February 2007
Volume 48, Issue 2
Free
Immunology and Microbiology  |   February 2007
Spantide I Decreases Type I Cytokines, Enhances IL-10, and Reduces Corneal Perforation in Susceptible Mice after Pseudomonas aeruginosa Infection
Author Affiliations
  • Linda D. Hazlett
    From the Department of Anatomy and Cell Biology, Wayne State University School of Medicine, Detroit, Michigan.
  • Sharon A. McClellan
    From the Department of Anatomy and Cell Biology, Wayne State University School of Medicine, Detroit, Michigan.
  • Ronald P. Barrett
    From the Department of Anatomy and Cell Biology, Wayne State University School of Medicine, Detroit, Michigan.
  • Jianhua Liu
    From the Department of Anatomy and Cell Biology, Wayne State University School of Medicine, Detroit, Michigan.
  • Yunfan Zhang
    From the Department of Anatomy and Cell Biology, Wayne State University School of Medicine, Detroit, Michigan.
  • Shahrzad Lighvani
    From the Department of Anatomy and Cell Biology, Wayne State University School of Medicine, Detroit, Michigan.
Investigative Ophthalmology & Visual Science February 2007, Vol.48, 797-807. doi:10.1167/iovs.06-0882
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      Linda D. Hazlett, Sharon A. McClellan, Ronald P. Barrett, Jianhua Liu, Yunfan Zhang, Shahrzad Lighvani; Spantide I Decreases Type I Cytokines, Enhances IL-10, and Reduces Corneal Perforation in Susceptible Mice after Pseudomonas aeruginosa Infection. Invest. Ophthalmol. Vis. Sci. 2007;48(2):797-807. doi: 10.1167/iovs.06-0882.

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      © 2016 Association for Research in Vision and Ophthalmology.

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Abstract

purpose. To determine the effects of blocking substance P (SP) interactions with its major receptor (NK1-R) using the antagonist spantide I in susceptible mice infected with Pseudomonas aeruginosa.

methods. Immunohistochemistry and enzyme immunosorbent assay (EIA) tested levels of SP in the cornea of B6 and BALB/c mice. B6 mice were treated with spantide, and after infection, slit lamp examination; clinical score; bacterial counts; and myeloperoxidase (MPO), RT-PCR, ELISA, and polymorphonuclear (PMN) cell chemotaxis assays were performed.

results. SP corneal levels were significantly elevated constitutively and after infection in the B6 more than in BALB/c mice. Spantide treatment of B6 mice significantly decreased the number of perforated corneas, bacterial counts, and PMNs. mRNA levels for type I cytokines (e.g., IFN-γ) as well as MIP-2, IL-6, TNF-α, and IL-1β (mRNA and protein) also were significantly reduced after spantide treatment. The type II cytokine IL-10 (mRNA and protein) was elevated, whereas TGF-β mRNA levels were unchanged after spantide treatment. PMN chemotaxis was induced by SP and other neuropeptides in vitro, but was not affected by spantide I. mRNA for neurokinin-1-receptor-1 (NK-1R) was detected in the normal and infected corneas and on macrophages (Mφs), but not on PMNs (unstimulated or stimulated with endotoxin [LPS]). Spantide treatment of Mφs reduced IL-1β after LPS+SP treatment but not after either alone.

conclusions. The SP antagonist Spantide provides a novel approach to reduce type 1 and enhance the type 2 cytokine IL-10 in the infected cornea of B6 mice, leading to a significant reduction in corneal perforation and improved disease outcome.

