Presence of Collagen IV in the Ciliary Zonules of the Human Eye : An Immunohistochemical Study by LM and TEM
University Hospital/University of Groningen, Department of Ophthalmology (LIL,RJvdW,JMMH), and University of Groningen, Department of Pathology and Laboratory Medicine, Section of Medical Biology (RJvdW,MJAvL), Groningen, The Netherlands
Correspondence to: Leonoor I. Los, University Hospital/University of Groningen, Dept.of Ophthalmology, PO Box 30,001, 9700 RB Groningen, The Netherlands. E-mail: l.i.los{at}ohk.azg.nl
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Summary |
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Key Words: collagen IV ciliary zonules immunohistochemistry
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Introduction |
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In unfixed anatomic specimens, the ciliary zonules are visible as a transparent structure between the lens and ciliary body. Anatomic and histological studies showed that fibers running from the ciliary body towards the lens form the main structural component of the zonules. The nature of these fibers has been elucidated by various techniques. Rotary shadowing showed their morphology to resemble that of beads on a string (Mayne et al. 1991; Ren et al. 1991
; Wallace et al. 1991
). It was shown that it is possible to increase the distance between individual beads by stretching of the zonules before fixation (Ren et al. 1991
). This aspect corresponds with the elasticity of the tissue in vivo.
Intrinsic components of the zonular fibers have been identified by biochemical and immunohistochemical (IHC) techniques. Fibrillar components include at least eight different microfibrillar glycoproteins (see review by Chan and Choi 1995), amongst others, fibrillin (Keene et al. 1991
), a component of the microfibrils and microfibril-associated glycoprotein (MAGP), located on the beaded structures (Gibson and Cleary 1987
; Henderson et al. 1996
). Interestingly, both chondroitin sulfate proteoglycans (CS PGs; Kielty et al. 1996
) and heparan sulfate proteoglycans (HS PGs; Inoue 1995
) may be associated with fibrillin, whereas the former may even be an essential structural component of the beads (Kielty et al. 1996
).
The zonular fibers are surrounded by a coating consisting of non-fibrillar components, including glycosaminoglycans (hyaluronan), proteoglycans (PGs) (Chan and Choi 1995; Chan et al. 1997
; Kielty et al. 1996
), laminin (Marshall et al. 1992
), and fibronectin (Goldfischer et al. 1985
). Chan et al. (1997)
demonstrated that hyaluronan and CS PGs probably form large aggregates, as they do in cartilage. The presence of this coating explains the behavior of the zonules as a barrier to macromolecules. The latter is reflected in early anatomic descriptions of (virtual) spaces enclosed by different zonular layers, i.e., Hannover's channel (Hannover 1845
), located between the anterior and posterior zonular layers, and Petit's channel (Petit 1728
), enclosed by the posterior zonules and anterior hyaloid lamina. Early anatomists visualized these virtual spaces by injecting substances into them, e.g., egg white (Berger 1887
) or air (Petit 1728
). Under pathological conditions in the living eye, virtual spaces may sometimes be observed when abnormal substances (pigment, proteins, or blood) have become lodged inside (Berger 1887
).
In the course of our postembedding TEM IHC studies of the human vitreous body and retina, we observed that the zonules were stained intensely by anti-collagen IV antibodies. Because this is a novel finding, we decided to explore it in more detail.
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Materials and Methods |
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Postembedding Procedure
Embedding in Technovit 8100
Specimens were fixed by immersion in 2% paraformaldehyde in 0.1 M phosphate buffer for 1 hr at 4C. After removal of small parts of the globes, specimens were fixed for an additional 4 hr in fresh fixative. Specimens were washed overnight in 6.8% sucrose in PBS, briefly in bidistilled water, dehydrated through acetones (30100%), and infiltrated with Technovit 8100 (T8100; Heraeus Kulzer, Wehrheim, Germany). After infiltration with T8100 A (without accelerator) at 4C, specimens were transferred to 20C for infiltration with T8100 A + B (with accelerator) and to 4C for polymerization.
Sections of 34 µm for LM evaluation were cut on a Jung microtome and stained with toluidine blue. Areas were selected for LM or TEM IHC. Blocks selected for TEM evaluation were trimmed and cut on a Sorvall ultramicrotome. Thin sections (100 nm) thus obtained were mounted on formvar-coated grids, subjected to the IHC procedures described below, contrasted with uranyl acetate in 25 cp methylcellulose, and evaluated in a Philips 201 TEM operated at 80 kV.
