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From the Department of Anatomy and Cell Biology, Wayne State University School of Medicine, Detroit, Michigan.
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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-
) as well as MIP-2, IL-6, TNF-
, and IL-1ß (mRNA and protein) also were significantly reduced after spantide treatment. The type II cytokine IL-10 (mRNA and protein) was elevated, whereas TGF-ß 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 (M
s), but not on PMNs (unstimulated or stimulated with endotoxin [LPS]). Spantide treatment of Ms reduced IL-1ß 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.
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- production or endogenous absence of IL-12, resulting in reduced IFN- 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- production through IL-12-independent mechanisms, resulting in less corneal destruction. Previous work9 confirmed that IL-18 regulates IFN- 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- in the infected BALB/c cornea. Evidence has been provided that in the infected cornea NK cells are the source of IFN-, 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- 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 responses11 principally by regulating the function of cells such as dendritic cells (DCs) and macrophages (Ms), 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.
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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 
-gauge needle. A 5-µL aliquot containing a 1.0 x 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 Ms, 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 Ms, 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 M 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-, IL-12, IL-18, IFN-, and IL-1ß) and anti-inflammatory cytokines (TGF-ß 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 ß-actin.
Nested Real-Time PCR
To detect NK-1R in M 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 .
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After spantide I or PBS treatment, protein levels for IL-1ß 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-1ß, 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-1ß 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 M. The reported sensitivity of these assays was <8.0 pg/mL for SP, <3.0 pg/mL for IL-1ß, 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-; Thermo Spectronic, Pittsford, NY) and units of MPO/cornea were calculated. One unit of MPO activity is equivalent to 
2 x 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 M 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.
Ms were induced into the peritoneal cavity of B6 mice by IP injection of 3% thioglycollate20 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 Ms 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-1ß production by the M. Supernatants were harvested after 18 hours and IL-1ß 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.
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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- levels (Fig. 4C) was detected at 1 day pi (P = 0.55); however, spantide I-treatment significantly reduced levels of corneal TNF- mRNA at 3 and 5 days pi when compared with the PBS-treated group (P = 0.005 and 0.049, respectively). TNF- 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- mRNA (Fig. 4F) was not detected in normal B6 mouse cornea and spantide I-treatment did not significantly change the levels of IFN- detected at 1 day pi when compared with PBS-treatment (P = 0.84). In contrast, significant reduction in mRNA levels for IFN- 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).
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s (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 Ms and localized the NK-1R, confirming that it was expressed at the protein level on these cells (Figs. 9B 9C) .
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s 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-1ß 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-1ß production. Stimulation with LPS significantly elevated IL-1ß 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-1ß production when compared with LPS alone (P = 0.04). The addition of spantide I to the combined stimulants significantly reduced IL-1ß production (P = 0.009), suggesting the role of SP/NK-1R interaction in M production of IL-1ß. |
Discussion |
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, IL-6, IL-1,22 IL-12, and IFN-.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- 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 Ms, eosinophils, lymphocytes, and DCs and acts by binding to the NK-1R. SP specifically activates NF-
B, 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-.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-) mRNA levels; the latter two shown previously to be associated with poor disease outcome in B6 mice.7 31 IL-1ß also was reduced at both the mRNA and protein levels. IL-1ß has been shown to be of critical importance to the susceptible response of B6 mice and use of antibody neutralization,22 an IL-1ß inhibitor,32 or caspase-1-deficient mice33 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- 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- 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 Ms was essential in the regulation of IFN- 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 M-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 chemoattractant40 and we16 have shown that MIP-2 recruits PMNs into the P. aeruginosa-infected B6 cornea. IL-1, produced by monocytes, Ms, and corneal epithelial cells, also regulates PMN influx into tissues41 and activates antigen-presenting cells (APCs) and inflammatory cells.42 In B6 mice, Rudner et al.,22 showed that prolonged elevation of IL-1ß in P. aeruginosa corneal infection upregulates MIP-2, enhancing PMN influx into the cornea and inducing perforation. Others43 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-1ß 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-1ß-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 Ms.12 13 In the study reported herein, Ms 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-1ß in some systems, we also tested Ms by LPS stimulation with or without spantide treatment. LPS and SP potentiated IL-1ß production by Ms and spantide reduced the effect, providing an explanation of the reduction of IL-1ß 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 M 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 Ms,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.
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Footnotes |
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Submitted for publication July 28, 2006; revised October 17, 2006; accepted December 15, 2006.
Disclosure: L.D. Hazlett, None; S.A. McClellan, None; R.P. Barrett, None; J. Liu, None; Y. Zhang, None; S. Lighvani, None
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. 
1734 solely to indicate this fact.
Corresponding author: Linda D. Hazlett, Department of Anatomy and Cell Biology, 540 E. Canfield Ave., Detroit, MI 48201; lhazlett{at}med.wayne.edu.
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and TNF- are required for IL-12-induced development of Th1 cells producing high levels of IFN- in BALB/c but not C57BL/6 mice. J Immunol. 1998;160:1708–1716. in Pseudomonas aeruginosa corneal infection. Invest Ophthalmol Vis Sci. 2002;43:419–424. production and resistance to Pseudomonas aeruginosa infection. Eur J Immunol. 2005;35:1567–1575.[CrossRef][ISI][Medline][Order article via Infotrieve]This article has been cited by other articles:
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  Z. Zhou, R. P. Barrett, S. A. McClellan, Y. Zhang, E. A. Szliter, N. van Rooijen, and L. D. Hazlett Substance P Delays Apoptosis, Enhancing Keratitis after Pseudomonas aeruginosa Infection Invest. Ophthalmol. Vis. Sci., October 1, 2008; 49(10): 4458 - 4467. [Abstract] [Full Text] [PDF]
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S. A. McClellan, Y. Zhang, R. P. Barrett, and L. D. Hazlett Substance P Promotes Susceptibility to Pseudomonas aeruginosa Keratitis in Resistant Mice: Anti-inflammatory Mediators Downregulated Invest. Ophthalmol. Vis. Sci., April 1, 2008; 49(4): 1502 - 1511. [Abstract] [Full Text] [PDF]
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