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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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mRNA levels also were
decreased significantly in the cornea and in draining cervical lymph
nodes of mAb-treated mice. When CD28-/- animals were
tested, they exhibited a less severe disease response (no corneal
perforation) than wild-type B6 mice and had a significantly lower
delayed-type hypersensitivity response to heat-killed pseudomonas Ag.
These results support a critical role for B7/CD28 costimulation in
susceptibility to P. aeruginosa ocular
infection. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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37 times higher risk of developing bacterial
keratitis than individuals who use daily wear, rigid, gas-permeable
contact lenses (3). Because of its rapid progression and
the notorious resistance of P. aeruginosa to antibiotics,
the infection is difficult to treat and leads to blindness in the
infected eye, with the need for corneal transplantation.
Many clinical features of the disease are reproducible in rodent
models, including the rat (4) and mouse (5, 6). The mouse scarification model of Pseudomonas
keratitis has been well characterized, and the genetic susceptibility
of C57BL/6 (B6)3
(7) and related inbred strains (8) to eye
infection resulting in perforation of the cornea and blindness
correlated with a CD4+ T cell (Th1)-regulated
inflammatory response. That the tissue-destructive stromal inflammation
is mediated by T lymphocytes was evidenced by the decreased stromal
inflammation in mice that were mAb depleted of
CD4+ T lymphocytes or in which IFN- was
neutralized before corneal infection (7). In contrast, no
T effector cells were detected in the infected cornea of resistant, Th2
responder mice, such as BALB/c, that also exhibited a significantly
lower delayed-type hypersensitivity (DTH) than Th1 responder strains
(8).
The normal, uninflamed cornea, unlike most other tissue sites, lacks professional APC that are capable of presenting foreign Ag to CD4+ T cells (9). However, Langerhans cells (LC) are present in the adjacent conjunctival epithelium. Following experimental extended wear contact lens usage (10) or P. aeruginosa infection (11), LC migrate from the conjunctiva into the central cornea. LC resident in the conjunctival tissue are immature cells with limited APC function, but cytokines that are produced by infected tissue or the products of the pathogen itself can stimulate the migration and maturation of these cells (12). In this regard, cytokine mRNA transcripts are up-regulated early after P. aeruginosa corneal infection (13), but the products may be labile without T cell stimulation.
Maturational events that enhance the capacity of LC to present Ag to CD4+ T cells include increased expression of the CD80 and CD86 (B7 family) costimulatory molecules (14, 15). In a mouse model of HSV keratitis, immunostaining for CD80 (B7-1) was first detected at 3 days postinfection (p.i.), whereas CD86 (B7-2) was constitutively expressed on conjunctival LC. Early LC migration and B7-1 expression were independent of T cells, but T cells were required for the massive accumulation of B7-1+ LC in the cornea at the onset of inflammation (16).
To discern the role of B7/CD28 costimulation in P.
aeruginosa infection, we compared disease induced in mice
receiving B7-1/B7-2 mAb therapy before and after infection with
P. aeruginosa to disease induced in control mAb-treated
animals and in mice deficient for expression of CD28
(CD28-/-). Disease severity was significantly
reduced in B7-1/B7-2 mAb-neutralized mice and correlated with decreased
recruitment and activation of CD4+ T cells in
cornea and reduced mRNA transcript levels of IFN- in both cornea and
draining cervical lymph nodes (CLN) p.i. Disease also was reduced in
CD28-/- vs wild-type B6 mice, and this
correlated with a significantly reduced DTH response to pseudomonal Ag.
Collectively, these data support a critical role for B7/CD28 signaling
in the susceptibility response to P. aeruginosa ocular
infection.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female, B6, BALB/c, and CD28-/- mice on the B6 background were purchased from The Jackson Laboratory (Bar Harbor, ME) at 8 wk of age. Mice were housed in accordance with the National Institutes of Health guidelines, and all procedures in this study conformed to the Association for Research in Vision and Ophthalmology’s Statement for the Use of Animals in Ophthalmic and Vision Research.
