Substance P Promotes Susceptibility to Pseudomonas aeruginosa Keratitis in Resistant Mice: Anti-inflammatory Mediators Downregulated

Abstract

purpose. Studies have shown that blocking substance P (SP) binding to neurokinin 1 receptor with spantide I prevents Pseudomonas aeruginosa–induced corneal perforation in susceptible C57BL/6 mice. This study tested the effect of SP injection on the resistance response (cornea heals) of BALB/c mice.

methods. The day before infection, mice were injected intraperitoneally with SP or PBS. Disease was graded by clinical score, slit lamp, plate count, real-time RT-PCR, and ELISA assays, and polymorphonuclear neutrophils (PMNs) were quantitated using a myeloperoxidase assay. In additional experiments, BALB/c mice were injected intraperitoneally with vasoactive intestinal peptide (VIP) antagonist and similarly analyzed.

results. Mice injected with SP exhibited worsened disease on days 1 to 7 after infection compared with controls. SP injection resulted in elevated PMN levels and viable bacterial counts in the cornea 3 and 5 days after infection. mRNA expression for NFκB and type 1 cytokines (e.g., IFN-γ), as well as for TNF-α, MIP-2, IL-18, IL-6, and IL-1β, were significantly elevated, whereas VIP and cytokines TGF-β and IL-10 were significantly reduced. Differences in mRNA expression were selectively confirmed at the protein level by ELISA for NFκB, IL-1β, and IL-10. VIP antagonist treatment also resulted in exacerbated disease scores, elevated proinflammatory mediators, and reduced anti-inflammatory mediators.

conclusions. These data provide evidence that the neuropeptide SP, among its broad systemic effects, is a potent neuroimmunoregulator that promotes susceptibility in the resistant BALB/c mouse by overcoming the anti-inflammatory effects of VIP and IL-10 and that a balance between SP and VIP levels may be critical in disease resolution.

In humans, Pseudomonas aeruginosa keratitis is a rapidly developing, devastating disease that may lead to corneal perforation, with a higher incidence of disease in users of extended-wear contact lenses. ^{1 2} Experimental studies have focused on the inflammatory response of the host, including the participation of cytokines and chemokines, antigen-presenting cells (Langerhans cells and macrophages), polymorphonuclear neutrophils (PMNs), T cells, and natural killer (NK) cells in animal models of disease. ³ The use of Th1-type responder mice such as C57BL/6, which are classified as susceptible (cornea perforates), and Th2-type responder mice such as BALB/c, which are resistant (cornea heals), has provided valuable information regarding the disparate immune response to infection. ⁴ The upregulation of proinflammatory mediators such as IL-1β, ⁵ IL-12, and IFN-γ, ⁶ an inflammatory cell infiltrate including CD4⁺ T cells, persistent PMNs, and elevated numbers of viable organisms present in the cornea are characteristic features of the susceptible phenotype. ⁷ In contrast, resistant mice exhibit lower and more tightly regulated levels of IFN-γ, ⁸ cellular infiltrate, and viable bacteria in the cornea, ⁷ with elevated levels of the anti-inflammatory cytokine IL-10. ⁹ In addition to these differences, susceptible B6 compared with BALB/c mice also express higher levels of the proinflammatory neuropeptide substance P (SP), both constitutively and after infection. ¹⁰  

SP is the most widely studied member of the tachykinin family. It modulates a number of immune functions such as leukocyte activation, cytokine production, plasma extravasation, ^{11 12} and adhesion molecule upregulation. ¹³ We have previously shown that after infection, IFN-γ expression in the BALB/c mouse cornea is regulated by SP and its interaction with its physiologically preferred receptor, NK-1R, on NK cells. ¹⁴ More recently, we have shown that treatment of the susceptible B6 mouse with the SP antagonist, spantide I, which blocks SP–NK-1R interaction, improves disease outcome by reducing Th1 cytokine levels and elevating IL-10 levels. ¹⁰ In addition, we have found that the anti-inflammatory neuropeptide vasoactive intestinal peptide (VIP), which is found in higher concentration in the resistant BALB/c cornea, has an effect similar to that seen with spantide I when given exogenously to susceptible B6 mice. ¹⁵ These studies support the hypothesis that crosstalk between the nervous and immune system is mediated by soluble products such as neuropeptides or cytokines interacting with specific receptors present on immune cells and that SP and VIP not only act as mediators of the crosstalk, they are biologically involved in the interaction. 

