Fig. 2B delineate the area of the central cornea.)
In B6 mice, PMN also were present in the anterior chamber and within
the peripherally intact corneal epithelium. At 3–5 days p.i., the
corneal epithelium had thinned in BALB/c mice (data not shown). PMN
remained plentiful throughout the cornea and within the anterior
chamber. Free bacteria were rarely observed within the corneal stroma.
In B6 mice, the epithelium was completely denuded by 3 days p.i. (data
not shown). The corneal stroma was centrally thinned (approximately
half of normal), and PMN were numerous at the limbus (juncture of
cornea and contiguous conjunctiva and sclera) and in the anterior
chamber. Compared with BALB/c mice, fewer PMN were seen in the central
cornea. By 5 days p.i., corneal stromal thinning remained markedly
severe in B6 mice, and numerous PMN were observed throughout the
central cornea. Corneal tissue began healing in all BALB/c mice by 5–7
days p.i. Some corneas from these mice were assigned a +1 ocular
disease grade by 7 days p.i. (Fig. 1
). Corneas from these mice appeared
morphologically normal, and only a few PMN were observed within the
peripheral and/or central corneal tissue (data not shown).
Alternatively, other corneas from BALB/c mice showed a +2 ocular
disease grade. Fig. 2C shows the cornea of a BALB/c mouse
with a +2 ocular disease grade at 7 days p.i. The epithelium had begun
to resurface and PMN, and other inflammatory cells were observed in the
corneal stroma. In the stroma, collagen fibers appeared intact and
regularly spaced, and PMN were seen in the adjacent anterior chamber.
In contrast, by 7 days p.i., corneas from B6 mice had perforated as a
result of the extensive stromal collagen destruction (Fig. 2D). No epithelium was present in the central cornea of
these mice. PMN and other inflammatory cells remained numerous
throughout the cornea and anterior chamber, and the iris was
hyperemic.
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Histopathology results strongly suggested that susceptible vs
resistant mice would differ either in kinetics and/or in the number of
PMN that infiltrate the cornea after bacterial challenge. Therefore, we
next used an established MPO assay to estimate total corneal PMN number
in the two experimental groups of mice. No MPO activity was detected in
uninfected corneal tissue from either mouse strain (data not shown).
Similar amounts of enzyme activity were detected in BALB/c and B6 mouse
cornea from 1 to 3 days p.i. (Fig. 3) In
BALB/c mice, peak MPO activity was observed at 3 days p.i., whereas MPO
levels continued to increase in B6 mice through 7 days p.i. (last time
point examined in these studies). Significant differences in MPO
activity between BALB/c and B6 mice were detected at both 5 and 7 days
p.i. (p = 0.001 and 0.008 at 5 and 7 days p.i.,
respectively).
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To determine whether delayed infiltration of PMN into the superior
one-third of the central cornea of B6 mice was associated with impaired
clearance of bacteria from affected tissue, viable bacteria were
quantitated in BALB/c and B6 corneal tissue from 1 to 9 days p.i. The
mean log10 number of viable bacteria per BALB/c
and B6 cornea at each time p.i. is shown in Fig. 4. No difference in bacterial number was
observed between resistant and susceptible mice at 1 and 3 days p.i.
After 3 days p.i., BALB/c mice began to clear the invading pathogen
from corneal tissue. Only 2 log units of bacteria/cornea remained in
this experimental group by 9 days p.i. In contrast, reduction of ocular
load below 6 log units of bacteria was not observed in B6 mice until 9
days p.i. Significant differences between BALB/c and B6 mice were found
from 5 to 9 days p.i. (p = 0.0041, 0.0016, and
0.0023 at 5, 7, and 9 days p.i., respectively).