Keratitis caused by Pseudomonas aeruginosa develops rapidly and may lead to corneal perforation, with a higher incidence of disease associated with extended-wear contact lens use. 1 2 Experimental studies have shown that Th1 responder mouse strains (e.g., C57BL/6) are susceptible (cornea perforates), whereas Th2 responder strains (e.g., BALB/c) are resistant (i.e., the cornea heals) 3 after bacterial infection. Th1 response development with IFN-γ production is often dependent on the presence of IL-12 and the T cell’s ability to respond to the cytokine. 4 5 6 IL-12 has been detected in the cornea of susceptible B6 mice by RT-PCR and protein analyses. 7 In that study, we also found that in B6 mice, either sustained IL-12-driven IFN-γ production or endogenous absence of IL-12, resulting in reduced IFN-γ mRNA levels, leads to corneal perforation (susceptibility). 7 In contrast, studies using similar approaches failed to detect IL-12 in the infected cornea of BALB/c mice that control infection and restore corneal clarity (Rudner X et al. IOVS 2001;42:ARVO Abstract 2767), 8 suggesting that these animals regulate IFN-γ production through IL-12-independent mechanisms, resulting in less corneal destruction. Previous work 9 confirmed that IL-18 regulates IFN-γ production in the cornea of resistant BALB/c mice and that either its neutralization or endogenous absence results in increased bacterial load and corneal perforation. More recently, 10 we have shown an interrelationship between the neuropeptide substance P (SP) and production of IFN-γ in the infected BALB/c cornea. Evidence has been provided that in the infected cornea NK cells are the source of IFN-γ, express the NK-1R, and participate in the regulation of polymorphonuclear cell (PMN) infiltration. Evidence also has been provided that SP regulates production of IFN-γ indirectly through the regulation of IL-18 and directly through interaction with the NK-1R on NK cells. Collectively, the data demonstrate a unique link between neuropeptide regulation of the innate immune response and in resistant mice, protection against P. aeruginosa-induced corneal perforation. 
Alternately, evidence also indicates that SP plays an important role in augmenting inflammatory responses 11 principally by regulating the function of cells such as dendritic cells (DCs) and macrophages (Mφs), via the NK-1R. 12 13 SP, a product of both nerves and lymphocytes, is present in many areas of the central and peripheral nervous system. In this regard, the cornea is one of the most densely innervated tissues in the body and is richly supplied by both sensory and autonomic nerve fibers. 14 Recently, the distribution of neuropeptides, including SP, was elegantly shown in the human cornea, 14 but limited information is available for the mouse cornea, before or after infection. In the present study, we examined the distribution of SP in the mouse cornea and a disparate distribution of the neuropeptide in susceptible (more) B6 versus resistant (less) BALB/c mice was documented. Furthermore, treatment of B6 mice with spantide I, an SP antagonist, decreased type I cytokines, led to elevated IL-10 levels, and reduced corneal perforation after bacterial infection. The evidence strongly supports the tenet that SP augments the inflammatory response after P. aeruginosa infection in susceptible mice and provides insight into the mechanisms by which it contributes to poor disease outcome. 
Methods
Mice
Female, 8-week-old C57BL/6 (B6) and BALB/c mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and housed in accordance with the National Institutes of Health guidelines. Humane animal care conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Infection
P. aeruginosa strain 19660, purchased from the American Type Culture Collection (Manassas, VA), was prepared as described elsewhere. 15 Mice (n = 5/group/time) were anesthetized, and the left central cornea was scarified with a 25
\({5}/{8}\)
-gauge needle. A 5-μL aliquot containing a 1.0 × 106-CFU/μL bacterial suspension was applied to the wounded cornea and disease graded 1, 3, and 5 days postinfection (pi). 16  
Immunostaining
The left cornea of B6 and BALB/c mice (n = 3/group/time) was infected with P. aeruginosa. Before infection and at 1 and 7 days pi, eyes were removed, frozen in optimal cutting temperature (OCT; Tissue-Tek; Sakura Finetek, Torrance, CA) compound and sectioned at a thickness of 10 μM. The sections were incubated with primary rabbit anti-SP antibody (1:5000; Chemicon, Temecula, CA) and a biotinylated secondary goat anti-rabbit antibody (1:400; BD-PharMingen, San Diego, CA) for 1 hour each. Representative sections were observed and photographed with digital imaging (Axiophot with Axiocam; Carl Zeiss Meditec, Inc., Oberkochen, Germany). 
Peritoneal Mφs, harvested as described later, were plated (500,000/well) and allowed to adhere to chamber slides overnight (Laboratory-Tek; Nalge Nunc International, Naperville, IL). After nonadherent cells were removed, the slides were fixed for 2 minutes in cold acetone and air dried. Nonspecific staining was blocked with minimum essential medium (MEM) containing 5% fetal bovine serum (FBS) and 5% normal goat serum for 30 minutes. After the cells were washed in 0.1 M phosphate buffer, the primary antibody, a rabbit polyclonal specific for NK-1R (diluted 1:500 in blocking agent; Novus Biologicals, Littleton, CO) was applied for 1 hour. Slides were washed and endogenous peroxidase activity quenched in 0.3% hydrogen peroxide for 30 minutes. The secondary antibody, a biotin-conjugated goat anti-rabbit IgG (1:500; Jackson ImmunoResearch, West Grove, PA) was added and incubated with the sections for 1 hour. For both cornea and Mφs, after the sections were washed, extravidin horseradish peroxidase (HRP; 1:100, Sigma-Aldrich) was incubated with the sections for 30 minutes. Metal-enhanced diaminobenzidine (DAB; Fisher Scientific, Pittsburgh, PA) was used to visualize NK-1R-positive staining. Sections were visualized, and digital images were captured with a confocal laser scanning microscope (TSC SP2; Leica Microsystems, Mannheim, Germany). 
Controls for both cornea and Mφ staining were treated similarly but with omission of the primary antibody. 
Spantide Treatment
B6 mice (n = 5/group/time/assay) were injected intraperitoneally (IP) with 36 μg/mouse of synthetic spantide I (>99% purity; Bachem, King of Prussia, PA) on days −1 and 0 (day of infection) and daily through 5 days pi. 17 Control mice were similarly injected with vehicle (PBS <0.05 EU/mL endotoxin; Mediatech, Herndon, VA). Infected corneas were collected at 1, 3, and 5 days pi for real-time PCR detection of mRNA levels of cytokines. Normal, uninfected corneas also were harvested to determine basal levels of the target cytokines. In separate experiments, infected corneas (n = 5/group/time/assay) were harvested at 3 and 5 days pi and used for ELISA analysis, viable bacterial enumeration, and MPO quantitation. 
Real-Time PCR
Normal, uninfected, and infected corneas (n = 5/group/time) from spantide I versus PBS-treated B6 mice were removed at 1, 3, and 5 days pi. Corneas were stored in RNA stabilizer (RNAlater; Ambion Inc., Austin, TX) at −70°C. Total corneal RNA was extracted (RNA STAT-60; Tel-Test, Friendsville, TX) per the manufacturer’s instructions and used to produce a cDNA template for PCR reaction. One microgram of each RNA sample was reverse transcribed by M-MLV reverse transcription (Invitrogen, Carlsbad, CA) simultaneously in a 20 μL volume. cDNA products were diluted 1:25 with diethylpyrocarbonate (DEPC)-treated H2O and 2 μL of each cDNA dilution used for real-time PCR (20 μL reaction volume). mRNA levels of proinflammatory cytokines (MIP-2, IL-6, TNF-α, IL-12, IL-18, IFN-γ, and IL-1β) and anti-inflammatory cytokines (TGF-β and IL-10) were detected by real-time PCR (MyiQTM Single Color Real-Time PCR Detection System; BioRad, Hercules, CA). Master mix (iQTM SYBR Green Supermix; BioRad) was used for the PCR reaction with primer concentrations of 0.25 μM. After the preprogrammed hot-start cycle (3 minutes at 95°C), the parameters used for PCR amplification were 10 seconds at 95°C. 10 seconds at 59°C, and 30 seconds at 72°C. These cycles were repeated 40 times. These PCR parameters were used for all primers. The differences (x-fold) in cytokine and chemokine expression were calculated after normalization to β-actin. 
Nested Real-Time PCR
To detect NK-1R in Mφ samples, 2 μL of sample cDNA was combined with 2.5 U Taq DNA polymerase (Invitrogen), 0.02 mM dNTPs, 0.5 μg of each external primer (NK1R-450bp), and PCR buffer containing 2.5 mM MgCl2 in a total volume of 50 μL. Thirty-five cycles were run, with 95°C denaturation, 60°C annealing, and 72°C extension temperatures, with the first three cycles having extended denaturation and annealing times. One microliter of a 1:20 dilution of the previous PCR reaction mixture was added to the second reaction tube containing internal primers (NK1R-432bp) and appropriate concentrations of the other constituents. Fifteen microliters of each amplified sample was electrophoresed on ethidium bromide-stained agarose gels and visualized under UV illumination. All primer pair sequences used for real-time PCR are shown in Table 1
ELISA
SP protein levels were measured in normal and infected corneas from B6 and BALB/c mice (n = 5/group/time). Normal corneas were collected at 1, 5, and 7 days pi and assayed individually for SP with a competitive enzyme immunoassay (EIA) kit (R&D Systems, Minneapolis, MN). Samples were homogenized in 250 μL normal saline with a micro tissue grinder (Fisher Scientific, Itasca, IL). Samples were centrifuged at 5000g for 10 minutes and an aliquot of each supernatant assayed for SP protein. Duplicate experiments were performed and the results reported as picograms per milliliter ± SEM. 
After spantide I or PBS treatment, protein levels for IL-1β and IL-10 also were determined with ELISA kits (R&D). Infected corneas (n = 5/group/time) were harvested at 3 and 5 days pi. For detecting IL-1β, the corneas were individually homogenized in 1.0 mL PBS with 0.5% hexadecyltrimethylammonium bromide (HTAB) with a glass micro tissue grinder and centrifuged at 13,000g for 10 minutes. Supernatants were diluted 1:10 and 50 μL was used to assay for IL-1β levels. In a separate experiment, B6 corneas (n = 5/group/time) were treated similarly, and, at 3 and 5 days pi, individual corneas from each group were homogenized in 130 μL PBS with 0.1% Tween-20 and protease inhibitors (Roche, Indianapolis, IN). Fifty microliters of undiluted sample supernatant was used to assay for IL-10 levels. ELISA analysis was performed similarly on supernatants from lipopolysaccharide (LPS) and SP stimulated Mφ. The reported sensitivity of these assays was <8.0 pg/mL for SP, <3.0 pg/mL for IL-1β, and <4.0 pg/mL for IL-10. 
Quantitation of Corneal PMNs
A myeloperoxidase (MPO) assay was used to quantitate PMNs in the cornea of spantide I- versus PBS-treated B6 mice (n = 5/group/time) at 3 and 5 days pi. Corneas were removed, homogenized in 1.0 mL of 50 mM phosphate buffer (pH 6.0) containing 0.5% HTAB (Sigma-Aldrich), freeze-thawed four times, and centrifuged and 0.1 mL added to 2.9 mL of 50 mM phosphate buffer containing o-dianisidine dihydrochloride (16.7 mg/100 mL) and 0.0005% hydrogen peroxide. Change in absorbance at 460 nm was read on a spectrophotometer (Helios-α; Thermo Spectronic, Pittsford, NY) and units of MPO/cornea were calculated. One unit of MPO activity is equivalent to ∼2 × 105 PMN/mL. 18  
Quantitation of Viable Bacteria