The aim of the present study was to directly test the regulatory function of SP in promoting a Th1-dominant (susceptible) response by administering exogenous SP to resistant BALB/c mice. We show that disease response is worsened and that increases in cellular infiltrate, bacterial number, proinflammatory cytokines/chemokines, NFκB activation, and reduced levels of VIP and IL-10 occur, resulting in corneal perforation. 

Methods

Mice

Female 8-week-old 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 (ATCC, Manassas, VA), was prepared as described. ¹⁶ Mice (n = 5/group per time) were anesthetized, and the left central cornea was scarified with a 25 ⅝-gauge needle. A 5-μL aliquot containing a 1.0 × 10⁶ CFU/μL bacterial suspension was applied to the wounded cornea, and disease was graded at 1, 3, 5, and 7 days postinfection (p.i.). ¹⁷  

SP and VIP Antagonist Treatment

BALB/c mice (n = 5/group per time/assay) were injected intraperitoneally with 100 μL PBS containing 15 μM SP (Sigma Chemical, St. Louis, MO) on days −1, 0 (day of infection), and daily through 7 days p.i. In similar experiments, BALB/c mice (n = 5/group per time/assay) were injected intraperitoneally with 100 μL PBS containing 10 μg VIP antagonist (Bachem, San Carlos, CA) according to the same injection schedule as for SP. Control mice were similarly injected with PBS. Infected corneas were collected at 1, 3, 5, and 7 days p.i. for real-time RT-PCR detection of mRNA levels of cytokines/chemokines, NFκB, and VIP. Normal, uninfected corneas also were harvested to determine basal levels of the target genes. In separate experiments, infected corneas were harvested at 3 and 5 days p.i. and were used for ELISA analysis, viable bacterial enumeration, and myeloperoxidase (MPO) quantitation. Mice treated with VIP antagonist were harvested at 5 days p.i. to test selected gene and protein expression levels. 

Quantitation of Corneal PMNs

An MPO assay ¹⁸ was used to quantitate PMNs in the corneas of SP- and PBS-treated BALB/c mice (n = 5/group per time) at 3 and 5 days p.i. Corneas were removed, homogenized in 1 mL of 50 mM phosphate buffer (pH 6.0) containing 0.5% HTAB (Sigma), freeze-thawed four times, and centrifuged, and 0.1 mL was 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 approximately 2 × 10⁵ PMN/mL. ¹⁹  

Quantitation of Viable Bacteria

Bacteria were quantitated at 3 and 5 days p.i. in individual infected corneas of BALB/c mice (n = 5/group per time) after SP or PBS treatment. Each cornea was homogenized in 1.0 mL sterile saline containing 0.25% BSA; 0.1 mL of the corneal homogenate was serially diluted 1:10 in the same solution, and selected dilutions were plated in triplicate on Pseudomonas isolation agar (Difco, Detroit, MI). Plates were incubated overnight at 37°C, and the number of viable bacteria was counted. Results are reported as log₁₀ number of CFU/cornea ± SEM. 

Real-Time RT-PCR

Normal, uninfected, and infected corneas (n = 5/group per time) from SP- and PBS-treated BALB/c mice were removed at 1, 3, 5, and 7 days p.i. Corneas from VIP antagonist-treated corneas and their PBS-treated controls were harvested at 5 days p.i. Corneas were stored (RNAlater; Ambion Inc., Austin, TX) at −70°C. Total corneal RNA was extracted using RNA STAT-60 (Tel-Test, Friendsville, TX) according to the manufacturer’s instructions and were used to produce a cDNA template for PCR reaction. One microgram of each RNA sample was reverse transcribed (M-MLV Reverse Transcriptase; Invitrogen, Carlsbad, CA) simultaneously in a 20-μL volume. cDNA products were diluted 1:25 with diethylpyrocarbonate-treated H₂O, and 2 μL each cDNA dilution was used for real-time RT-PCR (20-μL reaction volume). mRNA levels of proinflammatory cytokines (MIP-2, IL-6, TNF-α, IL-18, IFN-γ, and IL-1β), anti-inflammatory cytokines (TGF-β and IL-10), and NFκB were detected by real-time RT-PCR (MyiQTM Single Color Real-Time PCR Detection System; Bio-Rad, Hercules, CA). Supermix (iQTM SYBR Green; Bio-Rad) was used for 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, and these cycles were repeated 40 times. ¹⁰  

For the detection of VIP mRNA, cDNA (prepared as described) was amplified for 40 cycles as follows: 15 seconds each at 94°C and 58°C and 45 seconds at 72°C. ²⁰ The fold differences in gene expression were calculated after normalization to β-actin. All primer pair sequences used for real-time RT-PCR are shown in Table 1 . 

