| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
regulates CD4+ T cell chemotaxis and indirectly enhances PMN persistence in Pseudomonas aeruginosa corneal infection
Department of Anatomy/Cell Biology, Wayne State University, Detroit, Michigan
Correspondence: Linda D. Hazlett, Ph.D., Wayne State University School of Medicine, Department of Anatomy/Cell Biology, 540 E. Canfield Avenue, Detroit, MI 48201. E-mail: lhazlett{at}med.wayne.edu
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
(MIP-1) in cell
infiltration into Pseudomonas aeruginosa-infected cornea
and subsequent disease was examined. Greater amounts of the chemokine
(protein and mRNA) were found in the infected cornea of susceptible B6
("cornea perforates") versus resistant BALB/c ("cornea heals")
mice from 1 to 5 days postinfection. Treatment of BALB/c mice with
recombinant (r) MIP-1 exacerbated disease and was associated with an
increased number of neutrophils (PMNs) in the cornea. Treatment of
BALB/c mice with rMIP-1 also induced recruitment of activated
CD4+ T cells into the affected cornea, converting resistant
to susceptible mice. Depleting CD4+ T cells in r-treated
BALB/c mice significantly decreased PMNs in cornea tissue, suggesting
that T cells regulate persistence of PMNs at this site. In B6 mice,
administration of neutralizing MIP-1 polyclonal antibody
also significantly reduced PMN numbers and pathology. Collectively,
evidence is provided that MIP-1 directly contributed to
CD4+ T cell recruitment and indirectly to PMN persistence
in the infected cornea.
Key Words: immunity • bacterial infection • chemokines • neutrophils
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Previous studies have suggested that susceptibility (corneal perforation) to P. aeruginosa challenge involves dysregulation of the local host inflammatory response, including persistence of neutrophils (PMNs) and elevated levels of interleukin (IL)-1 and macrophage inflammatory protein (MIP)-2 [10 , 11 ]. Alternatively, resistant mice (e.g., those that can restore corneal integrity after infection) rapidly down-regulate these responses after clearance of the invading pathogen from the cornea. Moreover, it has also been demonstrated in B6 mice that the presence of CD4+ helper T cell 1-type T cells is important in corneal-tissue destruction [12 ]. Further investigation into the role of CD4+ T cells in pathogenesis has shown that T cells can be found only in the corneas of susceptible but not resistant mice, from 3 to 7 days postinfection (p.i.), the latter time associated with extensive corneal thinning and/or perforation in susceptible animals [13 ]. Based on these data and other studies which have shown that products released by activated T cells can enhance recruitment of PMNs to inflamed tissues [14 , 15 ], we postulated that in the susceptible mice, amplification and persistence of the PMN response in the cornea could be, in part, mediated by the presence of T cells in the cornea.
Various chemokines have been identified that stimulate leukocyte
chemotaxis in vitro and elicit accumulation of specific inflammatory
cells in vivo. MIP-1, a member of the CC subfamily of chemokines, is
produced by activated T cells, macrophages, Langerhans cells, PMNs, and
B cells [16
, 17
]. In vitro chemotaxis
studies and MIP-1 receptor analyses have indicated that this
chemokine acts on T cells, B cells, macrophages, and basophils
[18
, 19
]. Evidence also suggests that
MIP-1 might attract PMNs to inflammatory sites
[20
21
22
23
]. Current studies tested whether MIP-1
expression in susceptible mice contributes to irreversible
corneal-tissue destruction after P. aeruginosa challenge. A
complementary approach using both susceptible and resistant murine
phenotypes was taken to better assess the role of MIP-1 in the
disease response.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Ocular response to infection
Ocular disease was graded macroscopically after corneal
challenge using the following established scale [24
]: 0,
clear pupil 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
with sealed lids, mice were anesthetized with isoflurane, and sterile
phosphate-buffered saline (PBS) was applied to the eyelids to permit
their careful partial opening without inducing corneal perforation.
Five mice from each experimental group [recombinant (r) MIP-1- vs.
vehicle-treated or MIP-1 pAb- vs. normal rabbit serum (NRS)-treated
mice] 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 [14
].