Bacteria were quantitated at 3 and 5 days pi in individual infected corneas of B6 mice (n = 5/group/time) after spantide I or PBS treatment. Each cornea was homogenized in 1.0 mL sterile saline containing 0.25% BSA. The corneal homogenate (0.1 mL) was serially diluted 1:10 in the same solution, and selected dilutions were plated in triplicate on Pseudomonas isolation agar (Difco; Fisher Scientific). Plates were incubated overnight at 37°C, and the number of viable bacteria counted. Results are reported as log10 number of CFU/cornea ± SEM. 
Peritoneal PMN and Μφ Isolation
PMNs were induced into the peritoneal cavity of B6 mice and harvested as described. 19 Briefly, mice were injected IP with 1.0 mL of a 9% casein solution and again 24 hours later. PMNs were lavaged from the peritoneal cavity 3 hours after the second injection, washed, and separated from other cells by ultracentrifugation. The PMNs (95% viable by trypan blue exclusion) were counted and resuspended to 10,000 to 20,000 cells per well for chemotaxis assay. 
Μφs were induced into the peritoneal cavity of B6 mice by IP injection of 3% thioglycollate 20 and harvested by peritoneal lavage 5 days later. The cell suspension was diluted to 1.3 million cells per well and plated, and nonadherent cells removed 5 hours after incubation at 37°C. Adherent cells were used for the in vitro stimulation assay, immunostaining, and PCR detection of the NK-1R, as just described. 
Chemotaxis
A 48-well microchemotaxis chamber (Neuroprobe, Bethesda, MD) with a 3-μm pore-sized filter separating the upper and lower chambers was used to test the chemotactic effect of SP, vasoactive intestinal peptide (VIP), and secretoneurin (SN) using MIP-2 as the positive control. Migration medium (RPMI 1640 and 0.5% BSA) provided a negative control. Thirty microliters each of MIP-2 (0.001 μg/mL; R&D Systems); RPMI 1640 and 0.5% BSA; or SP, VIP, or SN (each at 10−6 M) were placed into the chamber lower wells and 50 μL of a PMN cell suspension (10,000–20,000 cells/well) was placed in the upper wells and incubated at 37°C with 5% CO2 for 30 to 45 minutes. Nonmigrated cells were wiped off the back side of the filter after staining (Diff-Quick; Harleco Corp., Gibbstown, NJ). The stained filter was placed on a microscope slide and allowed to dry. Migrated cells were counted in six fields for each group, and the data were expressed as the mean number of migrating cells ± SEM. 
To elucidate the role of the NK-1R in PMN chemotaxis to SP, the assay was repeated and spantide I (10−8 M; Sigma-Aldrich) was combined with the PMNs in the upper wells to block the NK-1R. Cell migration was determined similarly. 
In Vitro Stimulation
Adherent Mφs from B6 mice were incubated at 37°C for 18 hours in DMEM/5% FBS containing 0.5 μg LPS, 0.1 μM SP, or a combination of both. Identical wells were treated as described earlier with the addition of spantide I (0.5 μM) to assess the effect of SP and NK-1R interaction on IL-1β production by the Μφ. Supernatants were harvested after 18 hours and IL-1β levels determined by ELISA, as described earlier. 
Statistical Analysis
The difference in clinical score at each experimental time point between two-groups was tested by the Mann-Whitney test. An unpaired, two-tailed Student’s t-test was used to determine the statistical significance of real-time PCR, ELISA, bacterial plate counts, chemotaxis, and MPO assays. Data were considered significantly different at P < 0.05. All experiments were repeated at least once with the same number of mice to ensure reproducibility and data from a single representative experiment are shown, unless indicated otherwise. 
Results
SP Immunohistochemistry
The spatial and temporal distribution of SP in the normal cornea of B6 versus BALB/c mice and after infection with P. aeruginosa was assessed by immunostaining (Fig. 1) . SP containing nerve fibers were more intensely stained in the normal, uninfected cornea (epithelium) of B6 (Fig. 1A)compared with BALB/c (Fig. 1B)mice. The staining intensity for SP was greater subepithelially and in the superficial stroma in the cornea of B6 (Fig. 1C)versus BALB/c (Fig. 1D)mice at 1 day pi. At 7 days pi, the central stroma of the B6 mouse cornea showed more intense labeling for SP containing nerve fibers (Fig. 1E)than that of the BALB/c mice (Fig. 1F)
SP Protein
To confirm the immunostaining data, corneas from B6 and BALB/c mice were analyzed by EIA for levels of SP protein (Fig. 2) . Normal, uninfected B6 corneas contained significantly higher levels of endogenous neuropeptide than BALB/c corneas (P = 0.02). The level of SP in the infected corneas of B6 mice also was significantly elevated over the level detected in the infected BALB/c cornea at days 1 and 7 pi (P = 0.0001 for both). No significant difference was detected between the two mouse strains at 5 days pi (P = 0.126). 
Disease Response, Bacterial Load, and PMN Infiltration
Based on these data, susceptible B6 mice were treated with spantide I, an NK-1R antagonist, to block SP interaction with its major receptor. After treatment, the severity of disease was observed and graded (Fig. 3A) . No significant difference in disease severity was observed between spantide I and PBS-treated groups at 1 day pi (P = 0.74). At 3 and 5 days pi, spantide I-treated mice had significantly less severe ocular disease than did the PBS-treated mice (P = 0.05 and 0.004, respectively). Slit lamp photomicrographs were taken of representative eyes from PBS- (Fig. 3B)and spantide I- (Fig. 3C)treated mice at 5 days pi. Perforation was clearly indicated in the PBS-treated eye (Fig. 3B) , whereas the spantide-treated eye, although opaque, had not perforated. 
We next tested whether spantide I treatment effected the number of viable bacteria present in the cornea at 3 and 5 days pi (Fig. 3D) . The bacterial load in the corneas of spantide I-treated mice was significantly less (∼0.5 log) than in PBS-treated mice at both 3 and 5 days pi (P = 0.001 and 0.006, respectively). Because the spantide I-treated mice had less severe disease scores and fewer viable bacteria present in the cornea at later times pi, we also quantitated the number of PMNs in the infected cornea using an MPO assay. Corneas from spantide I-treated mice contained significantly fewer PMNs than the corneas of PBS-treated mice at 3 and 5 days pi (Fig. 3E ; P = 0.0001 and 0.011, respectively). 
Real-Time PCR
To further test the effects of spantide I treatment on mRNA expression of proinflammatory and Th1 type cytokines and chemokines after P. aeruginosa infection, normal, uninfected corneas, as well as corneas at 1, 3, and 5 days pi were analyzed by real-time PCR. The kinetics of mRNA expression for each cytokine are shown in Figure 4 . Levels of MIP-2 mRNA (Fig. 4A)were significantly reduced in the corneas of spantide I- versus PBS-treated B6 mice at 3 and 5 days pi (P = 0.03 and 0.005, respectively). There was no significant difference found at 1 day pi (P = 0.1) and no constitutive expression detected in normal cornea. A significant reduction in IL-6 mRNA expression (Fig. 4B)was detected at 1, 3, and 5 days pi in spantide I-treated mouse corneas when compared with corneas from PBS-treated mice (P = 0.002, 0.02, and 0.0006, respectively). IL-6 was not detected in mRNA from normal B6 mouse cornea. No difference in TNF-α levels (Fig. 4C)was detected at 1 day pi (P = 0.55); however, spantide I-treatment significantly reduced levels of corneal TNF-α mRNA at 3 and 5 days pi when compared with the PBS-treated group (P = 0.005 and 0.049, respectively). TNF-α was constitutively expressed in normal B6 cornea, but was six times less than the peak levels found at 3 days pi. mRNA for IL-12 (Fig. 4D)was not detected in the normal B6 cornea, was similar between spantide I- and PBS-treated mice at 1 and 3 days pi (P = 0.37 and 0.92, respectively) and was significantly reduced after spantide I treatment at 5 days pi (P = 0.02). Treatment with spantide I significantly reduced the level of IL-18 mRNA (Fig. 4E)at 1 day pi compared with the PBS-treatment group (P = 0.02), but no difference in IL-18 mRNA levels was detected at 3 or 5 days pi (P = 0.47 and 0.92, respectively). No signal for IL-18 was detected in the normal B6 cornea. IFN-γ mRNA (Fig. 4F)was not detected in normal B6 mouse cornea and spantide I-treatment did not significantly change the levels of IFN-γ detected at 1 day pi when compared with PBS-treatment (P = 0.84). In contrast, significant reduction in mRNA levels for IFN-γ was seen in the cornea at 3 and 5 days pi after spantide I-treatment when compared with corneas of PBS-treated mice (P = 0.0006 and 0.02, respectively). 
IL-1β mRNA and Protein Levels
Because IL-1β plays an important role in P. aeruginosa keratitis, levels of IL-1β mRNA were similarly tested and the amount of IL-1β protein in the cornea after infection quantitated by ELISA (Fig. 5) . No expression of IL-1β was detectable in mRNA from normal, uninfected corneas of B6 mice and spantide I significantly reduced IL-1β mRNA levels at 3 and 5 days pi (P = 0.002 and 0.001, respectively) with no significant difference noted between the spantide I- and PBS-treated groups at 1 day pi (P = 0.13). IL-1β protein levels also were significantly reduced in spantide I- versus PBS-treated mice at both 3 and 5 days pi (P = 0.0001 and 0.001, respectively). 
Real-Time PCR and ELISA Analysis
TGF-β mRNA levels were detected in the normal B6 mouse cornea, but did not differ significantly between infected corneas from PBS- versus spantide I-treated mice at any time point tested (P = 0.56, 0.44, and 0.1 at 1, 3, and 5 days pi, respectively; Fig. 6 ). IL-10 mRNA was not detected in the normal cornea, but significantly elevated mRNA levels were found in the corneas of spantide I- versus PBS-treated mice at 1 and 5 days pi (P = 0.05 and 0.001, respectively). There was no significant difference in IL-10 levels at 3 days pi (P = 0.63). ELISA analysis showed no significant difference in the amount of IL-10 protein after spantide I treatment at 3 days pi (P = 0.1) but IL-10 protein was significantly higher in corneas from spantide I-treated mice at 5 days pi (P = 0.05) when compared with levels in the corneas from the PBS-treated group. 
Chemotaxis of PMN to Neuropeptides
Because SP was differentially expressed with significantly higher levels detected in B6 mice before and after infection, and because others have shown a chemotactic function, 21 a Boyden chamber assay was used to determine the chemotactic potential of several neuropeptides including SP, VIP, and SN on PMNs isolated from B6 mice. MIP-2, a potent PMN chemoattractant, was used as the positive control and medium alone as the negative control. As shown in Figure 7A , SP was chemotactic for PMNs isolated from B6 mice, and the number of migrating cells was significantly elevated compared with media (P < 0.0001). SP also had a greater chemotactic effect on PMNs when compared with either of the other neuropeptides tested, although the increase was not statistically significant (P = 0.13 for both VIP and SN). To test whether the NK-1R played a role in the chemotactic effect of SP on the PMNs, spantide I was added to the chamber wells containing the PMN to block the NK-1R (Fig. 7B) . No significant difference in chemotaxis was observed by addition of spantide I (MIP-2, P = 0.41; SP, P = 0.82; IL-1β, P = 0.53; and SP+IL-1β, P = 0.85) suggesting that the NK-1R does not play a role in chemotaxis induced by SP, nor through SP directly potentiating the effects of IL-1β. 
Real-Time PCR for NK-1R