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To determine whether PMN infiltration into P.
aeruginosa-infected corneas was correlated with local corneal
C-X-C chemokine production, the amount of MIP-2 and KC mRNA was
determined in uninfected and infected corneal tissue from BALB/c and B6
mice using RNase protection assays. Data from two separate experiments
are shown in Tables I and
II. Neither MIP-2, nor KC mRNA
transcripts were detected in uninfected corneal tissue. Initially (6 h
p.i.), low levels of MIP-2 mRNA were found in the cornea of BALB/c
mice, whereas MIP-2 was not detected in the cornea of B6 mice at this
time under the assay conditions tested (Table I). Comparable amounts of
MIP-2 transcripts were found in corneal tissue of both mouse strains
from 12 h to 3 days p.i. A peak in the level of MIP-2 transcript
expression was found in both mouse strains by 1 day p.i. By 5 days
p.i., corneal MIP-2 mRNA levels began to decline in resistant BALB/c
mice. Alternatively, the amount of MIP-2 transcripts detected in B6
mouse cornea remained elevated through 5 days p.i. Conversely, the
kinetics and amounts of KC mRNA detected in infected BALB/c and B6
corneal tissue were similar at all time points examined (6 h to 5 days
p.i.) (Table II). The levels of KC mRNA peaked at 1 day p.i. and
declined thereafter in both groups of mice.
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In addition to mRNA quantitation, the amount of MIP-2 and KC
protein also was determined in uninfected and infected corneal tissue
by ELISA. Data from a representative experiment are shown in Figs. 5 and 6.
Neither MIP-2 nor KC was detected in uninfected corneal tissue. A low
level of MIP-2 was detected in corneal tissue from BALB/c and B6 mice
by 12 h p.i. (Fig. 5). The amount of detectable MIP-2 protein
peaked at 3 days p.i. in BALB/c mice and slowly declined thereafter. In
contrast, the amount of MIP-2 protein in B6 cornea continued to
increase through 5 days p.i. (the last time point tested).
Significantly greater amounts of MIP-2 protein were detected in B6 vs
BALB/c mouse cornea from 1 to 5 days p.i. (p =
0.0021, 0.0049, and 0.0017 at 1, 3, and 5 days p.i., respectively). In
contrast to MIP-2, the amount of KC protein was elevated in both groups
of mice by 12 h p.i. (Fig. 6). Peak KC levels were detected in B6
and BALB/c mouse cornea at 12 h and 1 day p.i., respectively.
Analogous to the KC mRNA data described above, the corneal levels of KC
protein declined after 12 h to 1 day p.i. Statistically greater
amounts of KC were found only at 1 day p.i. in B6 mice
(p = 0.013), whereas no differences in corneal
KC protein were found when KC levels were analyzed in a second similar
experiment (data not shown).
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The above studies suggested that the ability to restore corneal
clarity following P. aeruginosa challenge is associated with
down-regulation of MIP-2 expression and ultimately reduction in PMN
number. Therefore, to ascertain the biological relevance of these data,
we next tested whether systemic administration of rMIP-2 exacerbated
corneal disease in resistant BALB/c mice. For these studies, BALB/c
mice were injected i.p. with 1.0 µg of rMIP-2 (or vehicle for control
mice) at 1 and 3 days p.i. Mean clinical scores from rMIP-2- or
vehicle-treated mice were calculated and are shown in Fig. 7. Similar ocular disease grades were
observed in both experimental groups at 1 day p.i. (before rMIP-2 or
vehicle treatment). By 3 days p.i., mice treated with rMIP-2 had
significantly higher ocular disease grades (p =
0.049). While vehicle-treated mice did not progress past a +2 ocular
disease grade throughout the experiment, the corneal tissue of mice
injected systemically with rMIP-2 perforated (+4) by 5–7 days p.i.
(p = 0.008 and 0.001 at 5 and 7 days p.i.,
respectively).