Quantitation of corneal chemokine and cytokine mRNA levels
Ribonuclease protection assays were used to quantitate corneal
levels of MIP-1, MIP-2, and IL-1ß mRNA. Generation and use of the
MIP-2 and IL-1ß cDNA clones has been described [10
,
11
]. The MIP-1 cDNA clone used was generated by
reverse transcriptase (RT)-polymerase chain reaction (PCR) using total
RNA from P. aeruginosa-infected corneas as the template for
the RT reaction. PCR primers were designed using MacVector Software
(Oxford Molecular, Madison, WI) to amplify nucleotides 21–357 of
murine MIP-1 (accession number X12531) [25
].
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-1, MIP-2, and
IL-1ß antisense-strand riboprobes were generated from the respective
cDNA clones by in vitro transcription. Likewise, unlabeled-sense-strand
RNA was created to produce a standard curve to quantitate the amount of
MIP-1, MIP-2, or IL-1ß RNA in corneal tissue.
For the quantitation of MIP-1, 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. For studies involving
the quantitation of corneal MIP-2 and IL-1ß mRNA, corneal tissue was
collected at 3 and 5 days p.i. from B6 mice treated with either
MIP-1 pAb or NRS. After collection, samples were flash-frozen in
liquid nitrogen and stored at -70°C until extraction of mRNA. Three
corneas were pooled for each individual sample. Total mRNA was
extracted from corneal tissue using RNazol B (Tel-Test, Friendsville,
TX) according to the manufacturer’s instructions. Five micrograms of
total mRNA from each sample were hybridized overnight at 56°C to 300
pg of the respective riboprobes. Similarly, various concentrations of
the unlabeled sense strand standard (6.2–150 pg) were hybridized to
the same amount of riboprobe. After hybridization, samples were
digested with 1,000 U of T1 nuclease (Gibco-BRL, 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 MDX Persen S II
densitometer and Image Quant densitometric software (Molecular
Dynamics, Sunnyvale, CA). This experiment was performed at least three
times to ensure reproducibility of the data, and the results from a
representative experiment are presented below. Results are reported as
attomoles of mRNA per microgram of total corneal RNA.
Quantitation of corneal MIP-1 protein levels
MIP-1 protein levels were determined using an enzyme-linked
immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN). For
these studies, individual corneas were collected from mice before
infection and at 12 h and 1, 3, and 5 days p.i. as described
above. Three corneas were collected separately at each time point. The
total weight of each individual cornea was determined before storage in
0.5 mL of serum-free Dulbecco’s modified Eagle’s medium at -70°C.
Before analysis, samples were thawed and homogenized with a glass
Kontes pestle (Fisher Scientific, Itasca, IL). Samples were centrifuged
at 5,000 g for 10 min, and an aliquot of each supernatant
was assayed for MIP-1 protein. Based on a preliminary ELISA
experiment using P. aeruginosa-infected corneal tissue,
supernatants were diluted 1:5 in the kit’s assay diluent to permit
detection of the MIP-1 signal within the linearity of the standard
curve. The reported sensitivity of the MIP-1 ELISA was <1.5 pg/mL.
This experiment was performed in duplicate to ensure reproducibility of
the data, and the results from a single representative experiment are
presented below. Results are reported as picograms of MIP-1 per
milligram of corneal tissue.
rMIP-1 administration
Murine rMIP-1 was purchased from R&D Systems. The lyophilized
powder (10 µg) was reconstituted in 2.0 mL of PBS containing 0.1%
bovine serum albumin (BSA) as suggested by the manufacturer. BALB/c
mice (n=5) were anesthetized with isoflurane and then
injected i.p. with 0.2 mL (1.0 µg) of rMIP-1 on days 1 and 3 p.i. as described before [10
]. Control (vehicle-treated)
mice (n=5) were similarly injected with 0.2 mL of PBS
containing 0.1% BSA. In a separate experiment BALB/c mice
(n=5) were injected subconjunctivally with rMIP-1 (0.5
µg in 7 µL of PBS) on the day of infection and at 18 h p.i.