Because no difference in chemotaxis resulted from the addition of spantide I to PMN, real-time PCR was used to detect the NK-1R on the PMNs (Fig. 8) . As a positive control, mRNA was harvested from normal and 5 day pi corneas of B6 mice. PMN from B6 mice (unstimulated), or stimulated for 2 or 18 hours with LPS (0.5 μg/mL) did not express the NK-1R. Normal corneas had significantly more NK-1R expression than infected (P = 0.01), and both were significantly elevated compared with the cells alone (P = 0.0002). 
PCR also was used to confirm the presence of the NK-1R on Μφs (Fig. 9A) . mRNA for the NK-1R was detected on normal as well as stimulated (LPS) cells and in normal and infected B6 mouse corneas. Both in the LPS-stimulated cells and in the infected cornea, the level of mRNA appeared to decrease (Fig. 9A) . We also immunostained Μφs and localized the NK-1R, confirming that it was expressed at the protein level on these cells (Figs. 9B 9C)
IL-1β Production
To test further whether the effect of spantide I on the reduction of disease severity is due to interference with the binding of SP to its major receptor, NK-1R, peritoneal Μφs were stimulated for 18 hours with LPS, SP, or a combination of both, with or without spantide I (Fig. 9D) . The level of the proinflammatory cytokine IL-1β was then assayed in the culture supernatant by ELISA. Treatment of the cells with PBS, or SP either with or without spantide I resulted in no detectable IL-1β production. Stimulation with LPS significantly elevated IL-1β production over PBS levels (P < 0.0001), but addition of spantide I had no significant effect (P = 0.93). The combination of LPS and SP resulted in a significant increase in IL-1β production when compared with LPS alone (P = 0.04). The addition of spantide I to the combined stimulants significantly reduced IL-1β production (P = 0.009), suggesting the role of SP/NK-1R interaction in Μφ production of IL-1β. 
Discussion
The toxic effects of Gram-negative bacterial endotoxins such as LPS are mediated by generation of endogenous proinflammatory cytokines such as TNF-α, IL-6, IL-1, 22 IL-12, and IFN-γ. 23 Neuropeptides are also endogenous factors that mediate immune responses and cytokine production. The stimulation of sensory nerves either from the environment or via axon reflexes induces vasodilation, causing leakage of plasma proteins from the circulation to extravascular spaces resulting in edema formation. 24 In addition, granulocytes adhere to endothelial cells and migrate into inflamed tissues. 25  
The cornea is one of the most densely innervated tissues in the body and is abundantly supplied by sensory and autonomic nerve fibers. Interest in corneal innervation has become of increased importance because of the clinical observation that corneal nerves are routinely injured after refractive surgical procedures or after corneal diseases, such as bacterial keratitis. The human cornea contains several neuropeptides, including SP, 14 the most extensively studied and potent member of a family of neuropeptides called the tachykinins, which exhibit preferential binding to one of three receptors called neurokinin receptors (NK-R). 
In the present study immunocytochemistry and EIA revealed that the murine cornea contained SP nerve fibers; that the distribution of SP was disparate, with B6 over BALB/c mice; and that SP at the protein level was increased after infection in both groups, but for the most part with higher levels in B6 mouse cornea. An exception to this was seen at 5 days pi. At this time, SP levels peaked in the BALB/c mouse cornea, which has been shown to correlate with peak levels of NK cell IFN-γ production and disease resolution. 10  
SP is a modulator of neuroimmune regulation, in particular the immune functions of mononuclear phagocytes. 26 The biological responses to SP are mediated by the NK-1R, the SP-preferring receptor, a G-protein-coupled receptor bearing seven transmembrane domains. 27 SP is secreted by nerves and inflammatory cells such as Μφs, eosinophils, lymphocytes, and DCs and acts by binding to the NK-1R. SP specifically activates NF-κB, a transcription factor involved in the control of cytokine expression and stimulates monocytes to produce inflammatory cytokines such as IL-1, -6, and -12 and TNF-α. 28 29 30 Because of the higher levels of SP in the B6 cornea, we blocked its activity through the NK-1R by using the antagonist spantide I. Spantide treatment significantly reduced corneal perforation, bacterial count, and PMN infiltration of into the cornea. Antagonist injection also reduced significantly type 1 cytokine (IL-18, IL-12, and IFN-γ) mRNA levels; the latter two shown previously to be associated with poor disease outcome in B6 mice. 7 31 IL-1β also was reduced at both the mRNA and protein levels. IL-1β has been shown to be of critical importance to the susceptible response of B6 mice and use of antibody neutralization, 22 an IL-1β inhibitor, 32 or caspase-1-deficient mice 33 all resulted in better disease outcome. 
Treatment with the SP antagonist spantide II, with properties similar to those of spantide I, was used to study the importance and contribution that expression of SP receptors makes to the protective response after oral inoculation with a lethal dose of Salmonella. In contrast to studies reported herein, mice pretreated with the antagonist had significantly reduced survival rates compared with control mice. Antagonist treatment significantly reduced IL-12 p40 and IFN-γ expression, required for resistance to this pathogen, at mucosal sites. 17 In contrast, corneal perforation has been shown by this laboratory to be ameliorated by lowering IFN-γ levels in the B6 cornea, 31 consistent with the current results and providing a role hitherto unknown for SP in corneal immunity. Antagonist treatment not only lowered proinflammatory cytokines, but also resulted in upregulation of the anti-inflammatory mediator IL-10 at the mRNA and protein levels. These data are consistent with previous work in the resistant BALB/c model in which we showed that IL-10 production by Μφs was essential in the regulation of IFN-γ levels and that a balance between levels of the two cytokines was critical for protection. 34 Using the antagonist, we again saw an important role for IL-10 and its correlation with improved disease, when it was upregulated in the susceptible B6 mouse after spantide treatment. In contrast, Cole et al. 35 showed that knocking out IL-10 in B6 mice reduced perforation. The disparity in these data may reflect the possibility that other undetected effects of knocking out the IL-10 gene may have occurred. In addition, different strains of P. aeruginosa were found to cause different levels of IL-10 production, and both strains used differed from the strain of bacteria used in our studies. Contrary to these data and consistent with our own, several regulatory molecules such as IL-10 and -13, are termed Μφ-deactivating factors, 36 whose major role is to prevent the excessive production of proinflammatory mediators. 
SP initiates numerous inflammatory reactions including PMN chemoattraction and activation. 37 PMN are necessary for eradication of microbial pathogens, but they also destroy tissue by proteolytic enzyme release. PMN infiltration is controlled by local production of proinflammatory mediators (e.g., IL-8). 38 It also has been shown that SP induces IL-8 synthesis (fourfold) in human corneal epithelial cells and elicits PMN chemotaxis. 39 In the mouse, MIP-2 (functional homologue of IL-8) is a potent PMN chemoattractant 40 and we 16 have shown that MIP-2 recruits PMNs into the P. aeruginosa-infected B6 cornea. IL-1, produced by monocytes, Mφs, and corneal epithelial cells, also regulates PMN influx into tissues 41 and activates antigen-presenting cells (APCs) and inflammatory cells. 42 In B6 mice, Rudner et al., 22 showed that prolonged elevation of IL-1β in P. aeruginosa corneal infection upregulates MIP-2, enhancing PMN influx into the cornea and inducing perforation. Others 43 have used a murine air-pouch model to investigate PMN infiltration in wild-type (wt) versus NK1 knockout (−/−) mice. The PMN response to exogenous IL-1β was significantly attenuated in −/− versus wt mice, and the response to SP also was reduced by 50%. Therefore, we also tested the role of SP and other neuropeptides against a known PMN chemotactic agent, MIP-2, and found that all three neuropeptides tested over medium elicited PMN chemotaxis. Although SP appeared to have a slightly greater chemotactic ability than the other neuropeptides tested, it was not significantly greater. In contrast, investigating the effects of these nervous system-derived mediators on the migratory behavior of human peripheral blood-derived mononuclear cells used to generate DCs, revealed contrasting responses in migration of the cells in Boyden chamber assays. Responses of the DCs to neuropeptides depended on the maturation state of the cell. Peripheral neuropeptides directly attracted immature DCs to peripheral nerve fibers where high concentrations of the peptides arrested the mature cells. 21  
We also tested PMN chemotaxis in the presence or absence of the antagonist and found that the addition of SP in the presence of spantide I did not result in any difference in cell chemotaxis, suggesting that the chemotactic effects of SP on PMN were not mediated by interaction with the NK-1R. This notion led us to test unstimulated as well as LPS-stimulated PMNs for the presence of the NK-1R at the mRNA level. Both the normal and infected cornea served as positive controls, yet no detection of the NK-1R on PMNs at the mRNA level was observed. These data suggest that SP potentiates the chemotaxis of PMN via mechanisms other than interaction with the NK-1R. In this regard, others have shown that SP is a “pure” chemoattractant, in that it can elicit chemotaxis without activating PMNs, 44 consistent with our results. Alternatively, in another model, Cao et al., 45 compared PMN accumulation in normal and inflamed skin using NK1R−/− mice. Results demonstrated that although SP induced edema in wt mice, it could not by itself induce PMN accumulation in normal mouse skin. However, the same study showed that SP potentiated IL-1β-induced PMN accumulation in vivo in wt mice, indicating an ability of SP to indirectly increase PMN accumulation at inflamed sites. 
Evidence indicates that SP plays an important role in augmenting inflammatory responses, principally by high-affinity binding to the NK-1R present on human NK cells, 46 DCs, and Mφs. 12 13 In the study reported herein, Μφs also were tested for the presence of the NK-1R which was detected both constitutively as well as after LPS stimulation and in the normal and infected B6 mouse cornea. We confirmed the RNA data and showed the presence of the receptor using immunocytochemistry. Because SP can potentiate IL-1β in some systems, we also tested Μφs by LPS stimulation with or without spantide treatment. LPS and SP potentiated IL-1β production by Mφs and spantide reduced the effect, providing an explanation of the reduction of IL-1β seen in vivo in our study after antagonist treatment. Because PMNs were reduced in the MPO assay after spantide treatment, we hypothesize that it is the Μφ that, at least in part, controls PMN influx into the cornea. These data in the susceptible mouse are consistent with those in a previous study of resistant BALB/c mice that were depleted of Μφs, 34 resulting in dysregulation of PMNs detected in the cornea by MPO assay. 
In summary, we have provided evidence to support the important immunomodulatory role of SP in augmenting inflammation in the cornea after P. aeruginosa infection in susceptible mice and have provided mechanisms by which it does so, contributing to perforation. 
 