Bacterial preparation
P. aeruginosa strain 19660 was purchased from American Type Culture Collection (Manassas, VA) and were maintained and prepared as described previously (7, 8). The stock culture was maintained on peptone tryptic soy broth (PTSB) slants (PTSB solidified with 1.7% agar; Difco, Detroit, MI) at 4°C, and fresh slants were prepared every 2 wk. Cultures were grown in PTSB at 37°C on a rotary shaker at 150 rpm for 18 h to an approximate OD of 1.6 at 540 nm, centrifuged at 6000 x g for 10 min at 15°C, and washed with, then resuspended in 5 ml of sterile saline to a concentration of 1.0 x 108 CFU/µl. For infection, two dilutions (1/10 each) were made in sterile saline for a final concentration of 1.0 x 106 CFU/µl.
Infection and ocular response
Mice were anesthetized, and the left cornea was scarified with a 25 5/8-gauge needle as described previously (7, 8). A 5-µl aliquot containing a 1.0 x 106 CFU/µl suspension of bacteria was applied to the wounded cornea, and ocular disease was evaluated daily up to 7 days p.i. Corneal disease was graded as previously described (17): 0 = clear or slight opacity, partially or fully covering the pupil; +1 = slight opacity, fully covering the anterior segment; +2 = dense opacity, partially or fully covering the pupil; +3 = dense opacity, covering the entire anterior segment; and +4 = corneal perforation or phthisis. Mean clinical scores were calculated by summation of the scores for each group (n = 5/group/time) of mice divided by the total number of mice scored at each time point.
LC in epithelial flat mounts
At 1, 4, and 6 days p.i., epithelia were harvested (n = 5/group/time) and stained with ADPase as previously described (18, 19). Briefly, enucleated eyes were placed in 0.02 M EDTA-PBS buffer, pH 7.2, at 37°C for 3 h. Corneal and conjunctival epithelium was separated from the stroma, fixed in cacodylate-buffered formaldehyde for 20 min at 4°C, and washed four times with cold 0.1 M cacodylate buffer. Epithelial sheets were incubated in ADPase substrate solution or in dihydroxyphenylalanine-oxidase (control) containing ADPase buffer, 2% lead nitrate, and ADPase (5 mg/ml; Sigma) for 15 min at 37°C. Sheets were washed four times with Tris-maleate buffer, pH 7.2, and developed for 5 min in ammonium sulfide (1/10). Sheets were washed again with Tris-maleate buffer, transferred to a glass slide, and coverslipped in glycerol. Representative areas were photographed at x25 on an Axiophot microscope (Carl Zeiss, Morgan Instruments, Cincinnati, OH) and printed at x200, and LC were counted (18). No stained LC were detected in dihydroxyphenylalanine-oxidase treated tissues.
Dual Ab staining
B7-1 expression was assessed in B6 vs BALB/c corneal epithelial flat mounts by dual staining with mAbs DEC-205 (clone NLDC-145, American Type Culture Collection), specific for LC, and B7-1 (clone 16-10A1; PharMingen, San Diego, CA), specific for B7-1. B7-2 staining of LC was tested by staining with the specific mAb B7-2 (clone GL-1, PharMingen). A rat anti-human HLA-DR5 (American Type Culture Collection clone SFR3-DR5, IgG2b) was used here and in the immunostaining studies reported below as a nonspecific control for B7-1 mAb, as it provided the same results as the recommended control mAb, hamster anti-keyhole limpet hemocyanin (clone Ha4/8, PharMingen). Epithelia were removed (as described above) at 3 and 6 days p.i. from B6 and BALB/c (n = 5/group/time) mice and at 5 days p.i. from B7-1/B7-2 and HLA-DR5 mAb-treated (described below) mice (n = 5/group) and were fixed in 2% paraformaldehyde/0.1 M cacodylate buffer at 4°C for 15 min. Sheets were washed four times at 4°C in 0.1 M cacodylate buffer for 10 min each time and were blocked with 0.1 M PBS containing 1% BSA (Sigma). Sheets were incubated in DEC-205 mAb (1/2) at 4°C overnight, washed in PBS/BSA, and incubated for 1 h at 37°C in FITC-conjugated goat anti-rat IgG (1/100; Jackson ImmunoResearch, West Grove, PA). For B7-1 staining, the same epithelial sheets were washed for 15 min in PBS/BSA, blocked with rat IgG (1/10; Jackson ImmunoResearch) for 1 h, and incubated for 2 h at 37°C with biotin-conjugated B7-1 mAb (1/25). Three PBS/BSA washes (10 min each) were performed before incubation in streptavidin-conjugated rhodamine (1/40; Jackson ImmunoResearch) at 37°C for 1 h. B7-2 staining was performed as described for B7-1. Sheets were placed on glass slides in a mountant containing 10 mg p-phenylenediamine, 0.01 M PBS, and glycerol. Representative areas were photographed with a Zeiss Axiophot microscope. Identical FITC- and rhodamine-stained fields were digitized with a SPOT digital camera (Sterling Heights, MI) and overlaid using MetaMorph (Universal Imaging, West Chester, PA). Dual-labeled cells were counted on at least eight fields (each at x200) for each group of mice as described above, and the number of positive cells was expressed as the total number of cells counted.