ELISA

After SP, VIP antagonist, or PBS treatment, protein levels for IL-1β and IL-10 also were determined using ELISA kits (R&D Systems, Minneapolis, MN). Corneas from infected mice (n = 5/group per time) were harvested at 3 or 5 days p.i., or both. Corneas were individually homogenized in PBS containing 0.1% Tween-20 with a glass microtissue grinder and were 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, BALB/c corneas (n = 5/group per time) were treated similarly, and at 3 or 5 days p.i., or both, 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. The reported sensitivity of these assays was less than 3.0 pg/mL for IL-1β and less than 4.0 pg/mL for IL-10. 

EIA Detection of VIP

After SP treatment, VIP protein levels were determined in BALB/c corneas with the use of a competitive enzyme immunoassay (EIA) kit (Peninsula Laboratories, San Carlos, CA). Individual corneas were collected at 3 and 5 days p.i. and homogenized in 250 μL normal saline. Samples were centrifuged at 5000g (10 minutes), and an aliquot of each supernatant was assayed in triplicate for VIP protein according to the manufacturer’s instruction. Assay sensitivity was 2 to 3 pg/mL. Results are expressed as pg/mL VIP ± SEM. 

Detection of Activated NFκB

After SP or PBS treatment, normal and infected corneas were harvested (1 and 5 days p.i.), and nuclear extracts were prepared using a commercially available kit (Active Motif, Carlsbad, CA). The protein content in the nuclear extracts was determined by the Bradford method, and 5 μg of each nuclear extract was assayed for the activated transcription factor NFκB (p65 subunit) with an ELISA-based kit per the manufacturer’s instructions (Active Motif). The sensitivity of this assay is reported to be less than 0.5 μg. 

Immunostaining

The presence of NK/NKT or CD4⁺ T cells was investigated by staining infected corneal tissue sections for the expression of asialo GM1⁺(NK cell), CD4⁺ (CD4⁺ T cell), or double-positive cells (NKT cell) using dual-label immunofluorescence staining and confocal laser scanning microscopy. Whole eyes were enucleated at 5 days p.i. from PBS- and SP-treated BALB/c mice (n = 3/group), embedded in optimal cutting temperature compound, snap frozen in liquid nitrogen, and stored at −20°C. Ten-micrometer-thick sections were collected on poly-l-lysine–coated slides, incubated at 37°C overnight, and then incubated in blocking agent (bovine serum albumin [2.5%; Sigma], goat IgG [1:100; Jackson ImmunoResearch, West Grove, PA], and donkey IgG [1:100; Jackson ImmunoResearch] in 0.01 M PBS) for 30 minutes at room temperature. After blocking for nonspecific antibody binding, the sections were incubated with primary antibodies, rat anti-mouse CD4 (1:10; BD PharMingen, San Diego, CA) and rabbit anti-mouse asialo GM1 (1:100; Wako Chemicals USA., Richmond, VA) for 1 hour, followed by CY3-conjugated goat anti-rabbit IgG (1:1500; Jackson ImmunoResearch) and CY5-conjugated donkey anti-rat IgG (1:1500; Jackson ImmunoResearch) for another hour. Sections were then incubated for 2 minutes with nuclear acid stain (SYTOX Green, 1:200,000; Fisher Scientific, Hanover Park, IL). Negative controls were treated similarly but without the primary antibodies. Sections were visualized, and digital images were captured with a confocal laser scanning microscope (TSC SP2; Leica Microsystems, Exton, PA). 

Statistical Analysis

The difference in clinical score at each experimental time point between two-groups was tested by the Mann-Whitney U test. An unpaired, two-tailed Student’s t-test was used to determine statistical significance of real-time RT-PCR, ELISA (protein and transcription factor activation), bacterial plate counts, and MPO assays. Data were considered significantly different at P < 0.05. All experiments were repeated at least once to ensure reproducibility, and data from a representative experiment are shown, unless indicated. 

Results

Disease Response, PMN Infiltration, and Bacterial Load

Based on previously published data ¹⁰ that showed blocking interaction of SP with its preferred receptor, NK-1R, using spantide improved disease outcomes in B6 mice, we sought to test the hypothesis that exogenous injection of SP in the resistant BALB/c mouse would exacerbate corneal disease. After infection, disease was graded through 7 days p.i. (Fig. 1A) , and significantly more severe disease was noted in the SP-treated group at 1, 3, 5, and 7 days p.i. (P = 0.04, P = 0.04, P = 0.0005, and P = 0.002, respectively) when compared with PBS-treated controls. Slit lamp photomicrographs, taken of representative eyes from both groups at 7 days p.i. (Figs. 1B 1C) , revealed a slight central opacity in the PBS-treated eye (Fig. 1B) , whereas perforation was seen in the eye after SP treatment (Fig. 1C) . 

