Expression and distribution of immunoglobulin G and its receptors in an immune privileged site: the eye

Abstract

It has recently been demonstrated that not only mature B lymphocytes, but also non-lymphoid cells, including cancer cells and neurons, express IgG. In the eye, an important immune privileged site, the presence of IgG has been ascribed to IgG entering the eye through breaches of the blood–ocular barrier. Here we demonstrate that the eye itself can produce IgG intrinsically. Applying immunohistochemistry, in situ hybridization, and RT-PCR, several intraocular structures were found to express proteins and mRNA transcripts of IgG heavy chains, light chains, V(D)J rearrangements, and enzymes required for V(D)J recombination. IgG receptors were also detected in the intraocular epithelium and endothelium. The extensive distribution of IgG and its receptors in intraocular structures indicates that locally produced IgG could play a significant role in maintaining the ocular microenvironment and protection of the eyes, and it might also be involved in the pathogenesis of age-related macular degeneration and some inflammatory diseases.

Fig. 1

Expression and distribution of IgG and its receptors in the normal human eye. a–h Positive immunostaining of Igγ (a, e), Igκ (c, g), and Igλ (d, h) antigens, as well as positive IGHG1 mRNA signals (b, f) are detected in the cytoplasm of the basal cells of the epithelial layer (arrow heads) and the cells of the endothelial monolayer (arrows) of the cornea. Weak positive signals are detectable in the tear film covering the outer surface of cornea. In e, f, g, h, the insert shows a higher magnification of the selected area. Scale bars, 10 µm (insert, 20µm). i–l Consecutive sections showing coexpression of Igγ (i), Igκ (k), Igλ (l) and IgG mRNA (j) in the ciliary body of the human eye. The same epithelial cell is indicated in consecutive sections by black arrow heads. Both ISH and IHC show positive staining in both layers of the ciliary epithelium, while only IHC and not ISH shows positive signals in the ciliary stroma (indicated by blue stars). Scale bars, 20 µm. m–p Consecutive sections of retina. Igγ, Igκ, Igλ, and IGHG1 mRNA are all detected in a number of ganglion cells (arrow heads) and in a few cells of the inner nuclear retinal layer (arrows). q FcRn was detected in the epithelium (arrow heads) and the microvascular endothelium (arrow) of ciliary body. r–t FcγRs expression in human eye. FcγRIII (CD16, r) and FcγRI (CD64, t) are extensively expressed in PE cells (inner layer, arrow heads), and FcγRII (CD32) is detectable in a limited number of cells in the ciliary stroma (s, arrows). Scale bars, 20 µm. NFL nerve fiber layer, GCL ganglion cell layer, IPL inner plexiform layer, INL inner nuclear layer, OPL outer plexiform layer

Fig. 2

IgG expression in the murine eye. a–c Positive IgG Fab a and IgG Fc c immunostaining and positive IGHG ISH signals b, distributed in the cytoplasm of corneal endothelial cells. Inserts show higher magnification. Scale bars, 20 µm (inserts, 20 µm). d–f Consecutive tissue sections showing IgG Fab d, IGHG mRNA e and IgG Fc f in the ciliary body of a C57 mouse. Positive IgG signals were found in the ciliary epithelial cells (black arrows). Scale bars, 20 µm. g–i Consecutive sections of the retina of an ICR mouse without melanin in its pigment epithelium. Positive IgG Fab g and Fc i immunoreactivity is detected in ganglion cells (arrows), the nerve fiber, and RPE (arrow heads). Positive IGHG mRNA signals are only detectable in ganglion cells and RPE cells. Serial sections show that IgG protein and mRNA colocalize in the cytoplasm of ganglion cells (see arrows for example). Scale bars, 20 µm. j–l Consecutive sections of the retina of a μMT mouse showing weak immunoreactivity in a few ganglion cells. Arrows indicate the same single ganglion cell in consecutive sections. Scale bars, 20 µm. NFL nerve fiber layer, GCL ganglion cell layer, IPL inner plexiform layer, INL inner nuclear layer, OPL outer plexiform layer, ONL outer nuclear layer, PCL photoreceptor cell layer, RPE retinal pigment epithelium

Fig. 3

RT-PCR amplification of IgG mRNA transcripts in the human eye. a IGHG1 (201 bp), VDJ_н (360–400 bp), Igκ (231 bp), and Igλ (223 bp) transcripts amplified from the cornea, ciliary body and retina of the normal human eye. AID transcripts are not detectable. No CD 19 band is identified. DEPC-treated water was used as a negative control. b RAG1 and RAG2 transcripts amplified from the normal human eye. R DNase treated RNA as template (negative control); C cDNA as template

Fig. 4

Results of sequence-blasting of IGHG1 mRNA transcripts amplified from human eye samples. The VDJ_н sequences of all three clones show high homology to the sequence of the V4-61 gene (mutation rate 2.7%)

Fig. 5

RT-PCR of murine eye samples. Both Igγ (482 bp) and Igκ (410 bp) transcripts are amplified from eye samples of ICR and µMT mice. No bands are observed in eye samples of SCID mice. Under identical conditions, the band intensity of µMT mouse appears much weaker than that of ICR mice. The band of ICR spleen sample shows the strongest intensity. In none of the eye samples CD 20 band is detected in eye samples. As a blank control, DEPC-treated water was used in place of RNA

Fig. 6

Results of sequence-blasting of three Igκ mRNA transcripts amplified from ICR mouse eye samples. The variable region of Igκ (VκJκ) of two clones showed 97.5% homology to that of bw20. VκJκ of the other clone showed high homology to that of cw9, with a mutation ratio of 2.16%