Dual labeling of CLN
Draining CLN ipsilateral to the infected eyes were removed at 1 and 6 days p.i. from B6 and BALB/c mice (n = 3/group/time), embedded in Tissue-Tek (Miles, Elkhart, IN), snap-frozen in liquid nitrogen, and stored at -20°C. Eight-micron sections were cut, mounted onto poly-L-lysine-coated slides (Polysciences, Warrington, PA), kept at 37°C overnight, and fixed in cold acetone for 2 min at -20°C. Slides were rinsed with 0.01 M PBS and incubated for 30 min with 1.0% BSA and 0.05% Tween-20.
Sections were incubated with DEC-205 mAb (1/200) for 1 h and washed, and goat anti-rat FITC-conjugated Ab (1/100; Jackson ImmunoResearch, West Grove, PA) was applied for 1 h. The second primary Ab, rabbit pAb, tested by Western immunoblot and found specific against P. aeruginosa strain 19660 heat-killed Ag (data not shown), was applied to the sections (1/25) and incubated for 1 h, followed by a donkey anti-rabbit rhodamine red x Ab (1/500; Jackson ImmunoResearch) for 1 h. Control sections were similarly treated with naive rabbit serum (NRS) and photography was performed as described above.
Monoclonal Ab neutralization
B6 mice (n = 5/group/time) were injected subconjunctivally and i.p. with neutralizing hamster and rat anti-mouse mAbs against B7-1/B7-2 (PharMingen). Intraperitoneal injections (50 µg of each mAb) were given every other day beginning on day 14 before P. aeruginosa challenge and ending 5 days p.i. Before challenge, anti-B7-2 (3 days, 10 µg/10 µl) and anti-B7-1 mAbs (1 day, 10 µg/10 µl) were injected subconjunctivally. Control B6 mice (n = 5/group/time) were injected with a nonspecific anti-HLA-DR5 mAb. Eyes were examined, and mean clinical scores were calculated.
Immunostaining
Infected eyes from B7 (B7-1/B7-2) or HLA-DR5 mAb-treated (n = 2/group/time) B6 mice were enucleated at 5 days p.i. Eyes were embedded in Tissue-Tek, as described above and previously (7, 8). Ten-micrometer sections were cut, mounted onto poly-L-lysine-coated slides (Polysciences), and incubated for 1 h with primary mAbs specific for CD4 (rat IgG2a, clone H129.19, 1/10) and CD25 (IL-2R, rat IgM, clone 7D4, 1/50; all from PharMingen). Sections were incubated with 0.3% hydrogen peroxide for 30 min, blocking endogenous peroxidase activity, and for 1 h with a biotinylated secondary Ab, anti-rat IgG2a (1:25; CD4) or anti-rat IgM (IL-2R, 1:100; all obtained from PharMingen). HRP-conjugated avidin (1:25 or 1:100; Zymed, San Francisco, CA) was incubated with the sections for 30 min before applying 3,3'-diaminobenzidine tetrahydrochloride (Pierce, Rockford, IL) for 10–15 min. Control sections were incubated similarly with HLA-DR5, as described previously (7, 8).