An MPO assay tested whether SP treatment had an effect on PMN infiltration into the infected cornea (Fig. 1D) . Corneas from SP-treated mice contained significantly more PMNs than the corneas of PBS-treated mice at both 3 and 5 days p.i. (P = 0.05 and P = 0.002, respectively). We also tested the effect of SP treatment on the number of viable bacteria present in the cornea (Fig. 1E) . Bacterial load in the corneas of SP-treated mice was significantly higher at 3 and 5 days p.i. (P < 0.0001 for both) compared with viable bacteria isolated from PBS-treated mice. 

Real-Time RT-PCR

To further test the effects of SP treatment, we evaluated mRNA expression of Th1-type and proinflammatory cytokines/chemokines after P. aeruginosa infection. Normal, uninfected corneas and corneas at 1, 3, 5, and 7 days p.i. were analyzed by real-time RT-PCR; mRNA expression levels are shown in Figure 2A 2B 2C 2D 2E . IFN-γ (Fig. 2A)was not detected in the normal, uninfected cornea but was significantly increased in SP- compared with PBS-treated mice at 1 (P = 0.0003), 5 (P = 0.04), and 7 days p.i. (P = 0.02). No difference was detected between the two groups at 3 days p.i. (P = 0.30). A significant increase in IL-18 mRNA expression (Fig. 2B)was detected in SP-treated mice at 1, 3, 5, and 7 days p.i. (P < 0.0001, P = 0.004, P = 0.005, and P = 0.05, respectively) when compared with PBS-treated controls. No signal for IL-18 was detected in normal, uninfected samples. TNF-α (Fig. 2C)was constitutively expressed in normal, uninfected BALB/c cornea and was significantly elevated in SP-treated compared with PBS-treated controls at 3, 5, and 7 days p.i. (P = 0.005, P = 0.001, and P = 0.04, respectively). TNF-α mRNA corneal expression levels were similar in SP- and PBS-treated mice at 1 day p.i. (P = 0.45). mRNA expression of IL-6 (Fig. 2D)was not detected in the normal, uninfected cornea and was significantly higher in SP-treated mice only at 5 and 7 days p.i. (P = 0.003 and P = 0.05, respectively). No significant differences were detected between the two groups at days 1 or 3 p.i. (P = 0.1 and P = 0.07, respectively). MIP-2 mRNA expression (Fig. 2E)was not detected in the normal, uninfected BALB/c cornea. Expression of MIP-2 mRNA was significantly upregulated by SP treatment at 3, 5, and 7 days p.i. (P = 0.01, P = 0.003, and P = 0.03, respectively). MIP-2 levels were similar between SP- and PBS-treated mice at 1 day p.i. (P = 0.95) 

IL-1β mRNA and Protein Levels

Expression of IL-1β mRNA (Fig. 3A)was not detectable in the normal, uninfected cornea. IL-1β mRNA levels were significantly elevated in the corneas of SP-treated mice compared with PBS-treated mice at 3, 5, and 7 days p.i. (P = 0.007, P = 0.004, and P = 0.03, respectively). No significant difference was found between the two groups at 1 day p.i. (P = 0.76). IL-1β protein levels (Fig. 3B)were significantly higher in the corneas of SP-treated mice at 3 and 5 days p.i. compared with PBS-treated controls (P = 0.05 and P < 0.0001, respectively). 

Real-Time RT-PCR and ELISA Analysis

Levels of anti-inflammatory and Th2-type cytokines/chemokines also were analyzed by real-time RT-PCR (Figs. 4A 4B)and ELISA (Fig. 4C) . TGF-β mRNA (Fig. 4A)was constitutively expressed in the normal, uninfected cornea, and levels were significantly lower in the corneas of SP-treated mice than of PBS-treated mice at 1, 3, and 7 days p.i. (P = 0.001, P = 0.0006, and P = 0.05, respectively). No significant difference in TGF-β was detected between the two groups at 5 days p.i. (P = 0.45). 