Postembedding Anti-collagen IV Labeling (LM)
Sections were pretreated with 0.1% trypsin (Gibco; Paisley, Scotland) in Tris-HCl, pH 7.8, containing 0.1% CaCl2 for 15 min at 37C, washed in PBS, incubated in 0.1 M citric acid, pH 3, for 45 min at 37C, washed in PBS, pH 7.4, incubated in PBS/1% BSA/2% rabbit serum for 30 min at RT, incubated in primary antibody (goat anti-human type IV collagen; SBA, Birmingham, AL) diluted 1:50 in PBS/1% BSA-c for 2 hr at 37C, washed in PBS, incubated in 0.1% H2O2/PBS to block endogenous peroxidase for 20 min under dark conditions, incubated in peroxidised secondary antibody [RAGPO (rabbit antigoat peroxidase); Dako, Glostrup, Denmark] 1:40 in PBS/1% BSA-c, washed in PBS, incubated in AEC solution (Sigma Chemical; St Louis, MO), washed in distilled water, stained with hematoxylin, washed in running tap water, and embedded in glycerin gelatin (Merck; Darmstadt, Germany).
Postembedding Anti-collagen IV Labeling (TEM)
Thin sections were pretreated with 0.1% trypsin (Gibco) in Tris-HCl, pH 7.8, containing 0.1% CaCl2 for 15 min at 37C, washed in PBS, incubated in 0.1 M citric acid, pH 3, for 45 min at 37C, washed in PBS, pH 7.4, incubated in PBS/0.15% glycine/5% BSA/5% rabbit serum for 30 min at 37C, incubated in primary antibody (goat anti-human type IV collagen; SBA), diluted 1:100 in PBS/1% BSA-c for 2 hr at 37C first and then overnight at RT, washed in PBS, incubated in rabbit anti-goat IgG conjugated to 6-nm gold 1:100 (Aurion, Wageningen, The Netherlands) for 60 min at RT, washed in PBS, fixed in 2% glutaraldehyde (TAAB Laboratories; Aldermaston, UK)/PBS for 2 min, washed in bidistilled water, incubated in silver enhancement solution (Aurion R-gent enhancer) for 5 min at RT, washed in bidistilled water, and counterstained. A similar procedure was performed with monoclonal mouse antihuman type IV collagen (Biogenesis; Poole, UK) 1:1000. In this case the secondary antibody was goat anti-mouse IgG conjugated to 6-nm gold 1:150 (Aurion).
Pre-embedding Anti-collagen IV Labeling (TEM)
Tissue blocks (7 x 5 x 2 mm) were washed in PBS for 1 hr at 4C, incubated in primary antibody in PBS [either polyclonal anti-collagen IV antibody (SBA) 1:10 or monoclonal anti-collagen IV antibody (Biogenesis) 1:50] at 4C (first series) or RT (second series) overnight, washed in PBS at 4C or RT, respectively, incubated in secondary antibody, i.e., rabbit anti-goat IgG conjugated to 6-nm gold 1:20 (polyclonal primary antibody) overnight, and goat anti-mouse IgG conjugated to 6-nm gold 1:20 (monoclonal primary antibody) overnight, washed in PBS, prefixed in 2% glutaraldehyde for 1 hr, washed in cacodylate buffer, washed in bidistilled water, incubated in silver enhancement solution (Aurion R-gent enhancer) for 10 min at RT, fixed in OsO4 (Serva; Heidelberg, Germany) for 0 min, 15 min, or 2 hr at 4C, washed in bidistilled water, dehydrated through ethanols (50100%), and embedded in Epon 812 (Serva) according to the standard procedure. Blocks selected for TEM evaluation were trimmed and cut on a Sorvall ultramicrotome. Thin sections (100 nm) thus obtained were mounted on copper grids, contrasted with uranyl acetate and lead citrate, and evaluated in a Philips 201 TEM operated at 60 kV.
Controls for all labeling procedures (pre-embedding and postembedding) underwent the entire procedure, except for the incubation with a primary antibody. In postembedding experiments, an irrelevant antibody (mouse anti-CD 68) was used as an additional control.
ELISA
We evaluated the specificity of the polyclonal goat antihuman type IV collagen antibody (SBA) by ELISA. Plates were coated overnight at RT with 100 µl of either fibronectin or collagen IV in 0.1 M carbonate buffer, pH 9.6. The following concentrations were used: fibronectin 25 µg/ml, 2.5 µg/ml, 0.25 µg/ml, and 0 µg/ml; collagen IV 2.5 µg/ml, 0.25 µg/ml, 0.025 µg/ml, and 0 µg/ml. Anti-type IV collagen was added to the wells in concentrations of 1:500; 1:2000; 1:8000, and 0. Wells were incubated for 1 hr at RT. Wells were incubated by RAGPO (Nordic Immunology Labs; Tilburg, the Netherlands). Secondary antibody was used at a dilution of 1:5000 for 1 hr at RT. Primary and secondary antibodies were diluted in buffer consisting of 0.05 M Tris-HCl, pH 8.0, 0.30 M NaCl, 0.05% Tween-20, and 1% BSA. Substrate (TMB: 3,3',5,5'-tetramethylbensidine HCl; Roth Brunschwig Chemie, Amsterdam, The Netherlands) was added and the reaction was stopped by the addition of 2 N H2O2 after 10 min. All washes were done with buffer consisting of 0.025 M Tris-HCl, 0.15 M NaCl, and 0.05% Tween-20. Wells were scanned by SOFTMAX at 490 nm.