(total of 1.0 µg). Control (n=5) mice were similarly
injected with PBS containing 0.1% BSA. Time points chosen for
administration of rMIP-1 or vehicle were based on the mRNA and
protein data presented herein. Each rMIP-1 injection study was
performed in duplicate to ensure reproducibility of the data, and
representative results from a single experiment are presented. In
another experiment, rMIP-1 was injected subconjunctivally as
described above into CD4+ T cell-depleted [rat anti-mouse
CD4, GK1.5, immunoglobulin (Ig) G2b (ATCC)] BALB/c mice
(n=5), whereas control mice (n=5) were similarly
injected with recombinant protein and an irrelevant HLA-DR5 monoclonal
antibody [mAb (rat anti-human, SFR3-DR5, IgG2b, ATCC)], the latter as
described before [12
]. Briefly, anti-CD4 or irrelevant
mAb (0.5 mg) was injected i.p. the day before infection and at 2 days
p.i. [12
]. Both specific and irrelevant mAbs had been
tested by fluorescein-activated-cell-sorter analysis, and only the
specific mAb resulted in 99% depletion of the CD4+T cell
subset [12
].
MIP-1 pAb administration
Neutralizing pAb to murine MIP-1 was purchased from
PeproTech, Inc. (Rocky Hill, NJ). MIP-1 pAb was purchased in two
forms for the different injection strategies: antigen affinity-purified
pAb and protein A affinity-purified pAb. For the studies described
herein, B6 mice (n=5) were anesthetized with isoflurane and
injected subconjunctivally with 10 µg (in 10 µL of PBS) of antigen
affinity-purified neutralizing MIP-1 pAb at 1 day before P.
aeruginosa corneal infection. After subconjunctival administration
of pAb, this experimental group also received 150 µg (in 0.2 mL of
PBS) of protein A affinity-purified MIP-1 pAb i.p. and another 150
µg of protein A-purified MIP-1 pAb at 1 and 3 days after corneal
infection. Control mice (n=5) were similarly treated both
subconjunctivally and i.p. with preimmune rabbit IgG and NRS,
respectively. The neutralization experiments were performed in
duplicate to ensure reproducibility of the data, and representative
results from a single experiment are shown below.
Histopathology
Eyes were enucleated from three mice per experimental group
(rMIP-1- vs. vehicle-treated and MIP-1 pAb- vs. NRS/preimmune
rabbit IgG-treated mice) at 5 days p.i. Eyes were immersed in PBS,
rinsed, and fixed in 1% osmium tetroxide, 2.5% glutaraldehyde, and
0.2 M Sorenson’s phosphate buffer (pH 7.4) (1:1:1) at 4°C for a
total of 3 h. Eyes were transferred into fresh fixative after
1.5 h, washed, dehydrated in graded ethanols, and embedded in
Epon-araldite as previously described [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 microscope (Morgan Instruments, Inc., Cincinnati, OH)
using Ilford pan F film (Mobberley, Chesire, United Kingdom).
Quantitation of PMNs in corneas
A myeloperoxidase (MPO) assay was used to quantitate the total
number of PMNs in corneas p.i. [10
, 11
].
Corneas were collected at 3 and 5 days p.i. in MIP-1 pAB- versus
NRS-treated mice (n=3/group/time) and at 5 days p.i. in
rMIP-1-treated (subconjunctival injection) and CD4+ T
cell-depleted or anti-HLA-DR5 (control) mAb-treated mice
(n=5/group). 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 s on ice and freeze-thawed three times. After centrifugation, a
supernate aliquot (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 was
monitored for 5 min using a spectrophotometer. The slope of the line
was determined for each sample and used to calculate results as units
of MPO per cornea. The experiments were repeated once similarly, and
representative data from single experiments are provided below.
Immunocytochemistry
Infected eyes from rMIP-1- and vehicle-treated mice
(n=3 eyes/experimental group) and rMIP-1,
CD4+ T cell-depleted versus recombinant-injected and
HLA-DR5-treated control (sham treated) mice (n=2/group) were
enucleated at 5 days p.i. After rinsing in sterile PBS, eyes were
embedded in Tissue Tek optimal-cryogenic-temperature compound (Miles,
Elkhart, IN), snap-frozen in liquid nitrogen, and stored at -70°C.