Table 1.
 
Nucleotide Sequence of the Specific Primers Used in PCR Amplification
Table 1.
 
Nucleotide Sequence of the Specific Primers Used in PCR Amplification
Gene Primers Sense
β-Actin 5′-GAT TAC TGC TCT GGC TCC TAG C-3′ F
5′-GAC TCA TCG TAC TCC TGC TTG C-3′ R
MIP-2 5′-TGT CAA TGC CTG AAG ACC CTG CC-3′ F
5′-AAC TTT TTG ACC GCC CTT GAG AGT GG-3′ R
IL-6 5′-CAC AAG TCC GGA GAG GAG AC-3′ F
5′-CAG AAT TGC CAT TGC ACA AC-3′ R
TNF-a 5′-ACC CTC ACA CTC AGA TCA TCTT-3′ F
5′-GGT-TGT CTT TGA GAT CCA TGC-3′ R
IL-12 p40 5′-GGT CAC ACT GGA CCA AAG GGA CTA TG-3′ F
5′-ATT CTG CTG CCG TGC TTC CAA C-3′ R
IL-18 5′-GCC ATG TCA GAA GAC TCT TGC GTC-3′ F
5′-GTA CAG TGA AGT CGG CCA AAG TTG TC-3′ R
IFN 5′-CAG AGC CAG ATT ATC TCT TTC TAC CTC AGA C-3′ F
5′-CTT TTT CGC CTT GCT GTT GCT GAA G-3′ R
IL-1β 5′-CGC AGC AGC ACA TCA ACA AGA GC-3′ F
5′-TGT CCT CAT CCT GGA AGG TCC ACG-3′ R
TGF 5′-AAC AAT TCC TGG CGT TAC CTT-3′ F
5′-CTG CCG TAC AAC TCC AGT GA-3′ R
IL-10 5′-TGC TAA CCG ACT CCT TAA TGC AGG AC-3′ F
5′-CCT TGA TTT CTG GGC CAT GCT TCT C-3′ R
NK1-R 5′-TGG ACT CTG ATC TCT TCC TGC CCA ACA-3′ F
5′-GGA CCC AGA TGA CAA AGA TGA CCA CTT-3′ R
NK1-R 5′-CCA ACA CCT ACT CCA CCA ACA CTT CTG-3′ F
5′-GGA CCC AGA TGA CAA AGA TGA CCA CTT-3′ R
Figure 1.
 
SP immunostaining in B6 versus BALB/c mice before and after P. aeruginosa infection. More intense staining of SP containing nerve fibers was seen in the corneal epithelium of normal, uninfected B6 mice (A) than in BALB/c mice (B). At 1 day pi, B6 (C) versus BALB/c (D) mice showed more intense staining beneath the epithelium and in the superficial stroma of the central cornea. At 7 days pi, the intensity of staining was heavier in the central corneal stroma of B6 (E) versus BALB/c (F) mice. All controls were negative (data not shown). Magnification, ×65.
Figure 1.
 
SP immunostaining in B6 versus BALB/c mice before and after P. aeruginosa infection. More intense staining of SP containing nerve fibers was seen in the corneal epithelium of normal, uninfected B6 mice (A) than in BALB/c mice (B). At 1 day pi, B6 (C) versus BALB/c (D) mice showed more intense staining beneath the epithelium and in the superficial stroma of the central cornea. At 7 days pi, the intensity of staining was heavier in the central corneal stroma of B6 (E) versus BALB/c (F) mice. All controls were negative (data not shown). Magnification, ×65.
Figure 2.
 
EIA analysis of SP protein levels in B6 and BALB/c mouse cornea. SP protein levels were endogenously higher in B6 normal, uninfected corneas (P = 0.02) and were elevated significantly at 1 and 7 days pi (P = 0.0001 for both) when compared with BALB/c corneas. There was no significant difference between the two groups at day 5 pi. Results are reported as the mean pg/mL protein ± SEM (n = 5/group/time).
Figure 2.
 
EIA analysis of SP protein levels in B6 and BALB/c mouse cornea. SP protein levels were endogenously higher in B6 normal, uninfected corneas (P = 0.02) and were elevated significantly at 1 and 7 days pi (P = 0.0001 for both) when compared with BALB/c corneas. There was no significant difference between the two groups at day 5 pi. Results are reported as the mean pg/mL protein ± SEM (n = 5/group/time).
Figure 3.
 
(A) Ocular disease response in spantide I- versus PBS-injected B6 mice after P. aeruginosa infection. Ocular disease was graded at 1, 3, and 5 days pi for each group (n = 5/group/time, repeated once). Significant differences between the two groups were seen only at 3 and 5 days pi (P = 0.01 and P = 0.0008, respectively). Eight of 10 corneas in the PBS-treated group perforated by 5 days pi compared with 1 of 10 corneas in the spantide I-treated group. Results are reported as individual clinical scores (from two separate experiments). (B, C) Slit lamp photomicrographs of P. aeruginosa-infected eyes after spantide I treatment. Representative eyes from PBS- (B) and spantide I-treated mice (C) were photographed at 5 days pi (D). Viable bacterial counts in B6 mouse cornea after P. aeruginosa infection. Corneas from the spantide I-treated mice (n = 5/group/time) had significantly lower numbers of viable bacteria (∼ 0.5 log lower) at both 3 and 5 days pi (P = 0.001 and 0.006, respectively) compared with control mice (n = 5/group/time). Results are reported as log10 CFU/cornea ± SEM. (E) number of PMNs per cornea (mean ± SEM; n = 5/group/time) as determined by an MPO assay. Approximately 40% fewer PMNs were detected in the corneas of spantide I-treated mice than in the control mice at 3 and 5 days pi (P = 0.0001 and 0.01, respectively).
Figure 3.
 
