Immunization Against Oxidized Elastin Exacerbates Structural and Functional Damage in Mouse Model of Smoke-Induced Ocular Injury

Abstract

Purpose: Age-related macular degeneration (AMD) is the leading cause of blindness in Western populations. While an overactive complement system has been linked to pathogenesis, mechanisms contributing to its activation are largely unknown. In aged and AMD eyes, loss of the elastin layer (EL) of Bruch's membrane (BrM) has been reported. Elastin antibodies are elevated in patients with AMD, the pathogenic significance of which is unclear. Here we assess the role of elastin antibodies using a mouse model of smoke-induced ocular pathology (SIOP), which similarly demonstrates EL loss.

Methods: C57BL/6J mice were immunized with elastin or elastin peptide oxidatively modified by cigarette smoke (ox-elastin). Mice were then exposed to cigarette smoke or air for 6 months. Visual function was assessed by optokinetic response, retinal morphology by spectral-domain optical coherence tomography and electron microscopy, and complement activation and antibody deposition by Western blot.

Results: Ox-elastin IgG and IgM antibodies were elevated in ox-elastin immunized mice following 6 months of smoke, whereas elastin immunization had a smaller effect. Ox-elastin immunization exacerbated smoke-induced vision loss, with thicker BrM and more damaged retinal pigment epithelium (RPE) mitochondria compared with mice immunized with elastin or nonimmunized controls. These changes were correlated with increased levels of IgM, IgG2, IgG3, and complement activation products in RPE/choroid.

Conclusions: These data demonstrate that SIOP mice generate elastin-specific antibodies and that immunization with ox-elastin exacerbates ocular pathology. Elastin antibodies represented complement fixing isotypes that, together with the increased presence of complement activation seen in immunized mice, suggest that elastin antibodies exert pathogenic effects through mediating complement activation.

Figure 1.

Antibody production in response to immunization with elastin or oxidized elastin. ELISA analysis was performed, coating plates with elastin or oxidized elastin peptide. Serum at different concentrations (1:100 to neat) from animals immunized with elastin or oxidized elastin was used to probe for binding, which was visualized with corresponding anti-mouse IgG and IgM secondary antibodies. Values were background subtracted and averaged (n = 3). After immunization and smoke exposure, a significant immune response against ox-elastin could be detected, based on IgG and IgM binding, whereas the response against control elastin was more modest.

Figure 2.

Antibodies generated after immunization recognized specific elastin fragments. Elastin and ox-elastin peptides (15 µg/lane) were loaded per lane and visualized by silver stain or transferred to PVDF membranes and used for Wester blotting. Serum samples from control animals (no immunization, no smoke exposure) or serum samples from smoke-exposed animals immunized with control elastin or oxidized elastin were used. (A) Silver stain identified distinct fragments between 1 and 250 kDa, and Ponceau S staining confirmed comparable transfer of control elastin or oxidized elastin peptides. (B) When blots were probed with serum samples from animals and developed with secondary antibodies against mouse IgG, a distinct ∼72-kDa band was identified with serum samples from oxidized elastin immunized mice. (C) Blots were probed with serum samples from animals and developed with secondary antibodies against mouse IgM, which identified full-length elastin as well as a smear in the ∼72-kDa range.

Figure 3.

Immunization with oxidized elastin impairs contrast sensitivity. (A) Optomotor responses were analyzed in C57BL/6 mice over 6 months. Immunized elastin or oxidized elastin was exposed to 6 months of cigarette smoke. Contrast sensitivity was determined by measuring the contrast threshold at a fixed spatial frequency (0.131 cycles per degree) and speed (12 deg/sec) and expressed as threshold (percent contrast required for perception). We previously determined that this spatial frequency falls within the range of maximal contrast sensitivity for 9-month-old wild-type mice (data not shown). Smoke-exposed mice showed a significant reduction in contrast sensitivity compared with controls raised in room air, which was augmented in mice immunized with oxidized elastin (repeated-measures ANOVA: P = 0.01). (B) Contrast sensitivity of mice from panel A at 6 months was compared with nonimmunized room air–raised and nonimmunized smoke-exposed mice. Contrast sensitivity was affected by smoke exposure and immunization, with the effect of control elastin (P < 0.05) being less severe than that of oxidized elastin (P < 0.0001). Data are expressed as mean ± SEM (n = 5–9 per condition).

Figure 4.

Ultrastructural changes in mice following smoke exposure and elastin immunization. Electron micrographs of the RPE/BrM/choriocapillaris complex (RPE/BrM/CC) obtained from C57BL/6J mice exposed to 6 months of room air were compared with those exposed to 6 months of smoke in the absence (smoke – untreated) and presence of elastin immunization (smoke – elastin; smoke – ox-elastin). (A) In a control animal raised in room air, BrM exhibits an organized pentalaminar structure, consisting of RPE-BM (RPE basement membrane), ICL (inner collagenous layer), MEL (middle elastic layer), OCL (outer collagenous layer), and CC-BM (choriocapillaris basement membrane). (B) The RPE/BrM/CC in animals exposed to smoke exhibit pathologic changes, including a thickening of BrM, which becomes disorganized, losing its pentalaminar structure, at its thicker points. (C) The RPE/BrM/CC is similarly affected in mice immunized with control elastin when compared with mice that are smoke exposed but not immunized. (D) The RPE/BrM/CC in animals exposed to smoke exhibit and immunized with oxidized elastin exhibit more severe pathologic changes, including larger areas of BrM being disorganized, losing its pentalaminar structure and the middle elastic layer. Insets highlight some of the morphologic features of mitochondria with degraded outer membranes and disorganized cristae in mice exposed to smoke and oblong mitochondria, particularly in the ox-elastin group.