Frozen sections (10 µm) of cornea were cut on a Microm cryostat
(Fisher Scientific). For immunostaining, sections were fixed for 2 min
in cold acetone (-20°C) and then rinsed with 0.01 M PBS. Sections
were then covered with PBS containing 1% BSA and 0.05% Tween-20 in a
moist chamber for 30 min to block nonspecific binding. They were
incubated for 1 h with primary mAb specific for CD4 (rat IgG2a
clone H129.19 diluted 1:10 in PBS) or IL-2R (rat IgM clone 7D4 diluted
1:50 in PBS) (PharMingen, San Diego, CA). After incubation with the
primary mAb, sections were treated for 30 min with 0.3%
H2O2 to block endogenous peroxidase activity.
Control sections were treated with the nonspecific HLA-DR5 mAb
[12
, 13
]. This step was followed by
incubation of the sections for 1 h with the appropriate
biotinylated secondary mouse anti-rat antibody (for CD4, anti-rat IgG2a
at 1:25; for IL-2R, anti-rat IgM at 1:100) (PharMingen). After
incubation with the secondary antibody, sections were treated for 30
min with horseradish peroxidase-conjugated Z-avidin (Zymed, San
Francisco, CA). For visualization of immunoreactive cells, color was
developed by incubating the sections for 15 min with
3,3'-diaminobenzidine tetrathydrochloride (Pierce, Rockford, IL).
Between incubation steps, sections were rinsed with PBS and air-dried.
Statistical analysis
An unpaired, two-tailed Student’s t-test was used to
determine statistical significance for the mean clinical scores and
ELISA and MPO data. Mean differences were considered significant at the
confidence level of P 
0.05.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
mRNA and protein after P.
aeruginosa infection mRNA and protein were quantitated in the
corneas of susceptible B6 and resistant BALB/c mice before and at
various times after infection. The mRNA data from a representative
experiment are shown in Table 1
. Under the conditions tested, MIP-1 mRNA was not detected in
the corneas of either mouse strain before or at 6 h p.i. By
12 h after infection, a low level of mRNA expression was detected
in the corneas of BALB/c but not B6 mice. RNA for MIP-1 was readily
detected in the corneas of BALB/c and B6 mice at 1 day p.i., and the
expression of this chemokine peaked in both strains of mice at 3 days
p.i. Whereas the kinetics of MIP-1 expression were similar in B6 and
BALB/c mice, the corneas of susceptible B6 mice expressed two- to
ninefold more MIP-1 mRNA from 1–5 days p.i. The expression of
MIP-1 protein was examined by ELISA (Fig. 1
). The chemokine was not detected in the uninfected corneas of
either mouse strain or in the corneas of B6 mice at 12 h p.i. A
low level of MIP-1 protein (
5.0 pg/µg of corneal tissue) was
detected in the corneas of BALB/c mice at 12 h p.i.
(P=0.0019). MIP-1 protein was readily detected in the
corneas of B6 and BALB/c mice by 1 day p.i., and peak expression was
observed in both groups of mice at 3 days p.i. Similar to the mRNA
data, significantly higher levels of MIP-1 protein were detected in
the corneas of B6 mice from 1–5 days p.i. (P=0.0005,
0.0037, and 0.0071 at 1, 3, and 5 days p.i., respectively).
|
|
administration to resistant mice (mRNA and
protein) were observed in the corneas of susceptible versus resistant
mice after P. aeruginosa challenge, we next tested whether
these differences were biologically relevant to the observed P.
aeruginosa-induced corneal pathology. To do this, resistant BALB/c
mice (n=5/group) were treated with murine rMIP-1 in
PBS-BSA or with PBS-BSA vehicle alone as described in Materials and
Methods. Ocular disease was graded at 1 to 7 days p.i. Similar ocular
disease grades were observed in the recombinant- versus
vehicle-injected groups at 1 day p.i. (Fig. 2
; representative i.p. injection data are shown). Ocular disease was
more severe in rMIP-1-treated mice from 3 to 7 days p.i.
(P=0.0021, 0.0353, and 0.0003 at 3, 5, and 7 days p.i.,
respectively). Corneal perforation was observed in all
rMIP-1-treated BALB/c animals by 7 days p.i., and this was similar
to the response of genetically susceptible B6 mice.
|
- and similar
vehicle-treated BALB/c mice histopathologically at 5 days p.i., and
these data are presented in Figure 3
. The corneas of mice treated with rMIP-1 (i.p. injection data
shown) lacked epithelium and marked stromal edema, and perforation also
were evident at this time p.i. (Fig. 3A
3B)
. Numerous inflammatory
cells were present throughout the stroma and filled the anterior
chamber of the recombinant-chemokine-treated animals. Although the
majority of the inflammatory cells appeared to be PMNs, mononuclear
cells also were a consistent feature in the corneas of these animals.