(A) Ocular disease response in spantide I- versus PBS-injected B6 mice after P. aeruginosa infection. Ocular disease was graded at 1, 3, and 5 days pi for each group (n = 5/group/time, repeated once). Significant differences between the two groups were seen only at 3 and 5 days pi (P = 0.01 and P = 0.0008, respectively). Eight of 10 corneas in the PBS-treated group perforated by 5 days pi compared with 1 of 10 corneas in the spantide I-treated group. Results are reported as individual clinical scores (from two separate experiments). (B, C) Slit lamp photomicrographs of P. aeruginosa-infected eyes after spantide I treatment. Representative eyes from PBS- (B) and spantide I-treated mice (C) were photographed at 5 days pi (D). Viable bacterial counts in B6 mouse cornea after P. aeruginosa infection. Corneas from the spantide I-treated mice (n = 5/group/time) had significantly lower numbers of viable bacteria (∼ 0.5 log lower) at both 3 and 5 days pi (P = 0.001 and 0.006, respectively) compared with control mice (n = 5/group/time). Results are reported as log10 CFU/cornea ± SEM. (E) number of PMNs per cornea (mean ± SEM; n = 5/group/time) as determined by an MPO assay. Approximately 40% fewer PMNs were detected in the corneas of spantide I-treated mice than in the control mice at 3 and 5 days pi (P = 0.0001 and 0.01, respectively).
Figure 4.
 
Real-time PCR analysis of corneal cytokine mRNA expression in spantide I- versus PBS-treated B6 mice after P. aeruginosa infection. (A) Corneal MIP-2 mRNA levels peaked at 5 days pi and were significantly decreased in the spantide I-treated mouse group at 3 and 5 days pi. No MIP-2 was detected in the normal, uninfected B6 corneas. (B) Corneal IL-6 mRNA levels peaked at 1 day pi and were significantly decreased in the spantide I-treated group at 1, 3, and 5 days pi. No IL-6 was detected in the normal cornea. (C) Corneal TNF-α mRNA levels peaked at 3 day pi and were elevated approximately six times over basal levels in normal corneal. Levels of IL-6 were significantly decreased in spantide I-treated groups at 3 and 5 days pi. (D) Corneal IL-12 mRNA levels peaked at 3 days pi and were significantly decreased in the spantide I-treated mouse group at 5 days pi; no IL-12 was detected in the normal cornea. (E) Corneal IL-18 mRNA levels peaked at 1 day pi and were significantly decreased in the spantide I-treated mouse group at 1 day pi. No IL-18 was detected in the normal cornea. (F) Corneal IFN-γ mRNA levels peaked at 5 days pi and were significantly decreased in the spantide I-treated group at 3 and 5 day pi. No IFN-γ was detected in normal corneas. All data are mean ± SEM; n = 5/group/time.
Figure 4.
 
Real-time PCR analysis of corneal cytokine mRNA expression in spantide I- versus PBS-treated B6 mice after P. aeruginosa infection. (A) Corneal MIP-2 mRNA levels peaked at 5 days pi and were significantly decreased in the spantide I-treated mouse group at 3 and 5 days pi. No MIP-2 was detected in the normal, uninfected B6 corneas. (B) Corneal IL-6 mRNA levels peaked at 1 day pi and were significantly decreased in the spantide I-treated group at 1, 3, and 5 days pi. No IL-6 was detected in the normal cornea. (C) Corneal TNF-α mRNA levels peaked at 3 day pi and were elevated approximately six times over basal levels in normal corneal. Levels of IL-6 were significantly decreased in spantide I-treated groups at 3 and 5 days pi. (D) Corneal IL-12 mRNA levels peaked at 3 days pi and were significantly decreased in the spantide I-treated mouse group at 5 days pi; no IL-12 was detected in the normal cornea. (E) Corneal IL-18 mRNA levels peaked at 1 day pi and were significantly decreased in the spantide I-treated mouse group at 1 day pi. No IL-18 was detected in the normal cornea. (F) Corneal IFN-γ mRNA levels peaked at 5 days pi and were significantly decreased in the spantide I-treated group at 3 and 5 day pi. No IFN-γ was detected in normal corneas. All data are mean ± SEM; n = 5/group/time.
Figure 5.
 
Real-time PCR and ELISA analysis of corneal IL-1β mRNA and protein levels after P. aeruginosa infection. (A) IL-1β mRNA levels peaked at 5 days pi and were significantly decreased in the spantide I-treated group at 3 and 5 days pi. No IL-1β was detected in the normal cornea. (B) IL-1β protein levels peaked at 3 days pi and were significantly decreased in the spantide I-treated group at 3 and 5 days pi. All data are mean ± SEM (n = 5/group/time).
Figure 5.
 
Real-time PCR and ELISA analysis of corneal IL-1β mRNA and protein levels after P. aeruginosa infection. (A) IL-1β mRNA levels peaked at 5 days pi and were significantly decreased in the spantide I-treated group at 3 and 5 days pi. No IL-1β was detected in the normal cornea. (B) IL-1β protein levels peaked at 3 days pi and were significantly decreased in the spantide I-treated group at 3 and 5 days pi. All data are mean ± SEM (n = 5/group/time).
Figure 6.
 
Real-time PCR and ELISA analysis of corneal anti-inflammatory cytokine mRNA and protein levels after P. aeruginosa infection. (A) TGF-β mRNA levels peaked at 1 day pi, but there were no significant differences at any time point between spantide I-treated and PBS-treated groups. (B) Corneal IL-10 mRNA peaked at 3 days pi and was significantly higher in the spantide I-treated versus control group at 1 and 5 days pi. IL-10 was not detected in normal corneas. (C) IL-10 protein peaked at 3 days pi and was significantly higher in the spantide I-treated group 5 days pi. All data are mean ± SEM (n = 5/group/time).
Figure 6.
 
Real-time PCR and ELISA analysis of corneal anti-inflammatory cytokine mRNA and protein levels after P. aeruginosa infection. (A) TGF-β mRNA levels peaked at 1 day pi, but there were no significant differences at any time point between spantide I-treated and PBS-treated groups. (B) Corneal IL-10 mRNA peaked at 3 days pi and was significantly higher in the spantide I-treated versus control group at 1 and 5 days pi. IL-10 was not detected in normal corneas. (C) IL-10 protein peaked at 3 days pi and was significantly higher in the spantide I-treated group 5 days pi. All data are mean ± SEM (n = 5/group/time).
Figure 7.
 
Effect of neuropeptides on PMN migration. (A) SP, VIP. and SN significantly increased PMN migration compared with the media control. (B) The NK-1R antagonist spantide I had no effect on SP-induced PMN migration in the presence or absence of IL-1β. MIP-2 was the positive control. Data are expressed as mean ± SEM of the number of migrating PMNs.
Figure 7.
 
Effect of neuropeptides on PMN migration. (A) SP, VIP. and SN significantly increased PMN migration compared with the media control. (B) The NK-1R antagonist spantide I had no effect on SP-induced PMN migration in the presence or absence of IL-1β. MIP-2 was the positive control. Data are expressed as mean ± SEM of the number of migrating PMNs.
Figure 8.
 
Real-time PCR analysis of NK-1R mRNA levels. There was a moderate level of NK-1R mRNA expression in the normal B6 cornea, and by 5 days pi, a twofold decrease in NK-1R mRNA expression was seen in the infected cornea. Neither unstimulated PMN nor PMN stimulated with LPS had any detectable expression of NK-1R mRNA.
Figure 8.
 
Real-time PCR analysis of NK-1R mRNA levels. There was a moderate level of NK-1R mRNA expression in the normal B6 cornea, and by 5 days pi, a twofold decrease in NK-1R mRNA expression was seen in the infected cornea. Neither unstimulated PMN nor PMN stimulated with LPS had any detectable expression of NK-1R mRNA.
Figure 9.
 
(A) Real-time PCR analysis of NK-1R levels in B6 peritoneal Μφ. NK-1R was detected constitutively (lane 1) and after LPS stimulation of Μφ (lane 2) as well as in normal (lane 3) and infected (lane 4) corneas. Both LPS stimulation and infection of the cornea appeared to decrease the expression of the receptor. (B, C) Confocal images of NK1-R staining of unstimulated Μφ, confirming the mRNA data. Magnifications: (B) ×70; (C) ×430. (D) ELISA analysis of the effect of spantide I on IL-1β production by stimulated peritoneal Μφ. LPS significantly increased IL-1β protein levels in cultured peritoneal Μφ but did not differ after spantide treatment (P = 0.93); SP alone did not stimulate IL-1β expression with or without spantide treatment. SP potentiated LPS induced IL-1β expression (P = 0.009). Spantide I alone had no effect on IL-1 β expression. Spantide I did not significantly change LPS-induced IL-1β expression, but significantly reduced LPS induced IL-1β expression in the presence of SP (P = 0.01). PBS had no effect on IL-1β production with or without spantide.
Figure 9.
 