IL-10 mRNA levels were significantly reduced in SP- compared with PBS-treated mice at 1, 3, 5, and 7 days p.i. (P = 0.04, P = 0.002, P = 0.0001, and P = 0.02), respectively, whereas the uninfected cornea was negative for cytokine expression (Fig. 4B) . Protein levels for IL-10 (Fig. 4C)also were significantly reduced at 3 and 5 days p.i. in SP- compared with PBS-treated control mice (P = 0.002 and P < 0.0001, respectively). 

Real-Time RT-PCR and Transcription Activation Detection for NFκB

Constitutive mRNA expression for NFκB was detected in the normal, uninfected cornea and was significantly elevated in corneas of SP-treated mice at days 1, 3, 5, and 7 p.i. (P = 0.006, P < 0.0001, P = 0.004, and P = 0.004, respectively) compared with PBS-treated controls (Fig. 5A) . Activated NFκB was detected at very low levels in nuclear extracts from normal, uninfected corneas but was significantly upregulated in extracts of SP- compared with PBS-treated corneas at 1 and 5 days p.i. (P = 0.02 and P = 0.002, respectively; Fig. 5B ). 

Immunostaining

Merged images of anti-asialo GM1, anti-CD4, and nuclear acid (SYTOX Green; Fisher Scientific)-stained sections are shown for SP- (Fig. 6A)and PBS-treated corneas (Fig. 6B)at 5 days p.i. Figure 6C(SP-treated corneas) and Fig. 6D(PBS-treated corneas) show anti-asialo GM1 staining that appears qualitatively more intense in the SP- than in the PBS-treated cornea. No CD4⁺ cells were labeled in the corneas of either SP- (Fig. 6E)or PBS-treated (Fig. 6F)mice. Negative controls showing nuclear label only (omission of the primary antibodies) are shown for SP-treated (Fig. 6G)and PBS-treated (Fig. 6H)corneas, respectively. 

Real-Time RT-PCR for VIP

Because treatment of susceptible B6 mice with VIP has been shown to be protective, preventing perforation, ¹⁵ we next tested whether the effects seen after SP treatment resulted from a change in VIP levels in the cornea (Figs. 7A 7B) . VIP was constitutively expressed in the normal, uninfected corneas of BALB/c mice. Levels of VIP mRNA were significantly reduced in the SP-treated group at 1, 3, and 7 days p.i. when compared with PBS-treated controls (P = 0.02, P < 0.0001, and P = 0.05, respectively). Levels of VIP peptide were reduced in SP-compared with PBS-treated cornea at 3 and 5 days p.i. (Fig. 7B) , but the difference was significant only at 5 days p.i. (P = 0.25 and P = 0.0004, respectively). 

Disease Response after VIP Antagonist Treatment

To compare the effect of neutralizing VIP with SP treatment, mice were injected with 10 μg VIP antagonist on a schedule similar to that used for SP administration. Figure 8Ashows that disease scores between PBS- and VIP antagonist-treated mice did not differ at 1 day p.i. (P = 0.14), but VIP antagonist treatment resulted in significantly higher disease scores at 3 and 5 days p.i. (P = 0.003 and P < 0.0001, respectively) compared with PBS-treated controls. Slit lamp photomicrographs taken of representative eyes from both treatment groups at 5 days p.i. show slight central opacity in the PBS-treated eye (Fig. 8B)compared with perforation shown in the VIP-antagonist treated eye (Fig. 8C) . 

Real-Time RT-PCR and ELISA

Given that disease outcome (susceptibility) was similar in VIP antagonist-treated and SP-treated BALB/c mice compared with PBS-treated controls, selected proinflammatory and anti-inflammatory mediators were tested by real-time RT-PCR (Fig. 8D)and ELISA (Figs. 8E 8F)at 5 days p.i., when 90% of the corneas in the VIP antagonist-treated mice had been perforated. The pattern of gene expression in the infected corneas of VIP antagonist-treated mice was similar to that after SP treatment. (Fig. 8D) . mRNA for proinflammatory cytokines IL-18, IL-1β, and IFN-γ was significantly increased over that for PBS-treated controls (P = 0.005, P = 0.001, and P = 0.0003, respectively), and anti-inflammatory mediators IL-10 and TGF-β were significantly reduced (P = 0.007 and P = 0.0003, respectively) in VIP antagonist-treated compared with control-treated mice. 

ELISA analysis showed that VIP antagonist treatment also resulted in an increase in corneal IL-1β protein level (Fig. 8E ; P = 0.001) and a decrease in IL-10 protein level (Fig. 8F ; P = 0.002) compared with the levels found in PBS-treated controls. 