RT-PCR
Total RNA was isolated from corneas and CLN of B6 mice treated
with anti-B7-1/B7-2 vs anti-HLA-DR5 mAb (n = 5
mice/group) at 5 days p.i. and corneas from
CD28-/- and wild-type B6 mice
(n = 5/group/time) at 3, 5, and 7 days p.i. Total RNA
(50 ng) was reversed transcribed using Random primers (Life
Technologies, Grand Island, NY) and Sensiscript reverse transcriptase
(Qiagen, Valencia, CA) in the presence of 10 U of RNase inhibitor
(Promega, Madison, WI). Amplification of cDNA was conducted with Taq
polymerase (Life Technologies) in a GeneMate Thermal Cycler (ISC
BioExpress, Kaysville, UT). All primers were synthesized by Life
Technologies. The primer sequences were
5'-TGCATCTTGGCTTTGCAGCTCTTCCTCATGGC-3'and
5'-TGGACCTGTGGGTTGTTGACCTCAAACTTGGC-3' for IFN- (364 bp) and
5'-GTGGGCCGCTCTAGGCACCAA-3' and 5'-CTCTTTGATGTCACGCACGATTTC-3'
for 
-actin (539 bp). The cycling conditions used were 94°C for
45 s, 59°C for 30 s, and 72°C for 1 min for 35 cycles,
with a final extension at 72°C for 10 min. The same amount of RNA
(without RT reaction) was subjected to PCR amplification as a negative
control to verify the absence of DNA contamination in the RNA samples.
Twenty microliters of final PCR products were analyzed by
electrophoresis (1.2% agarose gel with ethidium bromide). Bands were
visualized under UV transillumination and quantitated using an
AlphaImager 2000 Documentation and Analysis system (
Innotech, San
Leandro, CA). Integrated density values (IDV) for the IFN- PCR
product were corrected for the amount of -actin on each sample. Data
are expressed as the mean IDV of three to five samples from
separate mice.
DTH assay
For Ag challenge, a similarly prepared culture (as described above, 1.0 x 108 CFU/µl) was heat-killed (60 min at 56°C) and diluted 1/10 in sterile saline to a final concentration of 1.0 x 107 CFU/µl. At 5 days p.i., 1.0 x 108 CFU heat-killed P. aeruginosa cells (10 µl in 0.01 M PBS) were injected into the ear of CD28-/- and wild-type B6 mice ipsilateral to the infected eye. PBS was injected similarly into the contralateral ear as a control. Ear thickness was measured with a dial pocket gauge (L.S. Starrett, Small Parts, Miami Lakes, FL) before and at 24 and 48 h after Ag challenge. DTH was calculated as previously described (20).
Statistical analysis
Unpaired Student’s t test was used to determine the significance of the mean clinical scores, ADPase, B7, and dual staining of LC, RT/PCR, and DTH data. A p = 0.05 confidence interval was used to determine the level of significance. All experiments were repeated at least once, and representative data typical of a single experiment are shown.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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At 3–7 days p.i. significant differences were observed in the mean clinical scores between the two groups of mice, with a milder course of disease (no grade above +2 throughout 7 days p.i.) in BALB/c mice. Initially (1 day p.i.), BALB/c mice had faint opacity covering the anterior segment (+1), while B6 mice exhibited faint to dense central opacity covering the pupil (+2; p = 0.0948). By 3–7 days p.i. BALB/c mice had slightly increased (+2) opacity. Disease in B6 mice was more severe (+3) during this time (p = 0.0001 at 3 and 5 days p.i., respectively), and, as expected, only the corneas of B6 mice perforated (+4) by 7 days p.i. (p = 0.0001) (7, 8).
ADPase staining
Because of the important role of APC in Ag presentation to T
cells, we next tested whether the number of LC infiltrating the cornea
p.i. differed between the two groups. The data are shown in Fig. 1
. A significant
(p = 0.0001) 2-fold greater number of LC was
seen in the cornea of B6 vs BALB/c mice at 1 day p.i. By 4 days p.i. LC
in cornea had increased in both groups, but was not significantly
different (p = 0.176). By 6 days p.i. LC were
increased slightly in BALB/c mice, but had doubled in number in corneal
flat mounts of B6 mice (p = 0.0005).