(A) Real-time PCR analysis of NK-1R levels in B6 peritoneal Μφ. NK-1R was detected constitutively (lane 1) and after LPS stimulation of Μφ (lane 2) as well as in normal (lane 3) and infected (lane 4) corneas. Both LPS stimulation and infection of the cornea appeared to decrease the expression of the receptor. (B, C) Confocal images of NK1-R staining of unstimulated Μφ, confirming the mRNA data. Magnifications: (B) ×70; (C) ×430. (D) ELISA analysis of the effect of spantide I on IL-1β production by stimulated peritoneal Μφ. LPS significantly increased IL-1β protein levels in cultured peritoneal Μφ but did not differ after spantide treatment (P = 0.93); SP alone did not stimulate IL-1β expression with or without spantide treatment. SP potentiated LPS induced IL-1β expression (P = 0.009). Spantide I alone had no effect on IL-1 β expression. Spantide I did not significantly change LPS-induced IL-1β expression, but significantly reduced LPS induced IL-1β expression in the presence of SP (P = 0.01). PBS had no effect on IL-1β production with or without spantide.
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Figure 1.
 
SP immunostaining in B6 versus BALB/c mice before and after P. aeruginosa infection. More intense staining of SP containing nerve fibers was seen in the corneal epithelium of normal, uninfected B6 mice (A) than in BALB/c mice (B). At 1 day pi, B6 (C) versus BALB/c (D) mice showed more intense staining beneath the epithelium and in the superficial stroma of the central cornea. At 7 days pi, the intensity of staining was heavier in the central corneal stroma of B6 (E) versus BALB/c (F) mice. All controls were negative (data not shown). Magnification, ×65.
Figure 1.
 
SP immunostaining in B6 versus BALB/c mice before and after P. aeruginosa infection. More intense staining of SP containing nerve fibers was seen in the corneal epithelium of normal, uninfected B6 mice (A) than in BALB/c mice (B). At 1 day pi, B6 (C) versus BALB/c (D) mice showed more intense staining beneath the epithelium and in the superficial stroma of the central cornea. At 7 days pi, the intensity of staining was heavier in the central corneal stroma of B6 (E) versus BALB/c (F) mice. All controls were negative (data not shown). Magnification, ×65.
Figure 2.
 
EIA analysis of SP protein levels in B6 and BALB/c mouse cornea. SP protein levels were endogenously higher in B6 normal, uninfected corneas (P = 0.02) and were elevated significantly at 1 and 7 days pi (P = 0.0001 for both) when compared with BALB/c corneas. There was no significant difference between the two groups at day 5 pi. Results are reported as the mean pg/mL protein ± SEM (n = 5/group/time).
Figure 2.
 
EIA analysis of SP protein levels in B6 and BALB/c mouse cornea. SP protein levels were endogenously higher in B6 normal, uninfected corneas (P = 0.02) and were elevated significantly at 1 and 7 days pi (P = 0.0001 for both) when compared with BALB/c corneas. There was no significant difference between the two groups at day 5 pi. Results are reported as the mean pg/mL protein ± SEM (n = 5/group/time).
Figure 3.
 
(A) Ocular disease response in spantide I- versus PBS-injected B6 mice after P. aeruginosa infection. Ocular disease was graded at 1, 3, and 5 days pi for each group (n = 5/group/time, repeated once). Significant differences between the two groups were seen only at 3 and 5 days pi (P = 0.01 and P = 0.0008, respectively). Eight of 10 corneas in the PBS-treated group perforated by 5 days pi compared with 1 of 10 corneas in the spantide I-treated group. Results are reported as individual clinical scores (from two separate experiments). (B, C) Slit lamp photomicrographs of P. aeruginosa-infected eyes after spantide I treatment. Representative eyes from PBS- (B) and spantide I-treated mice (C) were photographed at 5 days pi (D). Viable bacterial counts in B6 mouse cornea after P. aeruginosa infection. Corneas from the spantide I-treated mice (n = 5/group/time) had significantly lower numbers of viable bacteria (∼ 0.5 log lower) at both 3 and 5 days pi (P = 0.001 and 0.006, respectively) compared with control mice (n = 5/group/time). Results are reported as log10 CFU/cornea ± SEM. (E) number of PMNs per cornea (mean ± SEM; n = 5/group/time) as determined by an MPO assay. Approximately 40% fewer PMNs were detected in the corneas of spantide I-treated mice than in the control mice at 3 and 5 days pi (P = 0.0001 and 0.01, respectively).
Figure 3.
 
(A) Ocular disease response in spantide I- versus PBS-injected B6 mice after P. aeruginosa infection. Ocular disease was graded at 1, 3, and 5 days pi for each group (n = 5/group/time, repeated once). Significant differences between the two groups were seen only at 3 and 5 days pi (P = 0.01 and P = 0.0008, respectively). Eight of 10 corneas in the PBS-treated group perforated by 5 days pi compared with 1 of 10 corneas in the spantide I-treated group. Results are reported as individual clinical scores (from two separate experiments). (B, C) Slit lamp photomicrographs of P. aeruginosa-infected eyes after spantide I treatment. Representative eyes from PBS- (B) and spantide I-treated mice (C) were photographed at 5 days pi (D). Viable bacterial counts in B6 mouse cornea after P. aeruginosa infection. Corneas from the spantide I-treated mice (n = 5/group/time) had significantly lower numbers of viable bacteria (∼ 0.5 log lower) at both 3 and 5 days pi (P = 0.001 and 0.006, respectively) compared with control mice (n = 5/group/time). Results are reported as log10 CFU/cornea ± SEM. (E) number of PMNs per cornea (mean ± SEM; n = 5/group/time) as determined by an MPO assay. Approximately 40% fewer PMNs were detected in the corneas of spantide I-treated mice than in the control mice at 3 and 5 days pi (P = 0.0001 and 0.01, respectively).
Figure 4.
 
Real-time PCR analysis of corneal cytokine mRNA expression in spantide I- versus PBS-treated B6 mice after P. aeruginosa infection. (A) Corneal MIP-2 mRNA levels peaked at 5 days pi and were significantly decreased in the spantide I-treated mouse group at 3 and 5 days pi. No MIP-2 was detected in the normal, uninfected B6 corneas. (B) Corneal IL-6 mRNA levels peaked at 1 day pi and were significantly decreased in the spantide I-treated group at 1, 3, and 5 days pi. No IL-6 was detected in the normal cornea. (C) Corneal TNF-α mRNA levels peaked at 3 day pi and were elevated approximately six times over basal levels in normal corneal. Levels of IL-6 were significantly decreased in spantide I-treated groups at 3 and 5 days pi. (D) Corneal IL-12 mRNA levels peaked at 3 days pi and were significantly decreased in the spantide I-treated mouse group at 5 days pi; no IL-12 was detected in the normal cornea. (E) Corneal IL-18 mRNA levels peaked at 1 day pi and were significantly decreased in the spantide I-treated mouse group at 1 day pi. No IL-18 was detected in the normal cornea. (F) Corneal IFN-γ mRNA levels peaked at 5 days pi and were significantly decreased in the spantide I-treated group at 3 and 5 day pi. No IFN-γ was detected in normal corneas. All data are mean ± SEM; n = 5/group/time.
Figure 4.
 
Real-time PCR analysis of corneal cytokine mRNA expression in spantide I- versus PBS-treated B6 mice after P. aeruginosa infection. (A) Corneal MIP-2 mRNA levels peaked at 5 days pi and were significantly decreased in the spantide I-treated mouse group at 3 and 5 days pi. No MIP-2 was detected in the normal, uninfected B6 corneas. (B) Corneal IL-6 mRNA levels peaked at 1 day pi and were significantly decreased in the spantide I-treated group at 1, 3, and 5 days pi. No IL-6 was detected in the normal cornea. (C) Corneal TNF-α mRNA levels peaked at 3 day pi and were elevated approximately six times over basal levels in normal corneal. Levels of IL-6 were significantly decreased in spantide I-treated groups at 3 and 5 days pi. (D) Corneal IL-12 mRNA levels peaked at 3 days pi and were significantly decreased in the spantide I-treated mouse group at 5 days pi; no IL-12 was detected in the normal cornea. (E) Corneal IL-18 mRNA levels peaked at 1 day pi and were significantly decreased in the spantide I-treated mouse group at 1 day pi. No IL-18 was detected in the normal cornea. (F) Corneal IFN-γ mRNA levels peaked at 5 days pi and were significantly decreased in the spantide I-treated group at 3 and 5 day pi. No IFN-γ was detected in normal corneas. All data are mean ± SEM; n = 5/group/time.
Figure 5.
 