Discussion

The cornea, which is supplied by sensory and autonomic nerve fibers containing neuropeptides such as SP, VIP, and calcitonin gene-related peptide, is highly innervated. ²¹ SP has been described almost exclusively as a peptide of neuronal origin, with its expression increased or induced in various inflammatory and infectious diseases. ^{10 22} Nonetheless, studies in rodent models have demonstrated the production of SP by inflammatory cells such as macrophages and dendritic cells. ¹³ The biological response to SP is mediated by its major physiological receptor, the NK-1R, present on CD4⁺ and CD8⁺ T cells, ²³ B lymphocytes, ²⁴ monocytes/macrophages, ^{10 25} neutrophils, ²⁶ mast cells, ²⁷ and eosinophils ²⁸ and alludes to the importance of SP in the regulation of their function. 

Another neuropeptide, VIP, has recently been show to have potent anti-inflammatory properties in bacterial keratitis, suggesting that SP would have a disparate effect. To test the latter hypothesis and the mechanisms whereby SP might potentiate P. aeruginosa keratitis, resistant BALB/c mice were injected with exogenous SP. When compared with its use in control-treated infected mice, SP demonstrated enhanced corneal perforation, elevated numbers of viable bacteria, and elevated numbers of PMN. In this regard, PMN chemotaxis and activation are two of the numerous inflammatory events that are regulated by SP. ^{28 29 30} SP induces a rapid influx of PMNs into a site of infection, which occurs simultaneously with the translocation of P-selectin and the upregulation of E-selectin. By promoting vasodilatation and leukocyte/adhesion molecule interaction, SP appears to augment the extravasation, migration, and subsequent accumulation of leukocytes at the site of injury. ³¹ This effect was abrogated in bronchial epithelium by treatment either with antibodies to CD11a, CD11b, or ICAM-1 or with the NK-1R antagonist CP-96345, suggesting that SP has the capacity to modulate PMN–epithelial cell interactions by regulating adhesion molecule expression and that this interaction is mediated by NK-1R. ³²  

In our study, real-time RT-PCR or ELISA, or both, showed that SP upregulated Th1-type 1 and proinflammatory cytokines, including the chemokine MIP-2. This murine functional homologue of IL-8, a potent chemoattractant for PMNs, ³³ was significantly upregulated at 3, 5, and 7 days p.i., supporting previous work showing that IL-8 and MIP-2 induced PMN chemotaxis ³⁴ and recruitment to the infected cornea, ⁷ respectively. These data suggest that SP induces PMN recruitment indirectly through upregulation of the chemokine MIP-2, but they do not rule out the mere consequences of the vascular changes (plasma extravasation) produced by the neuropeptide. ³⁵  

mRNA expression levels for IFN-γ and IL-18 also were significantly up-regulated in the corneas of BALB/c mice after SP treatment. These data are consistent with past studies showing that tight regulation of IFN-γ production by SP, which regulates both IL-18 and NK cell production of IFN-γ, was necessary for effective bacterial killing and disease resolution in the BALB/c cornea. ⁸ In contrast, prolonged elevation of IFN-γ, as seen here in BALB/c mice after SP treatment or as shown previously for susceptible B6 mice, leads to corneal perforation. ¹⁶  

Because SP also is chemotactic for T cells, ¹¹ another potential source of IFN-γ, we performed immunolabeling to identify CD4⁺ cells in the infected cornea. No CD4⁺ T cells were detected in the cornea of SP- or PBS-treated mice, but numerous asialo GM1⁺ (NK cell marker) cells that also produce the cytokine were detected, with qualitatively more found in the SP-treated cornea. This is in contrast with a past study from this laboratory ³⁶ in which Langerhans cells, capable of antigen presentation, were induced into the cornea before P. aeruginosa infection in BALB/c mice. These, compared with control injected mice, exhibited corneal perforation accompanied by an increased number of B7-1+ (mature) Langerhans cells, CD4⁺and IL-2R⁺ T cells, enhanced delayed-type hypersensitivity (DTH), and increased mRNA levels for IFN-γ in cornea. 

