Copyright ©The Histochemical Society, Inc.

Presence of Collagen IV in the Ciliary Zonules of the Human Eye : An Immunohistochemical Study by LM and TEM

Leonoor I. Los, Roelofje J. van der Worp, Marja J.A. van Luyn and Johanna M.M. Hooymans

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


    Summary
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
The ciliary zonules of the eye are composed of fibrillar and non-fibrillar components. Fibrils provide tensile strength and elasticity, whereas non-fibrillar components serve as a coating surrounding the fibrils. This coating behaves as a barrier to macromolecules. The present light and transmission electron microscopic (LM and TEM) study identified collagen IV as a novel component of this coating. Collagen IV was demonstrated by pre-embedding and postembedding immunohistochemical (IHC) techniques using monoclonal and polyclonal antibodies. The specificity of the polyclonal anticollagen IV antibody was verified by ELISA. (J Histochem Cytochem 52:789–795, 2004)

Key Words: collagen IV • ciliary zonules • immunohistochemistry


    Introduction
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
THE CILIARY ZONULES of the eye form the suspensory ligament of the lens. They are anchored to the ciliary body on the one hand and to the lens capsule on the other. Their main function is to mediate changes in the shape and curvature of the lens. Stretching of the zonules induces flattening of the lens, and their relaxation enables the lens to return to its more spherical resting shape. These changes in lens shape are reflected in changes in lens power. The latter enables the eye to form a focused image of objects at various distances from the eye (accommodation).

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. 1991Go; Ren et al. 1991Go; Wallace et al. 1991Go). It was shown that it is possible to increase the distance between individual beads by stretching of the zonules before fixation (Ren et al. 1991Go). 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 1995Go), amongst others, fibrillin (Keene et al. 1991Go), a component of the microfibrils and microfibril-associated glycoprotein (MAGP), located on the beaded structures (Gibson and Cleary 1987Go; Henderson et al. 1996Go). Interestingly, both chondroitin sulfate proteoglycans (CS PGs; Kielty et al. 1996Go) and heparan sulfate proteoglycans (HS PGs; Inoue 1995Go) may be associated with fibrillin, whereas the former may even be an essential structural component of the beads (Kielty et al. 1996Go).

The zonular fibers are surrounded by a coating consisting of non-fibrillar components, including glycosaminoglycans (hyaluronan), proteoglycans (PGs) (Chan and Choi 1995Go; Chan et al. 1997Go; Kielty et al. 1996Go), laminin (Marshall et al. 1992Go), and fibronectin (Goldfischer et al. 1985Go). Chan et al. (1997)Go 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 1845Go), located between the anterior and posterior zonular layers, and Petit's channel (Petit 1728Go), 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 1887Go) or air (Petit 1728Go). 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 1887Go).

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.


    Materials and Methods
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
Immunohistochemistry
Human donor eyes (n=12, aged 12–77 years) were obtained from the Cornea Bank (Amsterdam, The Netherlands) after removal of the corneas for cornea transplantation. These 12 donor eyes were subjected to the following procedures: LM IHC (n=2; 12 and 56 years); TEM postembedding IHC (n=5; 21, 29, 64, 66, and 74 years), and TEM pre-embedding IHC (n=5; 46, 54, 73 and 77 (two donors) years).

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 (30–100%), 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 3–4 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 (50–100%), 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.


    Results
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
Observations described below apply to all donor eyes studied by a particular method.

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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Figure 1

LM. Human zonular fibers (donor, 12 years) stained with anti-collagen IV (labeled structures appear pink due to AEC staining; the pink rather than red aspect of AEC-positive structures is due to Technovit, the embedding medium). Ce, basement membrane of the ciliary epithelium; zo, zonules; bv, blood vessel walls. Bar = 50 µm.