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B7-2 staining (data not shown) confirmed the results of an
earlier report (16) that found B7-2 constitutively
expressed in uninfected mouse conjunctiva. We next tested whether B7-1
expression differed between the two groups. B7-1 immunostaining was not
detected in the cornea or conjunctiva until 1 day p.i. (data not shown)
in either group. Dual labeling, representative of staining in BALB/c
(data not shown) and B6 mouse cornea, is shown in Fig. 2 at 3 days p.i., a time when T cells
begin to infiltrate the cornea of B6 mice (7). Fig. 2
illustrates FITC label for the DEC-205-specific LC marker (Fig. 2A), rhodamine staining for B7-1 (Fig. 2B;
identical field as Fig. 2A), yellow-stained cells (Fig. 2C) that are dual labeled, and an HLA-DR5-reacted negative
control (Fig. 2D). All cells that express the DEC-205 Ag do
not stain for B7-1. Table I depicts the
quantitative data from this experiment. No significant difference in
the mean number of dual-labeled LC was seen in B6 vs BALB/c mice at 3
days p.i. (30% for both), but significantly (p
= 0.0001) more of these cells were present in B6 (24%) vs BALB/c
(18%) corneal epithelial flat mounts at 6 days p.i.
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LC migrate from cornea to draining CLN where they present
viral Ag to naive T cells (16). Whether bacterial Ag from
the infected cornea could be detected associated with DEC-205-positive
cells in draining CLN had not been examined. Therefore, CLN ipsilateral
to the infected eye in both groups of mice were removed at 1 and 6 days
p.i. Tissue sections were dual labeled with a DEC-205 mAb and
anti-Pseudomonas pAb. These data are shown in Fig. 3. Immunostaining for DEC-205 and that
for Pseudomonas Ag were observed in the outer and
paracortical regions of the CLN of B6 (Fig. 3, A and
B, respectively) and BALB/c (data not shown) mice at all
times examined. All DEC-205-positive cells did not dual label (Fig. 3C), suggesting that some may not have been associated with
Ag. Immunostaining was similar in distribution in CLN of B6 and BALB/c
mice, but was patchy and sparse in the latter group. Control sections
incubated with NRS were negative (Fig. 3D). Table II depicts quantitative data from this
experiment at 1 and 6 days p.i. Approximately 2-fold more dual-labeled
Ag-associated LC were present in the CLN of B6 vs BALB/c mice at both l
day (84 vs 33%) and 6 days (78 vs 37%) p.i.
(p = 0.0175 and p = 0.0001,
respectively).
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To determine the relevance of these data, B6 mice were
treated with B7-1/B7-2 or HLA-DR5 mAb before and after infection with
P. aeruginosa. Ocular disease was graded, and mean clinical
scores were calculated (1–5 days p.i.). These data are presented in
Fig. 4. At 1 day p.i. both groups had
similar disease grades (+1), but beginning on day 2, control mice began
to consistently show slightly increased opacity (+2). At 3, 4, and 5
days p.i., B7-1/B7-2 mAb-treated mice had a significantly milder course
of disease compared with control mice (p =
0.007, 0.004, and 0.002 at 3, 4, and 5 days p.i., respectively).
Representative eyes from both groups were photographed at 5 days p.i.
using a slit lamp, and the data are shown in Fig. 5. B7-1/B7-2 mAb-treated mice had slight
opacity covering the anterior segment (Fig. 5A), while
HLA-DR5 mAb-treated animals had dense opacity covering the anterior
segment (+3 score) or corneal perforation (Fig. 5B). To
confirm the neutralizing effects of mAb treatment, we examined B7-1 and
B7-2 immunostaining in the cornea of the two groups of treated mice at
5 days p.i. These data are shown in Table III. B7-1 and B7-2 expression in cornea
was reduced significantly (71% (p = 0.0009)
and 82% (p = 0.0001), respectively) in the
experimental vs control mAb-treated group, confirming the effectiveness
of the blockade.
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Previously we showed that depletion of
CD4+ T cells in B6 mice improved disease outcome
after P. aeruginosa infection (7). Therefore,
we tested whether T cells were affected by treatment with B7-1/B7-2 vs
HLA-DR5 mAb, contributing to the improved outcome in the B7-treated
animals. Representative examples of immunostaining for CD4 and IL-2R T
cell surface markers in B6 mice at 5 days p.i. are shown in Fig. 6. Qualitatively fewer T cells were
detected in the cornea of B7-1/B7-2 mAb-treated (Fig. 6B) vs
control Ab-treated B6 mice (Fig. 6A). A few of these cells
in anti-HLA-DR5-treated (Fig. 6C) mice were
IL-2R+, but no activated T cells were detected in
the cornea of B7-1/B7-2 mAb-treated mice (Fig. 6D). No
immunostaining was detected in the cornea of B6 mice when
anti-HLA-DR5 was substituted for either specific primary mAb (data
not shown).
