Figure 1.
Levels of pro-inflammatory cytokines in glaucomatous retinal tissue.
Figure 1 depicts the altered concentrations of the pro-inflammatory cytokines TNF-α, IL-1ß, IL-6, IL-8 and IFN-γ in detail. The arrowheads indicate the relative intensity (U) on the y-axis of each individual subject as sample means and the black bar summarizes the group means. All investigated pro-inflammatory cytokines were up-regulated in the glaucomatous group (glaucoma) compared to the healthy subjects (ctrl). (In some box-plots, the arrowheads of sample means overlaid.).
Figure 2.
IgG autoantibody accumulates in glaucomatous retina.
The image in A 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, IgG depositions are fuzzy, scattered and seem to be located somatically, intra- and extracellularly in presumably neurons (type 1). As depicted in C, 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 D (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 E (100× magnification of the arrowhead marked cell in D). This cell type was investigated further with appropriate CD markers to elucidate their origin. The image shown in F 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 G and H. A homogenous, cloudy IgG accumulation (green fluorescence) is shown in G, whereas scattered punctuated IgG dots could be seen in H. Scale bars in A–D, G and H are 50 µm, in E 20 µm and in F 10 µm. In F, G and H, DAPI was used as nuclei dye (blue). Abbreviations: inl: inner nuclear layer, onl: outer nuclear layer.
Figure 3.
Quantification of cell loss, IgG depositions and plasma cells in cross-sections of glaucomatous and healthy retina.
After immunostaining for IgG accumulations and hematoxylin counterstaining, as shown in figure 2, 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 (A), while the number of IgG deposits in relation to the number of remaining cells is significantly increased (B). Based on morphological features, plasma cells were only detectable in the glaucomatous group as indicated in C. * = p<0.05; ** = p<0.01.
Figure 4.
Evidence for B- and T-cells in glaucomatous retina.
After immunodetection with markers against CD27 (panel A) and CD3 (panel 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 C 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 D 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.
Table 1.
Summary of immunostainings.