Figure 5.

Masks overlying the morphologic features to be analyzed. Electron micrographs described in Figure 4 were used for analysis. Adobe Photoshop was used to generate masks for BrM, basal infolding, and mitochondria for further analysis in ImageJ. Masks highlight the differences in RPE/BrM obtained from C57BL/6J mice exposed to 6 months of room air when compared with those exposed to 6 months of smoke in the absence (smoke – untreated) and presence of elastin immunization (smoke – elastin; smoke – ox-elastin).

Figure 6.

Morphologic alterations in Bruch's membrane thickness and RPE basal infoldings in response to smoke and elastin immunization. Summary of alterations in Bruch's membrane thickness and basal infoldings obtained from EM images described in Figure 4. (A) BrM thickness was determined using the masks outlined in Figure 5, and the percent BrM along a given RPE cell that is damaged (exceed the normal thickness of BrM in age-matched room air–exposed mice) is established. A smoke and immunization effect could be established. (B) Basal infoldings were measured for both the lamellar portion and the open space in between. However, no consistent differences were observable. Data are expressed as mean ± SEM (n = 4–6 retinas per condition, representing 8–12 cells).

Figure 7.

Morphologic alterations in mitochondria in response to smoke and elastin immunization. Summary of alterations in mitochondrial features obtained from EM images described in Figure 4. (A) Mitochondrial area (area of RPE cells occupied by mitochondria) was determined from electron micrographs, indicative of mitochondrial swelling and biogenesis. (B) Mitochondrial position was determined from electron micrographs by determining their centroid coordinates as a percentage of the corresponding RPE length and thickness, respectively. Each centroid was subsequently assigned to one of four bins (basal, apical, basolateral, and central). Based on our previous publication demonstrating that mitochondria exhibit an apical shift from the basal to central compartment in response to smoke, only the central bin is depicted here, demonstrating a smoke and immunization dependent shift of mitochondria. (C) Mitochondria shape was assessed in ImageJ, binning shapes from 0 to 1 into 10 bins, with 1 being a perfect circle. The normalized total mitochondrial distribution across the 10 bins demonstrates a shift to more oblong mitochondria in control elastin and oxidized elastin-immunized mice. Data are expressed as mean ± SEM (n = ∼450 mitochondria per condition).

Figure 8.

Analysis of complement products in response to smoke and elastin immunization. (A) Equal amounts of RPE/choroid extracts (15 µg/lane) were loaded per lane, probed for C3 (Comptech), and band intensities quantified. Arbitrary values were established based on normalization with β-actin. Age-matched animals exposed to room air were compared with those raised in smoke and immunized with control or oxidized elastin. (B) C3a levels were detectable in the majority of animals exposed to smoke and immunized with oxidized elastin. (C) C3d and (D) C3dg levels were elevated and showed additivity by smoke and immunogen. (E) C3b levels were also elevated, but additivity between smoke and immunogen could not be established due to a larger variation between samples in the smoke + ox-elastin group and the difficulty of quantifying the bands accurately (partial overlap of C3b and C3dg). Data are expressed as mean ± SEM (n = 2–6 independent samples per condition).

Figure 9.

Analysis of IgG and IgM binding in RPE/choroid in response to smoke and elastin immunization. (A) Equal amounts of RPE/choroid extracts (15 µg/lane) were loaded per lane, probed for mouse IgG (top blot) and IgM (middle blot), and band intensities quantified. Arbitrary values were established based on normalization with β-actin (bottom blot). Age-matched animals exposed to room air were compared with those raised in smoke and immunized with control or oxidized elastin. (B) IgG and (C) IgM levels were elevated and showed additivity by smoke and immunogen. Data are expressed as mean ± SEM (n = 2–3 independent samples per condition).

Figure 10.

Analysis of binding of IgG subtypes in RPE/choroid in response to smoke and elastin immunization. (A) Equal amounts of RPE/choroid extracts (15 µg/lane) were loaded per lane; probed for mouse IgG1, 2a, 2b, and 3; and band intensities quantified. Arbitrary values were established based on normalization with β-actin. For each set of gel images, the respective top blot represents the IgG subclass and the bottom blot the respective β-actin. Age-matched animals exposed to room air were compared with those raised in smoke and immunized with control or oxidized elastin. (B) IgG2a and IgG3 levels were elevated and showed additivity by smoke and immunogen, whereas IgG1 and IgG2b were elevated by smoke but were unaffected by the type of immunogen exposure. Data are expressed as mean ± SEM (n = 2–3 independent samples per condition).