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). When compared with vehicle-treated
mice, the vessels in the rMIP-2-treated group were of larger luminal
diameter, thicker walled, and inflammatory cell cuffing in the adventia
was present. Similar differences between rMIP-2- and vehicle-treated
mice were observed at 3 days p.i. (data not shown). In contrast to
rMIP-2-treated mice, ocular disease was significantly reduced in
vehicle-treated mice. A cornea with a +1 ocular disease grade at 5 days
p.i. is shown in Fig. 8B. At this time p.i., the epithelium
was intact from limbus. Compared with corneas from rMIP-2-treated mice,
collagen fibrils were intact and regularly spaced and only slight
corneal edema was seen in the superficial stroma. Qualitatively fewer
PMN were seen throughout the corneal tissue of the vehicle-treated
animals.
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). Mice treated
with rMIP-2 vs vehicle demonstrated a significantly greater amount of
corneal MPO activity at both of the time points examined
(p = 0.008 and 0.0023 at 3 and 5 days p.i.,
respectively).
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As an alternative approach to test whether MIP-2 plays a
significant role in the pathogenesis of P. aeruginosa ocular
disease, susceptible B6 mice were treated with multiple injections of
neutralizing MIP-2 pAb or NRS after corneal infection. Ocular disease
was graded in MIP-2 pAb- and NRS-treated mice from 1 to 9 days p.i.
(Fig. 10). Comparable ocular disease
grades were observed in mice treated with MIP-2 pAb and NRS from 1 to 5
days p.i. By 7 days p.i., all of the corneas in the NRS-treated group
had perforated (+4 ocular disease grade). Four of five B6 mice treated
with MIP-2 pAb displayed +2 ocular disease grades up to 9 days p.i.
(termination of experiment) (p = 0.003 and
0.001 at 7 and 9 days p.i., respectively).
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shows the corneal
histopathology in MIP-2 pAb- and NRS-treated B6 mice at 6 days p.i.
(the time point where corneal perforation was first observed in
NRS-treated mice). In MIP-2 pAb-treated mice, the corneal epithelium
was slightly irregular and intact collagenous fibers were observed
throughout the superficial and deep stromal regions of the cornea.
Moreover, corneal swelling was modest in these mice. In contrast, the
epithelium was denuded and/or patchy in the NRS-treated group.
Likewise, this group of mice demonstrated extensive degradation of the
corneal stroma with total dissolution of stromal fibrillar collagen
bundles along with pronounced corneal edema. Numerous PMN were present
throughout the cornea and in the anterior chamber of the NRS-treated
mice. Qualitatively, corneal PMN appeared to be fewer in number in
MIP-2 pAb-treated vs NRS-treated mice.
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). Alternatively, treatment of mice
with MIP-2 pAb did not affect corneal bacterial load. The number of
viable bacteria isolated from MIP-2 pAb- and NRS-treated mice was
similar between 3 and 7 days p.i. (Fig. 13).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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For the current studies, progression of P.
aeruginosa-induced ocular disease was monitored in BALB/c and B6
mice from 1 to 7 days p.i. (Fig. 1). Previous studies demonstrated that
both BALB/c and B6 mice were susceptible to corneal challenge when the
infecting dose of P. aeruginosa strain 19660 (American Type
Culture Collection) was 108 CFU
(30). In the studies described herein, mice were infected
with 106 CFU (20). Using
106 CFU, all B6 mice displayed corneal
perforation by 7 days p.i. In contrast, by 7 days p.i., some variation
in the degree of corneal opacity (reflected by +1 to +2 ocular disease
grades) was observed in BALB/c mice. Nonetheless, it was apparent that
the cornea from these mice began to heal by 5–7 d p.i.
Histopathological examination of infected corneal tissue at 5–7 days
p.i. (Fig. 2) confirmed that corneal healing had begun in BALB/c and
that extensive stromal loss and perforation had occurred in B6
mice.
The histopathology also suggested that differences in the number and/or
kinetics of PMN existed between resistant (cornea heals) and
susceptible (cornea perforates) mice. Well-defined differences in the
spatial distribution of PMN in cornea were observed between BALB/c (PMN
distributed throughout the entire cornea) and B6 (superior central
cornea essentially devoid of PMN) mice from 16 h and 3 days p.i.
(Fig. 2, A and B and data not shown).