IFN-γ remains an important regulatory cytokine of host defense and plays a critical role in the enhancement of macrophage-derived mediators such as TNF-α, IL-6, and IL-1. ^{37 38 39} All these cytokines were elevated at the mRNA expression level (and the protein level for IL-1β) after SP treatment. In contrast, mRNA and protein expression levels in cornea for IL-10 were significantly reduced in SP compared with PBS-treated controls. In this regard, IFN-γ has been shown to downregulate the synthesis of anti-inflammatory mediators such as IL-10, ⁴⁰ and balance between levels of these two cytokines has been reported as critical in resistance to Pseudomonas keratitis. ⁹  

The transcription factor NFκB regulates many genes that encode for mediators of immune and inflammatory responses and can be upregulated by TNF-α, IL-1β, ⁴¹ or LPS, a major component of the outer membrane of P. aeruginosa. Because SP treatment significantly upregulated TNF-α (mRNA) and IL-1β (mRNA and protein) expression in the cornea after infection, we used real-time RT-PCR and ELISA to analyze mRNA levels of NFκB, namely its activated (phosphorylated) p65 subunit, and found significant increases over PBS treatment. Others ⁴² also have shown increased activated NFκB levels after murine macrophages and dendritic cells were exposed to SP, providing support to the tenet that SP promotes susceptibility to bacterial infection through the up-regulation of proinflammatory molecules by activation of this transcription factor. Nonetheless, because SP is likely to have broad systemic impact and multiple downstream effects, particularly after IP injection, the precise sequence of events that cause enhanced corneal pathology in our model are likely to be more complex. 

Just as the biological responses to SP are mediated by its receptor, many of the anti-inflammatory effects of VIP are regulated through interactions with its receptors. ⁴³ Thus, we next tested whether the anti-inflammatory effects of VIP could be abrogated by blocking VIP/receptor interaction with an antagonist, ⁴⁴ resulting in a worsened disease response, similar to that observed after injection of exogenous SP. Our findings confirmed that blocking VIP interaction with its receptor enhanced inflammation and resulted in perforation of BALB/c-infected corneas by 5 days p.i. This in turn was accompanied by elevations in mRNA expression for IL-18, IFN-γ, and IL-1β and reductions in mRNA expression for IL-10 and TGF-β, with protein levels similarly altered for IL-1β and IL-10. 

These studies provide evidence supporting an interactive neuroimmune axis in the cornea regulated by a network of cytokines and neuropeptides that synergistically determine disease outcome after P. aeruginosa infection. 

 Supported by National Institutes of Health Grants R01EY02986 and P30EY04068.

 Submitted for publication October 23, 2007; revised November 15, 2007; accepted February 18, 2008.

 Disclosure: S.A. McClellan, None; Y. Zhang, None; R.P. Barrett, None; L.D. Hazlett, 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.

 

Table 1.

 

Nucleotide Sequence of the Specific Primers Used for PCR Amplification

Gene	Nucleotide Sequence	Primer
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-α	5′-ACC CTC ACA CTC AGA TCA TCTT-3′	F
	5′-GGT-TGT CTT TGA GAT CCA TGC-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-γ	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-β	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
NF κB	5′-GCT TTG CAA ACC TGG GAA TA-3′	F
	5′-TCC GCC TTC TGC TTG TAG AT-3′	R
VIP	5′-GAA GCC AGA AGC AAG CCT CAG TTC CTG GCA-3′	F
	5′-CCT CAC TAC AGA AGG TGG TCC AAA GAG AGG CC-3′	R

 F, forward; R, reverse.

Figure 1.

 

(A) Ocular disease response in SP- and PBS-injected BALB/c mice after P. aeruginosa infection. Ocular disease was graded at 1 to 7 days p.i. (n = 10/group per time), and significant differences in clinical scores were seen at all times examined. (B, C) Slit lamp photomicrographs are shown of representative eyes from PBS-treated (B) and SP-treated (C) mice at 7 days p.i. (D) Number of PMNs per cornea (mean ± SEM) (n = 5/group per time) was determined by an MPO assay. Significantly more PMNs were detected in the corneas of the SP-treated than in control-treated mice at 3 and 5 days p.i. (E) Corneas from SP- and control-treated mice (n = 5/group per time) had significantly more viable bacteria at 3 and 5 days p.i.

Figure 2.

 

Real-time RT-PCR of corneal cytokine mRNA expression in SP- and PBS-treated BALB/c mice after P. aeruginosa infection. (A) IFN-γ mRNA levels were significantly increased in the SP-treated compared with the control mouse group at 1, 5, and 7 days p.i. (B) IL-18 mRNA levels were significantly increased in the SP-treated group at 1, 3, 5, and 7 days p.i. (C) TNF-α mRNA levels were significantly increased compared with controls in SP-treated mice at 3, 5, and 7 days p.i. (D) IL-6 mRNA levels were significantly increased in the SP-treated mice at 5 and 7 days p.i. (E) MIP-2 mRNA levels were significantly increased in the SP-treated group at 3, 5, and 7 days p.i. Only TNF-α mRNA was detected in the normal uninfected cornea. All data are mean ± SEM; n = 5/group per time.

