Retinal laser burn-induced neuropathy leads to substance P-dependent loss of ocular immune privilege

Abstract

Inflammation in the eye is tightly regulated by multiple mechanisms that together contribute to ocular immune privilege. Many studies have shown that it is very difficult to abrogate the immune privileged mechanism called anterior chamber-associated immune deviation (ACAID). Previously, we showed that retinal laser burn (RLB) to one eye abrogated immune privilege (ACAID) bilaterally for an extended period of time. In an effort to explain the inflammation in the nonburned eye, we postulated that neuronal signals initiated inflammation in the contralateral eye. In this study, we test the role of substance P, a neuroinflamatory peptide, in RLB-induced loss of ACAID. Histological examination of the retina with and without RLB revealed an increase of the substance P-inducible neurokinin 1 receptor (NK1-R) in the retina of first, the burned eye, and then the contralateral eye. Specific antagonists for NK1-R, given locally with Ag within 24 h, but not 3, 5, or 7 d post-RLB treatment, prevented the bilateral loss of ACAID. Substance P knockout (KO) mice retained their ability to develop ACAID post-RLB. These data support the postulate that substance P transmits early inflammatory signals from the RLB eye to the contralateral eye to induce changes to ocular immune privilege and has a central role in the bilateral loss of ACAID. The possibility is raised that blocking of the substance P pathway with NK1-R antagonists postocular trauma may prevent unwanted and perhaps extended consequences of trauma-induced inflammation in the eye.

Figure 1. DH response after ACAID induction in RLB treated mice

Change (Δ) in ear thickness is indicated on the ordinate. Experimental treatment of the mice in each group is indicated below each bar, on the abscissa. Mice received RLB treatment or not at the time indicated prior to ACAID induction, one week later, mice were immunized with OVA and the following week, the ear pinnae was challenged with OVA. Ipsilateral = black bars; and Contralateral = white bars. An asterisk, *, indicates a significant difference P≤0.05 between the groups. The time course to 21 days was preformed three times. Experiment at 68 days and 77 days was performed once.

Figure 2. Immune privilege in C57BL/6 or IL-6KO mice

Change (Δ) in ear thickness is indicated on the ordinate. Experimental treatment of the mice in each group is indicated under the abscissa (anterior chamber inoculation = a.c.; subcutaneous inoculation = s.c.; ear challenge = e.c.). Panel A. B6 mice. Panel B. IL-6KO mice. Briefly mice were treated with OVA 1 day after RLB treatment. One week later, mice were immunized with antigen and mice were challenged in the ear pinnae with OVA the following week. This experiment was performed twice with similar results.

Figure 3. Photomicrograph of retina sections stained with Neurokinin 1 receptor of mice that received RLB treatment or not

A. 3D Photomicrograph B. 3D cartoon representation. For figure 3A and B, green indicates NK1-R staining, and red indicates TOPRO 3 as a nuclear stain. Sections are from the eyes of either non-treated mice or mice that received RLB 6h, 72h, or 7 days prior to enucleation. All images are 400× magnification. The layers of the eye are indicated to the left of the photomicrograph. C. Green indicates NK1-R staining, and red indicates DAPI 3 as a nuclear stain. Cornea of naive or RLB treated mice 6h post-RLB. D. Quantification of NKR-1 expression in retina of RLB treated mice. Integrated Optical Density (IOD) is shown in fold increase for each group. All groups were repeated at least three times and where normalized to naïve (control) mice. Each IOD value from the individual groups was normalized to each of the controls and the fold increase calculated and plotted. Ipsilateral = black bars; Contralateral = white bars. The confocal analysis was performed three times.

Figure 4. DH response after ACAID induction with or without substance P antagonist in RLB treated mice

Change (Δ) in ear thickness is indicated on the ordinate (anterior chamber inoculation = a.c.; subcutaneous inoculation = s.c.). Experimental treatment of the mice in each group is indicated under the abscissa. A) Spantide I treatment over time. Mice were treated with OVA and substance P antagonist 1, 3, 5, or 7 days after RLB treatment. One week later, mice were immunized with antigen and mice were challenged in the ear pinnae with OVA the following week. Experiments on 1 and 7 days post RLB were performed a minimum of 3 times; induction of ACAID with Spantide I on 3 and 5 days post RLB was performed once. B. Spantide I treatment at different concentrations. Ipsilateral = black bars; Contralateral = white bars. Spantide I treatment 10⁻⁴ M= bar with check pattern, the dose curve was performed once. Spantide I treatment 10⁻³ M= bar with vertical line pattern, Spantide I treatment 10⁻² M= bar with diagonal line pattern. C) Spantide II treatment. Spantide II given with OVA a.c. 1 day post RLB. Figure shows combined results of two experiments. An asterisk, *, indicates a significant difference P≤0.05 between the groups.

Figure 5. DH response after ACAID induction in RLB treated B6 or substance P KO mice

Panel A. B6 mice. Panel B. Substance P KO mice. Change (Δ) in ear thickness is indicated on the ordinate. Experimental treatment of the mice in each group is indicated under the abscissa. Briefly, B6 or substance P KO mice were inoculated with OVA 1 day after RLB treatment. One week later, mice were immunized with antigen and mice were challenged in the ear pinnae with OVA the following week. Ipsilateral = black bars; Contralateral = white bars. An asterisk, *, indicates a significant difference P≤0.05 between the bars. The figure represents the combined results of two experiments.

Figure 6. DH response after ACAID induction in substance P treated mice

Change (Δ) in ear thickness is indicated on the ordinate. Experimental treatment of the mice in each group is indicated under the abscissa. Briefly, mice were treated with OVA and substance P peptide one week prior to being immunized with antigen. One week later mice were challenged in the ear pinnae with OVA. An asterisk, *, indicates a significant difference of P≤0.05 between the bars. The figure represents the combined results of two experiments.