Furthermore, an overall greater number of PMN were observed in the
cornea from B6 vs BALB/c mice at 5 and 7 days p.i. Use of an
established PMN MPO assay to quantitate total corneal PMN number
provided verification that B6 mice had a significantly greater number
of PMN in corneal tissue, but only at later times p.i. (Fig. 3). In
both BALB/c and B6 mice, PMN number correlated well with the degree of
corneal opacity and ocular integrity. In this regard, those mice
displaying +1 vs +2 ocular disease grades had fewer corneal PMN and
reduced ocular disease when compared with those mice with a +3 ocular
disease grade.
When bacterial load was quantitated in the cornea of BALB/c and B6 mice
p.i., a significantly greater number of viable bacteria were detected
in the cornea of B6 vs BALB/c mice from 5 to 9 days p.i. (Fig. 4). Two-
to 3-log fold differences in viable bacterial number were found between
resistant and susceptible mice at 7 and 9 days p.i., respectively.
These data suggested that there may be a functional defect in the
ability of PMN from B6 mice to kill P. aeruginosa. To
address this issue, we used established PMN phagocytic and microbicidal
assays (31) to test the functional integrity of peripheral
blood-derived PMN from B6 vs BALB/c mice. These experiments showed that
there was no difference in the ability of PMN from either mouse strain
to phagocytose and kill P. aeruginosa (data not shown),
hence ruling out a defect in PMN as the cause for persistence of
bacteria in the cornea of B6 mice. Alternatively, the histopathology
data presented herein suggested that delayed infiltration of PMN into
the superior central cornea of B6 mice may alter their ability to
remove bacteria from the affected tissue at later times p.i. (Fig. 2).
We hypothesize that this delay could allow for continued replication of
bacteria in B6 corneal tissue, as reflected by the observed increase in
bacterial load at later times p.i. We propose that a greater number of
PMN would subsequently be required to ultimately reduce the bacterial
load. Consequently, this increase and/or persistence of PMN could
directly contribute to the outcome of extensive stromal destruction
observed in B6 mice.
In susceptible B6 mice, aberrant regulation of the inductive and/or
resolution phases of the host-inflammatory response also could
contribute to the susceptible phenotype. In this regard, differential
regulation of the expression and secretion of one or more soluble
proinflammatory mediators may contribute to the disparate PMN response
in susceptible vs resistant mice. To test this, the expression of two
C-X-C chemokines (KC and MIP-2) was examined in corneal tissue of
BALB/c and B6 mice after P. aeruginosa challenge. Similar
amounts of KC mRNA and protein were found in the cornea of both strains
of mice from 12 h to 5 days after infection (Fig. 6 and Table II).
The early peak of KC expression (12 h to 1 day p.i.) correlated with
the initial infiltration of PMN into infected cornea, implying that KC
could be involved in the early corneal PMN response. However, the rapid
decrease of KC expression in B6 cornea by 3 days p.i. strongly suggests
that this chemokine is not critical for the persistence of PMN in the
cornea of susceptible mice.
In contrast to KC, MIP-2 levels (mRNA and protein) in cornea of
susceptible mice were associated with increased corneal PMN at later
times p.i. This increase correlated with the onset of corneal
ulceration and perforation (Figs. 1 and 5 and Table I). Alternatively,
timely down-regulation of corneal MIP-2 levels and PMN number in
resistant mice was followed by corneal healing and re-establishment of
ocular integrity. One potential mechanism by which MIP-2 could amplify
the local PMN inflammatory response has been proposed by both Gainet et
al. and Mercer-Jones et al. (32, 33). Their
studies showed that activated PMN produce MIP-2 (mouse) or IL-8 (human)
and that production of the respective chemokine by PMN produces an
autoamplification loop of PMN recruitment and activation at inflamed
sites. It is possible that such augmentation of the cyclic MIP-2/PMN
response in B6 mice could lead to eventual corneal ulceration and
perforation. Furthermore, data reported by Yan et al. (19)
support the conclusions drawn in the current study regarding MIP-2 and
KC. This group showed previously that treatment of mice with
neutralizing pAb to MIP-2, but not KC, significantly reduced
HSV-induced corneal inflammation and PMN responses.
