Enhanced insight into the autoimmune component of glaucoma: IgG autoantibody accumulation and pro-inflammatory conditions in human glaucomatous retina.

BACKGROUND: There is accumulating evidence that autoimmune components, such as autoantibodies and autoantibody depositions, play a role in the pathogenesis of neurodegenerative diseases like Alzheimeŕs disease or Multiple Sclerosis. Due to alterations of autoantibody patterns in sera and aqueous humor, an autoimmune component is also assumed in the pathogenesis of glaucoma, a common reason for irreversible blindness worldwide. So far there has been no convincing evidence that autoantibodies are accumulated in the retina of glaucoma patients and that the local immune homeostasis might be affected. METHODS AND RESULTS: Six human glaucomatous donor eyes and nine samples from donors with no recorded ocular disease were included. Antibody microarrays were used to examine the patterns of pro-inflammatory proteins and complement proteins. Analysis of TNF-α and interleukin levels revealed a slight up-regulation exclusively in the glaucomatous group, while complement protein levels were not altered. IgG autoantibody accumulations and/or cellular components were determined by immunohistology (n = 4 per group). A significantly reduced number of retinal ganglion cells was found in the glaucomatous group (healthy: 104±7 nuclei/mm, glaucoma: 67±9 nuclei/mm; p = 0.0007). Cell loss was accompanied by strong retinal IgG autoantibody accumulations, which were at least twice as high as in healthy subjects (healthy: 5.0±0.5 IgG deposits/100 cells, glaucoma: 9.4±1.9 IgG deposits/100 cells; p = 0.004). CD27(+) cells and CD27(+)/IgG(+) plasma cells were observed in all glaucomatous subjects, but not in controls. CONCLUSION: This work provides serious evidence for the occurrence of IgG antibody deposition and plasma cells in human glaucomatous retina. Moreover, the results suggest that these IgG deposits occurred in a pro-inflammatory environment which seems to be maintained locally by immune-competent cells like microglia. Thereby, glaucoma features an immunological involvement comparable to other neurodegenerative diseases, but also shows a multifactorial pathomechanism, which diverges and might be linked to the specific nature of both eye and retina

Evidence for B- and T-cells in glaucomatous retina.

<p>After immunodetection with markers against CD27 (panel <b>A)</b> and CD3 (panel <b>B)</b>, we detected several CD27⁺ cells in the rgcl of 4/4 glaucomatous retina as well as few CD3⁺ cells in one glaucoma subject by brown DAB deposition (indicated by the black arrows, scale bars 50 µm). Positive detection (white arrows, scale bars 10 µm) could be provided by the immunofluorescence approach for both CD markers in the rightmost images, while additional CD20 immunostaining in retina was always negative. Panel <b>C</b> shows the analysis of co-localization of CD27⁺ and IgG. As indicated in the rightmost images, the co-localized pixels of the individual fluorescence images for CD27 and IgG (middle images) were highlighted in white and identify this cell type clearly as a plasma cell. Furthermore, panel <b>D</b> depicts the co-localization of an IgG deposition in close contact to iba1⁺ microglia. The interaction between microglia and IgG could be understood functionally in relation to an opsonization. DAPI (blue): nuclei. Scale bars in panel C is 10 µm and in D 50 µm.</p

Quantification of cell loss, IgG depositions and plasma cells in cross-sections of glaucomatous and healthy retina.

<p>After immunostaining for IgG accumulations and hematoxylin counterstaining, as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057557#pone-0057557-g002" target="_blank">figure 2</a>, calculation of remaining neuronal cells in the retinal ganglion cell-layer to the retinal length revealed a significant lowering in the glaucomatous group compared to controls (<b>A</b>), while the number of IgG deposits in relation to the number of remaining cells is significantly increased (<b>B</b>). Based on morphological features, plasma cells were only detectable in the glaucomatous group as indicated in <b>C</b>. * = p<0.05; ** = p<0.01.</p

IgG autoantibody accumulates in glaucomatous retina.

<p>The image in <b>A</b> representatively shows the retinal morphology of healthy control subjects, characterized by an intact retinal architecture, comparatively large numbers of cells in the retinal ganglion cell-layer (rgcl) and few IgG depositions. In the glaucomatous subjects, we examined IgG positive immunoreactions by brown DAB staining (indicated by arrows) and were able to distinguish three major types of IgG accumulations. In <b>B</b>, IgG depositions are fuzzy, scattered and seem to be located somatically, intra- and extracellularly in presumably neurons (type 1). As depicted in <b>C</b>, more intensely stained, compact and smaller IgG depositions (type 2) were located on fragmented nuclei of apparently apoptotic neurons. The third type of IgG deposition in <b>D</b> (arrowhead) was larger than the other depositions (arrow), usually round or oval and the nucleus size was increased nearly twofold compared to the majority of the remaining nuclei in the rgcl. These cells showed characteristics of plasma cells with eccentric and heterochromatic nuclei, enhanced size, cartwheel or clock face morphology and immunoglobulin presence, as indicated in <b>E</b> (100× magnification of the arrowhead marked cell in <b>D</b>). This cell type was investigated further with appropriate CD markers to elucidate their origin. The image shown in <b>F</b> represents our immunofluorescent visualization of IgG antibody accumulations (arrows, red fluorescence) and detailed multi-focal images were acquired subsequently confirmed by different anti-human IgG antibodies in the following images <b>G</b> and <b>H</b>. A homogenous, cloudy IgG accumulation (green fluorescence) is shown in <b>G</b>, whereas scattered punctuated IgG dots could be seen in <b>H</b>. Scale bars in <b>A–D, G and H</b> are 50 µm, in <b>E</b> 20 µm and in <b>F</b> 10 µm. In <b>F, G and H</b>, DAPI was used as nuclei dye (blue). Abbreviations: inl: inner nuclear layer, onl: outer nuclear layer.</p

Summary of immunostainings.

<p>Beside IgG accumulation, no detection of CD3, CD20 or CD27 positive cells were made in retina of healthy subjects. Glaucomatous retina revealed thereby evidences of CD27⁺ cells, CD27⁺/IgG⁺ plasma cells and few hints for T-cells in two of four glaucomatous samples.</p