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Results |
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Postembedding Immuno-LM
The zonular fibers stained with polyclonal anti-type IV collagen antibodies. Known basement membranes in the same section served as positive internal controls and were stained. They include the lens capsule, basement membranes of the non-pigmented ciliary epithelium, and blood vessel walls (Figure 1)
. No staining of any of these structures was observed with monoclonal anti-collagen IV antibodies. This suggests an overall lower staining signal with the latter method.
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ELISA
Because labeling intensity with the polyclonal antibody was much higher than with the monoclonal antibody, we decided to explore whether the polyclonal antibody is really specific for collagen IV, or whether crossreactivity with other known components of the zonules could have occurred. Information provided by the supplier shows that the polyclonal anti-type IV collagen antibody has been tested for crossreactivity with other types of collagen but not for crossreactivity with fibronectin, which is a known component of the non-fibrillar coating of the zonules (Goldfischer et al. 1985). Therefore, we tested the antibody for crossreactivity with fibronectin by ELISA. Collagen type IV-coated wells, incubated with anti-collagen type IV polyclonal antibody (SBA), showed a positive concentration-dependent signal. Fibronectin-coated wells showed a negative signal for all concentrations used (Figure 5)
. The results show no indication at all of any crossreactivity between the polyclonal anti-collagen type IV antibody and fibronectin, and confirm the specificity of the antibody.
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Discussion |
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The observed staining of the human zonules by anti-collagen IV appears to be a specific finding, whereas labeling intensity depends on the type of antibody (monoclonal vs polyclonal) and the embedding method (pre-embedding vs postembedding) used. Our observations indicate that collagen IV is part of the non-fibrillar coating associated with the ciliary zonules, which means that it may play a role in the macromolecular barrier function of the zonules and also in other proposed functions of this coating. The latter include (Chan et al. 1997) mediating interactions between parallel zonular fibers and organizing them into larger fiber bundles, protecting the zonules from overstretching by forming a network around them with its own rigidity and elasticity, and a protective function against enzymatic breakdown because of its high negative charge.
Collagen IV is typically found in basement membranes. The zonules contain additional components that have also been observed in basement membranes. Some of these components, including hyaluronan and CS PGs, are not normally found in basement membranes (Chan et al. 1997) but can be demonstrated in those basement membranes into which the zonules insert themselves (Chan and Choi 1995
). Hyaluronan and CS PGs were demonstrated in the basement membane of the non-pigmented ciliary epithelium (Chan and Choi 1995
). Hyaluronan, but not CS PGs, was also found in the lens capsule (Chan and Choi 1995
). Heparan sulfate PG is generally found in basement membranes and has been described as a component of zonular fibrils but not of the coating surrounding them (Chan and Choi 1995
). This suggests that the non-fibrillar components associated with the zonules represent an extracellular matrix with a unique composition that has some elements in common especially with those basement membranes to which it attaches itself, but which differs in many respects from basement membranes in general.
Because the zonules themselves do not contain any cells, it is probable that the associated collagen IV is produced in adjacent cells. Candidates would be the lens epithelial cells, the (embryonic) hyaloid cells, and/or the non-pigmented epithelial cells of the ciliary body. Experiments in embryonic mouse (Sarthy 1993) and chick eyes (Dong et al. 2002
) show that extracellular matrix components, such as components of the retinal basal lamina, can be produced at sites different from their final destination. After their production, these components are "shed," e.g., into the vitreous body, and finally assembled into an extracellular matrix elsewhere. In these embryonic eyes, the main sources of (retinal) collagen IV are the lens epithelial cells and possibly the hyaloid cells. In adult eyes, the non-pigmented epithelial cells of the ciliary body are also highly probable candidates for the production of zonular collagen IV, because these cells are capable of producing quite a number of zonule components of both fibrillar and non-fibrillar nature (Rhodes et al. 1982
; Ohnishi et al. 1983
; Bennett and Haddad 1986
; Hanssen et al. 2001
).
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Acknowledgments |
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We wish to thank the Cornea Bank (Amsterdam, The Netherlands) for providing us with human donor eyes, the technicians at the Laboratory for Cell Biology and Electron Microscopy for advice and assistance, and Johan Bijzet for help with the ELISA procedure.
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Footnotes |
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Literature Cited |
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