In contrast, less corneal pathology and tissue destruction were
observed in vehicle-treated mice by 5 days p.i. (Fig. 3C
3D
; i.p.
injection data shown). The central corneal epithelium of these mice,
although thin, was resurfaced, and collagen fibers appeared essentially
intact and regularly spaced particularly in the mid to deep stroma.
However, epithelial down-growth and pockets of inflammatory cells were
still observed in a few areas of the central cornea. A moderate number
of PMNs but rare mononuclear cells were identified within the corneas
of vehicle-treated mice. Qualitatively, there appeared to be fewer
inflammatory cells in the corneas of vehicle- versus rMIP-1-treated
mice at 5 days p.i.
|
-treated mice. Because previous studies have shown that the
presence of CD4+ T cells in the cornea contributes to
corneal disease pathology [12
, 13
] and
treatment with rMIP-1 produced a similar exacerbation of corneal
disease, we next tested whether treatment with rMIP-1 induced
recruitment of CD4+, IL-2R+ T cells into the corneas of
resistant BALB/c mice. Figure 4
shows CD4 and IL-2R cell surface marker immunostaining in the
corneas of rMIP-1- and vehicle (representative i.p. injection data
shown)-treated mice at 5 days p.i. Positive immunostaining for the CD4
and IL-2R T cell surface markers was observed only in the corneas of
rMIP-1-treated mice (Fig. 4A)
. CD4-expressing cells were most
frequently detected paracentrally as cell clusters located beneath the
epithelium (Fig. 4A)
. Although CD4+ T cells were readily
detected in the corneas of rMIP-1-treated mice at 5 days p.i., only
a few cells stained positively for IL-2R (Fig. 4C)
. Corneal sections
from vehicle-treated BALB/c mice stained with IL-2R and sections from
an experimental group stained with the HLA-DR5 mAb were negative for
immunostaining (data not shown), similar to the corneas of a
vehicle-treated BALB/c mouse immunostained for the CD4 T cell marker
(Fig. 4B)
.
|
- versus vehicle-treated mice
and to test directly the role of the CD4+ T cell in this
outcome, an MPO assay was used. For this, BALB/c mice were injected
subconjunctivally with recombinant MIP-1 and then either depleted of
CD4+ T cells or treated with the irrelevant anti-HLA-DR5
mAb (sham depleted). In CD4+ T cell versus sham depleted
recombinant-injected BALB/c mice, MPO activity (7.2 vs. 25.5 U of MPO,
respectively) was significantly reduced (P=0.038).
Immunostaining confirmed that the corneas of CD4+ T
cell-depleted, rMIP-1-treated mice lacked T cells (data not shown)
and appeared similar to the corneas shown in Fig. 4B
, whereas
recombinant-injected, sham depleted mice exhibited positive
CD4+ T cell immunostaining (Fig. 4D)
.
MIP-1 pAb administration to susceptible mice
As an alternative complementary approach to test the biological
significance of MIP-1 in P. aeruginosa-induced corneal
pathology, susceptible B6 mice were treated before and after corneal
infection with a neutralizing MIP-1 pAb in an attempt to reduce
stromal destruction and perforation. By 5 days p.i., corneal
perforation (+4 ocular disease grade) was evident in B6 mice treated
with NRS/rabbit IgG (Fig. 5
). Alternatively, mice treated with MIP-1 pAb showed
significantly reduced ocular disease (+1–+2 ocular disease grades) by
5 days p.i. (P=0.0003). Figure 6
shows slit-lamp photomicrographs of representative eyes
photographed at 5 days p.i. Figure 6A
shows the eye of a B6 mouse
treated with MIP-1 pAb [+1 ocular disease grade (slight opacity
fully covering the entire anterior segment)], whereas Figure 6B
shows
the eye of an animal treated with NRS/rabbit IgG [+4 ocular disease
grade (corneal perforation)].
|
|
pAb or NRS/rabbit IgG were compared histopathologically.