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As we had previously correlated a Th1 response and increased
levels of IFN- with susceptibility in B6 mice (7), we
next used RT-PCR to test B7-1/B7-2 vs HLA-DR5 mAb-treated B6 mice for
mRNA transcript levels of IFN- in cornea and draining CLN at 5 days
p.i. The results are shown in Fig. 7 as a
representative agarose gel and a graph plotting the band IDV.
Neutralization of B7-1/B7-2 significantly reduced IFN- mRNA
expression in the cornea (51%; p = 0.04556) and CLN
(31%; p = 0.01778) compared with HLA-DR5 mAb
treatment.
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Because of the potential for anti-B7-1/B7-2 mAb itself to
provide signaling in interaction with CD28, we tested the response to
infection in CD28-/- vs wild-type B6 mice.
These data are presented in Fig. 8. At 1
day p.i., the responses of both groups were similar and in the +1 to +2
range. By 3–7 days p.i. CD28-/- mice had lower
disease grades, which differed significantly compared with that in
wild-type mice (p = 0.0054, p =
0.0014, and p = 0.0001 at 3, 5, and 7 days p.i.,
respectively). CD28-/- mouse corneas did not
perforate, while all the corneas of wild-type mice perforated by 5–7
days p.i. We also tested the responses of the two groups to heat-killed
bacterial Ag challenge at 5 days p.i. by measuring DTH. This assay was
an additional measure of host T cell responsiveness to P.
aeruginosa infection. These data are shown in Fig. 9. DTH measured at 24 and 48 h after
Ag challenge was significantly (p = 0.0002 and
p = 0.0106, respectively) elevated in wild-type B6 vs
CD28-/- mice.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We were also interested in whether P. aeruginosa Ag
associated LC in draining CLN could be detected in our bacterial model.
Dependent on the type of tissue in which they reside and the character
of the pathogen-related migration-inducing signals, DC have been shown
to reach lymph nodes within 4–48 h (23, 27, 28, 29, 30). The
cells can act in the inductive phase of an immune response as well as
APC for peripheral tissue-homing memory T cells (24, 31).
In both B6 and BALB/c mice, dual staining for bacterial Ag in
association with DEC-205+ cells was confirmed in
draining CLN at 1 and 6 days p.i., with a greater number of
dual-labeled cells at both times in susceptible vs resistant mice
(Table II
). These data suggest that LC may carry bacterial Ag from the
cornea to the CLN. They do not rule out the possibility that Ag drained
to CLN from the eye and associated with DEC-205-expressing cells in the
node. Nonetheless, these data do show that both groups of mice are
capable of carrying out an inductive response to pseudomonal Ag, but
are disparate regarding the number of cells engaged in this
process.
Among the maturational events that increase the capacity of LC to present Ag to CD4+ T cells is an increase in expression of the B7 family of costimulatory molecules (14, 15, 32). The capacity of B7 to deliver a costimulatory signal to T cells by binding to CD28 is well documented both in vitro and in vivo (33, 34, 35). Systemic treatment with mAb to B7 or with the ligand CTLA4-Ig can reduce the severity of T cell-mediated inflammatory processes such as experimental lupus (35) and autoimmune encephalomyelitis (36, 37). However, such studies addressing the differential functional role of B7-1 and B7-2 in these autoimmune models have resulted in conflicting data (38). These models led us to ask whether there were differences in the maturation of corneal LC in susceptible vs resistant mice, as evidenced by expression of B7 costimulatory molecules. In our studies, as in HSV keratitis, B7-1 was not detected in the mouse ocular epithelium until after infection. However, in our model, positively stained cells were detected 2 days earlier (1 vs 3 days) p.i. than reported in the HSV model (16). Although the total number of dual-labeled cells was decreased at 6 vs 3 day p.i., significantly more such cells were present at the latter time in B6 vs BALB/c cornea. This decrease in labeled cells may reflect corneal healing in BALB/c vs perforation in B6 mice, but this remains untested. Further, some, but not all, migrating LC in cornea expressed B7-1, suggesting that LC migration and B7-1 expression are independently regulated in bacterial as well as in ocular viral infection (16).
