Induced autoimmunity to heat shock proteins elicits glaucomatous loss of retinal ganglion cell neurons via activated T-cell-derived fas-ligand

Abstract

Glaucomatous optic neuropathy causes blindness through the degeneration of retinal ganglion cells (RGCs) and their axons, which comprise the optic nerve. Glaucoma traditionally is associated with elevated intraocular pressure, but often occurs or may progress with intraocular pressure in the normal range. Like other diseases of the CNS, a subset of glaucoma has been proposed to involve an autoimmune component to help explain the loss of RGCs in the absence of elevated intraocular pressure. One hypothesis involves heat shock proteins (HSPs), because increased serum levels of HSP autoantibodies are prominent in some glaucoma patients with normal pressures. In the first direct support of this hypothesis, we found that HSP27 and HSP60 immunization in the Lewis rat induced RGC degeneration and axon loss 1-4 months later in vivo in a pattern with similarities to human glaucoma, including topographic specificity of cell loss. Infiltration of increased numbers of T-cells in the retina occurred much earlier, 14-21 d after HSP immunization, and appeared to be transient. In vitro studies found that T-cells activated by HSP immunization induced RGC apoptosis via the release of the inflammatory cytokine FasL, whereas HSP immunization induced activation of microglia cells and upregulation of the FasL receptor in RGCs. In summary, our results suggest that RGC degeneration in glaucoma for selected individuals likely involves failed immunoregulation of the T-cell-RGC axis and is thus a disturbance of both proapoptotic and protective pathways.

Figure 1.

Apparent RGC loss in retinas of HSP-immunized rats. A, Sections of retina from control and HSP27- and HSP60-immunized animals demonstrating similarity of retinal layers: nerve fiber (NF), retinal ganglion cell (RGC), inner plexiform (IP), inner nuclear (IN), outer plexiform (OP), and outer nuclear (ON). Scale is the same for all three panels. B, Whole-mount preparations of retina from control (left), HSP27-immunized (middle), and HSP60-immunized (right) rats labeled with immunocytochemistry against the RGC-specific marker Brn3a. Plane of focus is the RGC layer. Scale is the same for all three panels. C, Quantification of RGC density near the neuroretinal rim in three animal groups. The difference between the mean cell counts of control and HSP-immunized groups was highly significant (p < 0.001). D, Mean peak RGC density plotted for individual retinas in our three groups of animals.

Figure 2.

RGC density measurements in the rat retina after immunostaining against Brn3a. A, An individual frame focused through the RGC layer from an actual montage from control retina used in counting. B, Low-magnification view of entire whole-mount montage of control retina showing orientation and location of the optic nerve head (ONH), superior vertical meridian and temporal horizontal meridian. C, False-color representation of the montage of the whole retina shown in B, with area centralis indicated (AC). White areas near AC correspond to region of highest RGC density on the color scale (cells/mm² × 1000). D, Density of Brn3a-labeled RGCs averaged across animals as a function of eccentricity in the temporal (left) and nasal (right) hemifields of the retina, measured at 0.1 mm intervals. E, Density of Brn3a-labeled RGCs from D pooled within 1 mm intervals as a function of eccentricity in temporal (left) and nasal (right) hemifields. For the temporal hemifield, t test comparison of the means at each interval indicated significance (p ≤ 0.01) for differences between control and HSP27 animals at each eccentricity except 3–4 mm (p = 0.85). The same comparison between control and HSP60 animals yielded significance at all eccentricities (p ≤ 0.02). For the nasal hemifield, t tests indicated significant differences between control and HSP27 retina only at 3–4 mm eccentricity (p < 0.01) and between control and HSP60 retina at 0–1 mm (p < 0.01) and 1–2 mm (p < 0.001).

Figure 3.

Axon density is reduced in optic nerve of HSP-immunized animals. A, High-magnification light micrograph of control nerve demonstrating typical distribution of RGC axons of a variety of diameters tightly packed into regular fascicles. B, Optic nerve of HSP27-immunized animal has modest decrease in axon density, distension of interfascicular regions (arrows), and appearance of multilaminar myelin sheaths for some axons (circles). C, Optic nerve from HSP60-immunized animal demonstrates severe axonal dropout, obvious gliosis within the interfascicular regions, axons with multilaminar myelin sheaths (circles), and the appearance of small, degenerated profiles (arrows). D, Quantification of axon density in three animal groups. The difference between the mean axon counts of control and HSP-immunized groups was highly significant (p < 0.001).

Figure 4.

Immunolabeling of retinal TCRαβ+ T-cells. The T-cells are marked with green circles in each retinal image (20×) for counting. A, Adjuvant, 21 d; B, HSP60, 21 d; C, adjuvant, 14 d; D, HSP27, 14 d. The images located at the right top corner of each panel represents the TCRαβ+ T-cell distribution patterns of entire retinas. Scale bar at lower left side of each panel, 100 μm.