Because persistence of MIP-2 expression in B6 mice appeared to be
associated with continued PMN infiltration and corneal perforation, we
next tested whether the resistance phenotype of BALB/c mice could be
altered by administration of rMIP-2. For these studies, systemic rMIP-2
administration was used in an attempt to alter disease outcome. This
route of injection was selected because others previously have shown
that serum proteins can readily extravasate into corneal tissue
following induction of a corneal inflammatory response (34, 35). This is due largely to the ingrowth from the limbus of
blood vessels into the normally avascular cornea. As vessels invade
inward, it is feasible to hypothesize that a serum-derived local
chemokine gradient (from the systemically administered rMIP-2) could be
established within the cornea that could contribute to the recruitment
of PMN. The rMIP-2 itself also may accentuate the angiogenic response
in cornea as described in studies reported by Keane et al.
(36). The data in Fig. 8 show that systemic administration
of rMIP-2 vs vehicle increased the size and frequency of blood vessels
infiltrating the peripheral cornea. In addition, there was an increase
in the number of inflammatory cells surrounding and within the lumen of
these vessels following rMIP-2 treatment. Thus, the data presented in
the current study provide structural evidence of the mechanism by which
a local chemokine gradient could be established in the cornea of
rMIP-2-treated mice.
Measurement of corneal MPO activity in rMIP-2- vs vehicle-treated mice
at 3 and 5 days p.i. confirmed that the systemic treatment protocol
described above increased the total number of PMN in cornea of
rMIP-2-treated mice (Fig. 9). Examination of corneal histopathology in
the two experimental groups of mice also showed that mice treated with
rMIP-2 had exacerbated corneal disease (Fig. 8). In combination, these
studies demonstrate that MIP-2 can regulate the PMN response in
P. aeruginosa-infected cornea and that an increase in the
number of this cell type correlates with extensive stromal tissue
destruction.
As an additional test of the in vivo biological role of MIP-2 in PMN
recruitment and corneal destruction, susceptible B6 mice were treated
with a neutralizing MIP-2 pAb or NRS after corneal challenge. Treatment
with MIP-2 pAb vs NRS was effective in reducing corneal PMN number,
stromal destruction, and perforation in B6 mice up to 9 days p.i.
without an increase in bacterial load (
Figs. 10–13). Because a large
number of bacteria remained in the cornea of MIP-2 pAb-treated mice, it
is unreasonable to assume that complete protection (e.g., total corneal
healing) against P. aeruginosa-induced stromal destruction
and perforation was achieved using pAb treatment alone. It may be
necessary to combine MIP-2 pAb with antibiotics to reduce both the host
response and bacterial load to increase the likelihood of corneal
healing after bacterial challenge.
In summary, our results directly demonstrate that MIP-2 is a mediator of corneal PMN infiltration. These studies suggest that prolonged expression of this chemokine in P. aeruginosa-infected cornea contributes to persistence of this cell in an infected B6 cornea and ultimately to corneal ulceration and perforation. In contrast, resistant BALB/c mice resolve corneal infection without extensive stromal destruction, and this correlates with their ability to eliminate the invading pathogen by attraction of a sufficient number of PMN to the site of infection. Following bacterial clearance, restoration of corneal clarity and ocular integrity in resistant mice is associated with cessation of MIP-2 expression and subsequent down-regulation of PMN migration into the cornea.
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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:
3 Abbreviations used in this paper: PMN, polymorphonuclear neutrophil, MIP, macrophage inflammatory protein; p.i., postinfection; pAb, polyclonal Ab; MPO, myeloperoxidase; B6, C57BL/6J; BALB/c, BALB/cByJ; NRS, normal rabbit serum.
Received for publication April 19, 1999. Accepted for publication November 4, 1999.
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