The requirement for costimulation of T effector cells at an
inflammatory site remains controversial. It has been suggested that
during acute infections, cytokines/chemokines that are produced by
cells (resident and inflammatory) within the lesion may supplant the
need for costimulation of effector T cells (39). In the
HSV model, replicating virus and viral Ag are detectable in the cornea
at 5 days p.i., whereas T cell-mediated inflammation is initiated at
around 10 days p.i. (16). This is in contrast to the
Pseudomonas model used herein, in which viable bacteria have
been detected in the cornea throughout 9 days of infection
(8) with a significantly greater bacterial load in
susceptible mice (8). Thus, in susceptible B6 mice,
activation of infiltrating effector T cells could occur in the inflamed
tissue site via other mechanisms besides B7 costimulation. To determine
the role of B7/CD28 pathway costimulation in our bacterial model,
systemic and intraocular mAb neutralization of B7-1/B7-2 was performed.
mAb-treated mice had a milder course of disease compared with control
Ab-treated animals and dual Ab staining confirmed that both B7-1- and
B7-2-positive cells were significantly reduced in cornea and CLN by the
Ab blockade. We also tested whether T cell recruitment to cornea was
affected and found that in B7-1/B7-2 mAb vs control treated mice, T
cell immunostaining for CD4- and IL-2R (activated)-positive cells was
significantly reduced qualitatively or absent, respectively, compared
with control Ab-treated mice. RT-PCR data also supported the importance
of costimulation through this pathway in the disease response, as mRNA
transcripts for IFN- were reduced in both cornea and CLN in
B7-1/B7-2 vs control mAb-treated mice. These data also are consistent
with an earlier report from this laboratory showing that neutralization
of IFN- correlated with reduction of corneal disease in B6 mice
(7). Reduction of other cytokines/chemokines also has been
reported using anti-B7 mAbs. Production of macrophage inflammatory
protein-1 induced following TCR-mediated T cell activation was
inhibited by coculture with anti-B7 mAbs, indicating that full
production of this C-C chemokine depended on interaction with B7 ligand
through CD28 (21).
Ligation of CD28 together with TCR stimulation can prevent cell death and stimulate cytokine production and proliferation as well as recruitment of other cells to inflammatory sites by producing chemokines, important mediators of chemotaxis in vivo (40, 41, 42, 43, 44). The role of CD28 is unique, because IL-2 does not provide costimulatory signals needed for chemokine production despite its ability to enhance expression of chemokine receptors (21). Due to the potential of the B7-1/B7-2 mAbs to initiate costimulatory signaling through CD28, we next tested CD28-/- mice. Ocular disease and DTH were reduced in these mice, and none of the corneas of deficient vs wild-type mice perforated p.i., confirming the mAb neutralization studies.
In summary, our findings strongly suggest that in the susceptibility response to P. aeruginosa, B7/CD28 costimulation appears critical in the induction of an inflammatory response resulting in corneal perforation and blindness.
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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: B6, C57BL/6; DTH, delayed-type hypersensitivity; LC, Langerhans cell; CLN, cervical lymph node; NRS, naive rabbit serum; PTSB, peptone tryptic soy broth; p.i., postinfection; IDV, integrated density value; DC, dendritic cells.
Received for publication July 17, 2000. Accepted for publication October 24, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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synergistically upregulates the granulocyte-macrophage-colony-stimulating factor-induced B7-1 expression in murine Langerhans cells. Br. J. Dermatol. 135:194.[Medline]
contributes to the anticryptococcal delayed-type hypersensitivity reaction and protection against Cryptococcus neoformans. J. Leukocyte Biol. 61:147.[Abstract]
in bronchoalveolar lavage fluid of allergic asthmatic patients. Am. J. Respir. Crit. Care Med. 153:1398.[Abstract]
production by synovial T cells. Clin. Exp. Immunol. 101:398.[Medline]
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