These data are shown in Figure 7
. Figures 7A
and 7B
show the cornea of a B6 mouse treated with
MIP-1 pAb. Although the epithelium in this experimental group was
essentially intact in the central cornea, there was evidence for some
epithelial down-growth into the stroma. A few inflammatory cells were
observed throughout the stromal and epithelial components of the
corneas of mice treated with MIP-1 pAb. Although some
disorganization of collagen fibrils was observed in the superficial
stroma just below the epithelium, the collagen fibrils in the mid to
deep stroma appeared regularly spaced and intact. In contrast, in B6
mice treated with NRS/rabbit IgG, the epithelium was completely denuded
from limbus to limbus (Fig. 7C
7D)
. Extensive stromal thinning, marked
corneal edema, and numerous PMNs were evident in the corneas of the
NRS/rabbit IgG-treated mice. Because the histopathology suggested that
treatment of resistant mice with MIP-1 pAb reduced infiltration of
PMNs into the cornea after P. aeruginosa challenge, the
amount of MPO activity was determined in the corneas of both
experimental groups of mice at 3 and 5 days p.i. Figure 8
shows that a significantly greater amount of MPO activity was
detected in the corneas of NRS/rabbit IgG- versus MIP-1 pAb-treated
mice at both times tested p.i. (P=0.033 and 0.019 at 3 and 5
days p.i., respectively).
|
|
pAb reduced infiltration of PMNs into infected corneal tissue, we also
tested whether one of the mechanisms by which MIP-1 causes corneal
PMN infiltration includes induction of local MIP-2 or IL-1ß
(potent-PMN-chemoattractant) expression. To do this, MIP-2 and IL-1ß
mRNA expression was measured in the corneas of susceptible mice treated
with MIP-1 pAb or NRS/rabbit IgG. Table 2
shows the levels of expression of these chemoattractants at 3 and
5 days p.i. Similar amounts of MIP-2 mRNA were detected in both
experimental groups at 3 days p.i. Conversely, the corneas of mice
treated with NRS/rabbit IgG versus MIP-1 pAb had a 1.7-fold greater
amount of MIP-2 mRNA at 5 days p.i. When the corneal expression of
IL-1ß was examined in these two experimental groups, mice treated
with NRS/rabbit IgG demonstrated an approximately twofold increase in
IL-1ß mRNA levels at both 3 and 5 days p.i.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
in
corneal-tissue destruction. We focused on MIP-1 versus other T
cell-attracting chemokines such as gamma interferon-inducible protein
10 or regulated on activation, normal T cell expressed and
secreted [17
] chemokines because previous studies
[28
] demonstrated significant up-regulation of MIP-1
in outbred mice p.i.
MIP-1 mRNA and protein levels were determined in the infected
corneas of susceptible ("cornea perforates") B6 and resistant
("cornea heals") BALB/c mice. Five days p.i. was selected as the
last time point for testing in these experiments because extensive
stromal thinning and, in some instances, perforation are observed in
susceptible animals at this time p.i. [10
]. In addition,
the presence of activated T cells is first observed in susceptible mice
at this time [12
, 13
]. As expected,
susceptible B6 mice expressed greater amounts of MIP-1 mRNA and
protein from 1 to 5 days p.i. The kinetics of mRNA and protein
expression closely paralleled each other in both strains of mice.
Although a decline in MIP-1 was observed in BALB/c mice by 5 days
p.i., these levels remained significantly elevated in B6 mice at this
time. Recent work by Charles et al. (30) has demonstrated similar
differences in MIP-1 expression in the central nervous systems of B6
and BALB/c mice infected with mouse adenovirus. In this model of
hemorrhagic encephalopathy, susceptible B6 mice produced significantly
more MIP-1 than resistant BALB/c mice.
To ascertain whether the differences in MIP-1 expression between B6
and BALB/c mice were biologically relevant, complementary experiments
were done using B6 and BALB/c mice. In the first studies, we tested
whether i.p. administration of rMIP-1 exacerbates corneal disease in
resistant BALB/c mice. The i.p. route of injection was used because
others have shown that serum proteins can readily extravasate into the
inflamed cornea from blood vessels that invade the normally avascular
cornea [31
, 32
]. Previous studies from our
laboratory also provide structural evidence that i.p. injection of the
low-molecular-weight chemokine MIP-2 establishes a chemokine gradient
in P. aeruginosa-infected corneal tissue
[10
]. Nonetheless, to assure the establishment of a
local chemokine gradient in the cornea, subconjunctival injection also
was used in this study and confirmed the data obtained from the
i.p.-injection studies. Furthermore, DiPietro et al.