Figure 5.

Effect of T-cell activation on RGC-5 cell survival. RGC-5 cells were cocultured with HSP activated T-cells for 24 h in serum-free medium. A, Formation of oligonucleosomal fragments was determined by 2% agarose gel electrophoresis. Lane 1: 100 bp DNA ladder as the size marker; Lane 2: HSP27 activated T-cells; Lane 3: HSP60 activated T-cells; Lane 4: IFA activated T-cells; Lane 5: BSA activated T-cells; Lane 6: RGC-5 cells alone; Lane 7: T-cells alone. B, Rate of RGC-5 cells apoptosis measured by FACS assessment of BRDU labeling as described in Materials and Methods in the presence of HSP60 activated T-cells. C, Rate of RGC-5 cells apoptosis measured by FACS assessment of BRDU labeling as described in Materials and Methods in the presence of HSP27 activated T-cells. D–F, Effect of conditioned medium obtained from HSP activated T-cells on apoptosis of RGC-5 cells. D, Formation of oligonucleosomal fragments was determined by 2% agarose gel electrophoresis. Lane 1: 100 bp DNA ladder as the size marker; Lane 2: Medium from HSP60 activated T-cells; Lane 3: Medium from HSP27 activated T-cells; Lane 4: Medium from IFA activated T-cells; Lane 5: Medium from BSA activated T-cells; Lane 6: Medium from RGC-5 cells alone; Lane 7: Medium from T-cells alone. E, Rate of RGC-5 cells apoptosis measured by FACS assessment of BRDU labeling as described in Materials and Methods in the presence of conditioned medium from HSP60 activated T-cells. F, Soluble FasL levels in the conditioned medium obtained from the activated T-cells, using ELISA. Similar results were obtained in three independent experiments.

Figure 6.

Effect of soluble FasL from HSP60 activated T-cells' conditioned medium on apoptosis of RGC-5 cells. A, Formation of oligonucleosomal fragments was determined by 2% agarose gel electrophoresis. Lane 1: 100 bp DNA ladder as the size marker; Lane 2–4: RGC-5 cells + medium from HSP60 activated T-cells before immunoprecipitation with FasL antibody; Lane 5: RGC-5 cells + medium alone. Lane 6–8: RGC-5 cells + medium from HSP60 activated T-cells after immunoprecipitation with FasL antibody; Lane 9: RGC-5 cells + medium from IFA activated T-cells; Lane 10: RGC-5 cells + medium from BSA activated T-cells; Lane 11–15: RGC-5 cells treated with IL-2 (50 ng/ml), IL-4 (10 ng/ml), IL-6 (10 ng/ml), TNF-α (50 ng/ml), TNF-β (50 ng/ml), respectively. Lane 16: RGC-5 cells treated with anti-FasL Ab (5 μg/ml) alone. B, Rate of RGC-5 cells apoptosis measured by FACS assessment of BRDU labeling as described in Materials and Methods in the presence of conditioned medium from HSP60 activated T-cells after immunoprecipitation with FasL antibody. Similar results were obtained in three independent experiments. C, Dose-dependent effect of recombinant human FasL on RGC-5 cells death. D, E, Expression of Fas in RGC-5 cells by Western blotting (D) and RT-PCR (E).

Figure 7.

Glial activation in the retina by HSP immunization. A, Expression of glial-acidic fibrillary protein (GFAP) in astrocytes of control (left panel) and HSP60-immunized (right panel) rats. Arrowheads in the left panel delineate capillaries outlined by GFAP-labeled astrocyte processes. Scale is the same for both panels. B, Retinal sections from control (left), HSP27-immunized (middle), and HSP60-immunized (right) animals immunostained for OX18 (green) and counter-labeled with the nuclear dye DAPI (blue). In control retina, OX18 labeling is localized discretely to microglia cell bodies (arrows) in the outer plexiform (OP) layer and to a lesser extent to microglia (bracket) in the inner nuclear layer (IN). In HSP27 retina, OX18 label is intensified in ramified microglial processes throughout the retina, most prominently in the outer nuclear (ON), inner plexiform (IP), and retinal ganglion cell (RGC) layers. Migrating microglial cells are identified by their elongated, elliptical cell bodies (arrowheads). In HSP60 retina, levels of OX18 have increased in individual microglia cells in the OP (arrows) and in the RGC and IP layers, where the microglia have become more ramified. Scale is the same for all three panels. C, Retinal sections from control (left), HSP27-immunized (middle), and HSP60-immunized (right) animals immunostained for FasR (red) and counter-labeled with the nuclear dye DAPI (blue). Label is minimal in control retina. In HSP27 retina, FasR is increased throughout the retina and is prominent in endothelial cells of blood vessels (BV) and in the large cell bodies of RGCs (arrowheads). In HSP60 retina, FasR label is less diffuse throughout the retina and is again prominent in RGC cell bodies (arrowheads). Scale is the same for all three panels.