 
Postembedding Immuno-TEM
The zonular fibers were intensely stained with polyclonal anti-type IV collagen antibodies. In addition, basement membranes in the same section, including the lens capsule, the basement membranes of the non-pigmented ciliary epithelium, and blood vessel walls were strongly positive (Figure 2) . Areas outside these structures, serving as internal negative controls, were negative. This supports the specificity of the staining. Again, the monoclonal anti-type IV collagen antibodies gave a much lower labeling intensity of both the zonules and the surrounding basement membranes (Figure 3) , confirming an overall lower staining signal.



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Figure 2

Postembedding TEM, polyclonal anti-collagen IV antibody (A–C). (A) Zonules (donor, 21 years); (B) vitreous body (vb), zonules (zo), and ciliary epithelium (ce) (donor, 64 years); (C) lens epithelium (le) and lens capsule (lc) (donor, 21 years). Note intense anti-collagen IV staining of zonular fibers (zo), lens capsule (lc), and basement membrane of the ciliary epithelium (ce). Bars = 1 µm.

 


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Figure 3

Postembedding TEM, monoclonal anti-collagen IV antibody (donor, 77 years). Zonules, detail. Note low staining intensity (compared with Figure 2) of the zonular fibers. Bar = 1 µm.

 
Pre-embedding immuno-TEM was undertaken to try to localize anti-collagen IV staining either to the zonular fibrils or to the coating material surrounding them. Staining intensity was somewhat lower compared with that in postembedding experiments with both the monoclonal and the polyclonal antitype IV collagen antibodies. In sections in which non-fibrillar material surrounding the zonular fibers could be identified, labeling was localized to this material and not to the fibers themselves (Figure 4) .



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Figure 4

Pre-embedding TEM [donor 77 (A) and 46 (B,C) years]. (A,B) polyclonal and (C) monoclonal anti-type IV collagen antibodies. (A) Overview; (B,C) details in which antibodies are preferentially located to the non-fibrillar material associated with the zonular fibers and not to the fibers themselves. Bars = 100 nm.

 
Control procedures in all pre-embedding and postembedding labeling experiments were negative (not shown).

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. 1985Go). 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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Figure 5

Using ELISA, specific binding of the anti-collagen IV antibody to collagen IV was demonstrated. No crossreactivity with fibronectin was found. (A) Plates coated with collagen IV (2.5 -> 0 µg/ml) and incubated with anti-collagen IV (1:500 -> 0) show a positive concentration-dependent signal. (B) Plates coated with fibronectin (25 -> 0 µg/ml) and incubated with anti-collagen IV (1:500 -> 0) show no signal for any of the concentrations used.

 

    Discussion
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
Previous studies by Reich et al. (1975)Go and Schmut et al. (1976)Go, who used disk electrophoresis to analyze bovine vitreous and zonules, found that collagen IV may be a component of the ciliary zonules. However, this observation was questioned later by Streeten (1982)Go, who suggested that zonular specimens could easily have been contaminated with basement membrane components of adjacent tissues, e.g., the ciliary body and the lens. Therefore, it was stated that a definitive answer to the question of whether collagen IV is a component of the zonules should be obtained by IHC. The present study addresses the above question using this technique and confirms the presence of collagen IV in the zonules.

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. 1997Go) 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. 1997Go) but can be demonstrated in those basement membranes into which the zonules insert themselves (Chan and Choi 1995Go). Hyaluronan and CS PGs were demonstrated in the basement membane of the non-pigmented ciliary epithelium (Chan and Choi 1995Go). Hyaluronan, but not CS PGs, was also found in the lens capsule (Chan and Choi 1995Go). 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 1995Go). 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 1993Go) and chick eyes (Dong et al. 2002Go) 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. 1982Go; Ohnishi et al. 1983Go; Bennett and Haddad 1986Go; Hanssen et al. 2001Go).


    Acknowledgments
 
Supported by the Rotterdamse Vereniging Blindenbelangen, Rotterdam, The Netherlands and by the Stichting OOG (Ondersteuning Oogheelkunde's Gravenhage), The Hague, The Netherlands.

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.


    Footnotes
 
Received for publication January 28, 2004;
    Literature Cited
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 

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