[33
] have recently demonstrated that MIP-1 induces an
angiogenic response in a corneal micropocket assay. The current studies
demonstrate both macroscopically and microscopically that P.
aeruginosa-induced ocular disease was exacerbated in BALB/c mice
treated with rMIP-1. Consistent with the susceptible phenotype
[10
, 11
, 26
], an increased
number of PMNs was observed in the corneas of rMIP-1- versus
vehicle-treated mice at 5 days p.i.
Although the predominant cellular infiltrate into the corneas of
rMIP-1- and vehicle-treated BALB/c mice was PMNs, mice treated with
MIP-1 consistently demonstrated a mononuclear cell infiltrate in
corneas. Further studies performed to determine the identity of the
mononuclear cells in BALB/c mice converted to the susceptible phenotype
showed that CD4+ T cells could be detected only in the
corneas of rMIP-1-treated mice at 5 days p.i. When corneal tissue
was immunostained for the T cell activation marker IL-2R, a few
CD4+ T cells stained positively. To determine whether
MIP-1 either directly or indirectly contributed to the persistence
of PMNs in the corneas at later times (5 days) p.i.,
recombinant-treated (subconjunctivally injected) mice were either
depleted or sham depleted of CD4+ T cells, and corneas were
analyzed for PMN number by MPO assay. CD4+ T cell-depleted
mice exhibited a significant decrease in corneal MPO activity which was
threefold less than in sham depleted controls. These data are evidence
that persistence of PMNs in cornea was due, at least in part, to an
indirect effect of MIP-1 and that T cells and/or their secreted
factors were direct contributors to this occurrence. In combination,
these data are consistent with prior P. aeruginosa-induced
keratitis studies in several aspects: (1) CD4+ T cells
could be detected only in the corneas of mice classified as susceptible
to P. aeruginosa corneal challenge [12
]; (2)
CD4+ T cells were readily detected in the corneas of these
mice by 5 days p.i. [12
, 13
]; (3) a few
activated, IL-2R-positive cells were detected in the T cell infiltrate
[13
]; and (4) the presence of activated T cells in
infected corneas at 5–7 days p.i. was associated with the persistence
of PMNs in the corneas of susceptible animals [12
].
Furthermore, similar studies using a herpes simplex virus (HSV)-induced
model of cornea infection showed that CD4+ T cells could
not be detected in the corneas of MIP-1-deficient mice. In addition,
the number of PMNs in the corneas of MIP-1-deficient mice infected
with HSV was reduced by >80% in comparison with wild-type controls
[14
].
As an additional means of evaluating the biological relevance of
MIP-1 in P. aeruginosa-induced corneal destruction,
susceptible B6 mice were administered pAb to MIP-1 before and after
corneal infection to reduce ocular disease. A combination of
subconjunctival and i.p. injections was used to increase the efficiency
of pAb delivery to the cornea. This treatment was effective in
preventing stromal scarring and corneal perforation. Similar enhanced
protection against HSV- and P. aeruginosa-induced corneal
disease has been described when a combination of subconjunctival and
i.p. pAb treatment (using either pAb to MIP-2 or IL-1ß) is
administered [10
, 11
, 26
,
34
]. Consistent with the amelioration of corneal
pathology in mice treated with MIP-1 pAb, we also found that these
animals had fewer PMNs in corneas at 3 and 5 days p.i.
As to the mechanism(s) by which MIP-1 enhances the recruitment of
PMNs into infected corneal tissue, various possibilities exist. Local
production of MIP-1 might establish a chemokine gradient that
directly acts on PMNs to induce their migration into the cornea.
Studies by Takano et al. [32
] support this hypothesis.
Using rat PMNs, their studies showed that rMIP-1 has PMN chemotactic
activity, although the potency of this chemokine activity is
significantly less than that of CINC-1 (rat analog of the PMN
chemoattractant MIP-2). In addition, other studies also have shown that
PMNs can produce MIP-1 protein and mRNA [33
],
suggesting that MIP-1-induced recruitment of PMNs might, in part,
take place in an autocrine manner. In addition, Bonecchi et al.