Figure 3.

 

Real-time RT-PCR and ELISA of corneal IL-1β after P. aeruginosa infection. (A) IL-1β mRNA levels were significantly increased in the SP-treated group at 3, 5, and 7 days p.i. compared with PBS-treated controls; no IL-1β was detected in the normal cornea. (B) IL-1β protein levels were significantly increased in the SP- compared with the PBS-treated group at 3 and 5 days p.i. Reported sensitivity of the assay was less than 3.0 pg/mL. All data are mean ± SEM; n = 5/group per time.

Figure 4.

 

Real-time RT-PCR and ELISA of corneal anti-inflammatory cytokines after P. aeruginosa infection. (A) TGF-β mRNA was constitutively expressed in the normal BALB/c cornea, and levels were significantly decreased in SP- compared with PBS-treated groups at 1, 3, and 7 days p.i. (B) Corneal IL-10 mRNA levels were significantly lower in the SP- compared with the PBS-treated control group at 1, 3, 5, and 7 days p.i. IL-10 was not detected in normal cornea. (C) IL-10 protein was significantly lower in the SP- than in the PBS-treated group at 3 and 5 days p.i. Reported sensitivity of the assay was less than 4.0 pg/mL. Data are mean ± SEM; n = 5/group per time.

Figure 5.

 

Real-time RT-PCR and ELISA detection of NFκB after P. aeruginosa corneal infection. (A) NFκB mRNA was constitutively expressed in normal cornea, and levels were significantly increased after SP treatment at 1, 3, 5, and 7 days p.i. compared with PBS-treated controls. (B) The amount of active NFκB (p65 subunit) was significantly higher in the nuclear fractions prepared from SP-treated corneas at 1 and 5 days p.i. than in PBS-treated control mice. Reported sensitivity of the assay was less than 0.5 μg. Data are mean ± SEM; n = 5/group per time.

Figure 6.

 

Dual immunofluorescent staining for asialo GM1 (NK cell marker) and CD4⁺ T cells in corneas of SP- and PBS-treated BALB/c mice at 5 days p.i. (A) Merged images of asialo GM1 (red), CD4⁺ T cell (blue), and SYTOX Green nuclear staining in SP-treated (A) and PBS-treated (B) corneas. Asialo GM1 positively stained NK cells were qualitatively increased after SP treatment (C) compared with the PBS-treated control (D). Neither the SP-treated cornea (E) nor the PBS-treated control (F) showed positively labeled CD4⁺ T cells. Negative controls showed only nuclear stain (primary antibodies were omitted) in SP-treated (G) and PBS-treated (H) control. Original magnification, ×60.

Figure 7.

 

Real-time RT-PCR and ELISA of corneal VIP after P. aeruginosa infection. (A) VIP mRNA levels were significantly decreased in the SP-treated group at 1, 3, and 7 days p.i. compared with PBS-treated controls. (B) VIP levels were reduced in corneas from the SP- and the PBS-treated groups at 3 and 5 days p.i. but were significant only at 5 days p.i. Reported sensitivity of the assay was 2 to 3 pg/mL. Data are mean ± SEM; n = 5/group per time.

Figure 8.

 

(A) Ocular disease response in VIP antagonist- compared with PBS-injected BALB/c mice after P. aeruginosa infection. Ocular disease was graded at 1, 3, and 5 days p.i. (n = 10/group per time). Significant differences between the two groups were seen at 3 and 5 days p.i., with more perforation in the VIP antagonist-treated group by 5 days p.i. Results are reported as individual clinical scores. (B, C) Slit lamp photomicrographs of P. aeruginosa-infected eyes after VIP antagonist treatment. Representative eyes from PBS-treated (B) and VIP antagonist-treated (C) mice are shown at 5 days p.i. (D) mRNA levels of proinflammatory cytokines IL-18, IL-1β, and IFN-γ were significantly elevated in VIP antagonist-treated compared with PBS-treated corneas at 5 days p.i. Levels of anti-inflammatory cytokines IL-10 and TGF-β were significantly decreased at 5 days p.i. in VIP antagonist-treated compared with control PBS-treated corneas. (E) Corneal protein levels of IL-1β were significantly elevated at 5 days p.i. in VIP antagonist-treated mice compared with PBS-treated controls. (F) Conversely, IL-10 corneal protein was significantly lower in mice receiving VIP antagonist than in those receiving PBS treatment.