Real-time PCR and ELISA analysis of corneal IL-1β mRNA and protein levels after P. aeruginosa infection. (A) IL-1β mRNA levels peaked at 5 days pi and were significantly decreased in the spantide I-treated group at 3 and 5 days pi. No IL-1β was detected in the normal cornea. (B) IL-1β protein levels peaked at 3 days pi and were significantly decreased in the spantide I-treated group at 3 and 5 days pi. All data are mean ± SEM (n = 5/group/time).
Figure 5.
 
Real-time PCR and ELISA analysis of corneal IL-1β mRNA and protein levels after P. aeruginosa infection. (A) IL-1β mRNA levels peaked at 5 days pi and were significantly decreased in the spantide I-treated group at 3 and 5 days pi. No IL-1β was detected in the normal cornea. (B) IL-1β protein levels peaked at 3 days pi and were significantly decreased in the spantide I-treated group at 3 and 5 days pi. All data are mean ± SEM (n = 5/group/time).
Figure 6.
 
Real-time PCR and ELISA analysis of corneal anti-inflammatory cytokine mRNA and protein levels after P. aeruginosa infection. (A) TGF-β mRNA levels peaked at 1 day pi, but there were no significant differences at any time point between spantide I-treated and PBS-treated groups. (B) Corneal IL-10 mRNA peaked at 3 days pi and was significantly higher in the spantide I-treated versus control group at 1 and 5 days pi. IL-10 was not detected in normal corneas. (C) IL-10 protein peaked at 3 days pi and was significantly higher in the spantide I-treated group 5 days pi. All data are mean ± SEM (n = 5/group/time).
Figure 6.
 
Real-time PCR and ELISA analysis of corneal anti-inflammatory cytokine mRNA and protein levels after P. aeruginosa infection. (A) TGF-β mRNA levels peaked at 1 day pi, but there were no significant differences at any time point between spantide I-treated and PBS-treated groups. (B) Corneal IL-10 mRNA peaked at 3 days pi and was significantly higher in the spantide I-treated versus control group at 1 and 5 days pi. IL-10 was not detected in normal corneas. (C) IL-10 protein peaked at 3 days pi and was significantly higher in the spantide I-treated group 5 days pi. All data are mean ± SEM (n = 5/group/time).
Figure 7.
 
Effect of neuropeptides on PMN migration. (A) SP, VIP. and SN significantly increased PMN migration compared with the media control. (B) The NK-1R antagonist spantide I had no effect on SP-induced PMN migration in the presence or absence of IL-1β. MIP-2 was the positive control. Data are expressed as mean ± SEM of the number of migrating PMNs.
Figure 7.
 
Effect of neuropeptides on PMN migration. (A) SP, VIP. and SN significantly increased PMN migration compared with the media control. (B) The NK-1R antagonist spantide I had no effect on SP-induced PMN migration in the presence or absence of IL-1β. MIP-2 was the positive control. Data are expressed as mean ± SEM of the number of migrating PMNs.
Figure 8.
 
Real-time PCR analysis of NK-1R mRNA levels. There was a moderate level of NK-1R mRNA expression in the normal B6 cornea, and by 5 days pi, a twofold decrease in NK-1R mRNA expression was seen in the infected cornea. Neither unstimulated PMN nor PMN stimulated with LPS had any detectable expression of NK-1R mRNA.
Figure 8.
 
Real-time PCR analysis of NK-1R mRNA levels. There was a moderate level of NK-1R mRNA expression in the normal B6 cornea, and by 5 days pi, a twofold decrease in NK-1R mRNA expression was seen in the infected cornea. Neither unstimulated PMN nor PMN stimulated with LPS had any detectable expression of NK-1R mRNA.
Figure 9.
 
(A) Real-time PCR analysis of NK-1R levels in B6 peritoneal Μφ. NK-1R was detected constitutively (lane 1) and after LPS stimulation of Μφ (lane 2) as well as in normal (lane 3) and infected (lane 4) corneas. Both LPS stimulation and infection of the cornea appeared to decrease the expression of the receptor. (B, C) Confocal images of NK1-R staining of unstimulated Μφ, confirming the mRNA data. Magnifications: (B) ×70; (C) ×430. (D) ELISA analysis of the effect of spantide I on IL-1β production by stimulated peritoneal Μφ. LPS significantly increased IL-1β protein levels in cultured peritoneal Μφ but did not differ after spantide treatment (P = 0.93); SP alone did not stimulate IL-1β expression with or without spantide treatment. SP potentiated LPS induced IL-1β expression (P = 0.009). Spantide I alone had no effect on IL-1 β expression. Spantide I did not significantly change LPS-induced IL-1β expression, but significantly reduced LPS induced IL-1β expression in the presence of SP (P = 0.01). PBS had no effect on IL-1β production with or without spantide.
Figure 9.
 
(A) Real-time PCR analysis of NK-1R levels in B6 peritoneal Μφ. NK-1R was detected constitutively (lane 1) and after LPS stimulation of Μφ (lane 2) as well as in normal (lane 3) and infected (lane 4) corneas. Both LPS stimulation and infection of the cornea appeared to decrease the expression of the receptor. (B, C) Confocal images of NK1-R staining of unstimulated Μφ, confirming the mRNA data. Magnifications: (B) ×70; (C) ×430. (D) ELISA analysis of the effect of spantide I on IL-1β production by stimulated peritoneal Μφ. LPS significantly increased IL-1β protein levels in cultured peritoneal Μφ but did not differ after spantide treatment (P = 0.93); SP alone did not stimulate IL-1β expression with or without spantide treatment. SP potentiated LPS induced IL-1β expression (P = 0.009). Spantide I alone had no effect on IL-1 β expression. Spantide I did not significantly change LPS-induced IL-1β expression, but significantly reduced LPS induced IL-1β expression in the presence of SP (P = 0.01). PBS had no effect on IL-1β production with or without spantide.
Table 1.
 
Nucleotide Sequence of the Specific Primers Used in PCR Amplification
Table 1.
 
Nucleotide Sequence of the Specific Primers Used in PCR Amplification
Gene Primers Sense
β-Actin 5′-GAT TAC TGC TCT GGC TCC TAG C-3′ F
5′-GAC TCA TCG TAC TCC TGC TTG C-3′ R
MIP-2 5′-TGT CAA TGC CTG AAG ACC CTG CC-3′ F
5′-AAC TTT TTG ACC GCC CTT GAG AGT GG-3′ R
IL-6 5′-CAC AAG TCC GGA GAG GAG AC-3′ F
5′-CAG AAT TGC CAT TGC ACA AC-3′ R
TNF-a 5′-ACC CTC ACA CTC AGA TCA TCTT-3′ F
5′-GGT-TGT CTT TGA GAT CCA TGC-3′ R
IL-12 p40 5′-GGT CAC ACT GGA CCA AAG GGA CTA TG-3′ F
5′-ATT CTG CTG CCG TGC TTC CAA C-3′ R
IL-18 5′-GCC ATG TCA GAA GAC TCT TGC GTC-3′ F
5′-GTA CAG TGA AGT CGG CCA AAG TTG TC-3′ R
IFN 5′-CAG AGC CAG ATT ATC TCT TTC TAC CTC AGA C-3′ F
5′-CTT TTT CGC CTT GCT GTT GCT GAA G-3′ R
IL-1β 5′-CGC AGC AGC ACA TCA ACA AGA GC-3′ F
5′-TGT CCT CAT CCT GGA AGG TCC ACG-3′ R
TGF 5′-AAC AAT TCC TGG CGT TAC CTT-3′ F
5′-CTG CCG TAC AAC TCC AGT GA-3′ R
IL-10 5′-TGC TAA CCG ACT CCT TAA TGC AGG AC-3′ F
5′-CCT TGA TTT CTG GGC CAT GCT TCT C-3′ R
NK1-R 5′-TGG ACT CTG ATC TCT TCC TGC CCA ACA-3′ F
5′-GGA CCC AGA TGA CAA AGA TGA CCA CTT-3′ R
NK1-R 5′-CCA ACA CCT ACT CCA CCA ACA CTT CTG-3′ F
5′-GGA CCC AGA TGA CAA AGA TGA CCA CTT-3′ R
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