[22
] recently have shown that in human PMNs, interferon
(IFN)-
causes up-regulation of CCR1 receptors on PMNs and induces
their chemotaxis, broadening the action of CC chemokines to include
PMNs. Alternatively, MIP-1 might indirectly cause the migration of
PMNs into P. aeruginosa-infected corneal tissue. In this
regard, MIP-1 has previously been shown to induce the expression of
different inflammatory mediators including the PMN chemoattractants
MIP-2 and IL-1ß. Therefore, to directly test the latter theory, we
measured the expression of MIP-2 and IL-1ß mRNA in the corneas of
mice treated with MIP-1 pAb or NRS/rabbit IgG. As predicted by our
hypothesis, these experiments demonstrated that mice treated with
NRS/rabbit IgG versus MIP-1 pAb expressed approximately twofold
greater amounts of both MIP-2 and IL-1ß in the infected corneas at 5
days p.i.
Although it is possible that MIP-1 induces PMN migration into the
infected cornea through both direct and indirect pathways, it also is
probable that the mechanism by which this occurs in this model of
infection is somewhat complex. Based on the data described herein, we
propose that the presence of CD4+ T cells in cornea tissue
and the release of various inflammatory mediators from this cell type
are responsible for the persistence of inflammation and corneal
perforation in susceptible mice. In the current studies, MIP-1
protein and mRNA were detected in the corneas of BALB/c and B6 mice.
Yet, as shown before, CD4+ T cells are detected routinely
only in the corneas of susceptible B6 mice [12
,
13
]. These data suggest that the significantly lower
amount of MIP-1 produced in the corneas of BALB/c mice (from 1 to 5
days p.i.) is insufficient to establish a strong enough chemokine
gradient to recruit T cells into the cornea. This theory was supported
by experiments in which rMIP-1 was administered to resistant BALB/c
mice. These data demonstrated that CD4+ T cells were
recruited to the corneas of chemokine-treated BALB/c mice, and CD4 T
cell depletion studies confirmed that the presence of these cells was
directly related to an increase in total PMNs in corneas at later times
p.i.
In summary, current studies provided strong evidence that increased
and/or persistent expression of MIP-1 in corneas after P.
aeruginosa challenge contributes to the development of the
susceptible phenotype. This response is associated with an increase in
PMNs as well as the appearance of activated CD4+ T cells in
the cornea. Based on the studies in B6 versus BALB/c mice and
rMIP-1-treated T cell or sham depleted mice, the current data
strongly suggest that factors released by activated T cells contribute
directly to the persistence of PMNs in the cornea and ultimately to
corneal scarring and/or perforation.
|
ACKNOWLEDGEMENTS |
|---|
Received April 1, 2001; revised July 17, 2001; accepted July 18, 2001.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
(MIP-1) and MIP-1 chemokines attract distinct populations of lymphocytes J. Exp. Med. 177,1821-1826 and MIP-2 are involved in T cell-mediated neutrophil recruitment J. Leukoc. Biol. 52,303-306[Abstract]
(MIP-1) in acute lung injury in rats J. Immunol. 154,4793-4802[Abstract]
and IL-2 are protective in the skin but pathologic in the corneas of HSV infected mice J. Immunol. 149,3023-3028[Abstract]
as a critical macrophage chemoattractant in murine wound repair J. Clin. Invest. 101,1693-1698[Medline]
This article has been cited by other articles:
|
|
 |
|
  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]
|
|
|
|
 |
|
 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]
|
|
|
|
 |
|
 Y.-J. Day, M. A. Marshall, L. Huang, M. J. McDuffie, M. D. Okusa, and J. Linden Protection from ischemic liver injury by activation of A2A adenosine receptors during reperfusion: inhibition of chemokine induction Am J Physiol Gastrointest Liver Physiol, February 1, 2004; 286(2): G285 - G293. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
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]
|
|
|
|
|
|
B. K. Ambati, A. Anand, A. M. Joussen, W. A. Kuziel, A. P. Adamis, and J. Ambati Sustained Inhibition of Corneal Neovascularization by Genetic Ablation of CCR5 Invest. Ophthalmol. Vis. Sci., February 1, 2003; 44(2): 590 - 593. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
| ||||||||||||||||||||||||||||||||||||||
