Interaction with Collagen IV Protects Lens Epithelial Cells from Fas-Dependent Apoptosis by Stimulating the Production of Soluble Survival Factors

Abstract

purpose. To investigate the sensitivity of lens epithelial cells (LECs) to Fas-dependent apoptosis and to determine the role of interaction with the extracellular matrix (ECM) in the regulation of Fas-dependent apoptosis.

methods. Sensitivity to Fas-mediated apoptosis of primary human LECs and HLE-B3 LECs cultured on different substrates was determined by Hoechst staining after incubation with Fas-stimulating or control IgM. Fas expression was determined by Western blot analysis. Effects of varied cell density and conditioned media from HLE-B3 cells cultured on different substrates on the Fas sensitivity of HLE-B3 cells cultured on tissue culture (TC) plastic were determined.

results. Primary LECs cultured as free-floating anterior capsulotomy specimens were resistant to Fas-dependent apoptosis. The LE cell line, HLE-B3, was sensitive to Fas-dependent apoptosis when cultured on TC plastic but not on lens capsule. Culture on collagen IV, but not on laminin, rendered HLE-B3 cells resistant to Fas-dependent apoptosis, although Fas was still expressed. Primary LECs cultured on TC plastic after migration from lens capsule explants were resistant to Fas-dependent apoptosis if the lens capsule and attached cells were still present, but not if the lens capsule had been removed. Conditioned medium from LECs cultured on collagen IV, but not TC plastic, protected cells cultured on TC plastic from Fas-dependent apoptosis. The protective effect of culture on collagen IV diminished with decreasing cell density.

conclusions. LECs are protected from Fas-dependent apoptosis by interaction with collagen IV. Soluble factors released by the LECs cultured on collagen IV protect LECs from Fas-dependent apoptosis.

The objective of this study was to investigate the sensitivity of LECs to Fas-dependent apoptosis, to develop novel therapeutic strategies designed to induce apoptosis in LECs and prevent posterior capsular opacification (PCO). PCO is the leading cause of visual loss after cataract surgery and results from a postoperative scarring reaction mediated exclusively by LECs. ¹ In modern cataract surgery, the opacified natural lens is removed from within its capsule, leaving the capsular bag intact to receive a synthetic intraocular lens (IOL). The capsular tissues are clear immediately after surgery, but later contract and opacify as the previously quiescent LECs undergo fibroblastic transformation, proliferate, and migrate over the posterior capsule. New IOL materials and designs have reduced but not eliminated PCO. ^{2 3} A reliable method of inducing apoptosis in LECs would prevent PCO. Modulation of the wound-healing response to cataract surgery so as to preserve normal lens capsular tissue compliance should also enhance the efficacy of flexible IOLs designed to reverse presbyopia. 

A variety of pharmacologic approaches to LEC knockout based on cytotoxic drug delivery have been explored, including intraoperative injection of ricin-conjugated monoclonal antibodies directed against LEC epitopes ⁴ and postoperative diffusion of thapsigargin from polymethylmethacrylate lens surfaces. ⁵ An inescapable problem for any fluid phase cytotoxic treatment is the potential for collateral damage caused by the drug fraction not delivered to target cells. A vacuum-ring–based system currently in development, which is designed to isolate the intracapsular compartment for irrigation with cytotoxic drugs during surgery, may be viable and safe, ⁶ but the potential for device failure and damage to nontarget cells would remain. Several IOL materials can be surface modified by covalent attachment of Fas ligand via polyethylene glycol spacer chains (Futter C, et al. IOVS 2002;43:ARVO E-Abstract 433). Spacer chains act to preserve biological activity, whereas covalent attachment prevents diffusion of ligand away from the IOL’s surface. IOLs implanted within the capsular bag are directly apposed to the LECs but not to other ocular cells. If LECs are Fas sensitive, they should be killed with little risk of toxicity to nontarget ocular cells. To explore the viability of this approach, we investigated the sensitivity of LECs to apoptosis induced by soluble anti-Fas antibody. 

Apoptosis or programmed cell death can be initiated by many stimuli, including growth factor withdrawal, UV or γ-irradiation, chemotherapeutic drugs, and cell surface “death” receptors of the TNF receptor superfamily. Fas is the best characterized of this family of receptors and, when activated by Fas ligand, induces apoptosis in susceptible cells. ⁷ The use of soluble Fas-ligand in the eye is attractive, as naturally occurring Fas-ligand is present in the aqueous humor of healthy eyes, and membrane-bound Fas ligand is constitutively expressed in uveal tissue. ⁸ Susceptibility to Fas-induced apoptosis is regulated by multiple factors, including interaction with the extracellular matrix (ECM) and the presence of cytokines and survival factors. These factors influence the response to Fas ligation by modulating the relative expression levels of pro- and antiapoptotic genes in the FLICE-inhibitory protein (FLIP), ⁹ BCl-2, ¹⁰ and inhibitors of apoptosis (IAP) gene families. ¹¹ LECs, which are isolated from any contiguous contact with other cell types within the lens capsule in a serum free environment, are dependent on soluble secreted factors from other LECs to survive. ¹² Many cell types are also dependent on ECM attachment for their continued survival ^{13 14} and in some cases apoptosis induced by detachment from the ECM has been shown to be Fas dependent. ¹⁵  

Several studies show that both primary LECs and LE cell lines express Fas and suggest that they are sensitive to apoptosis induced by Fas-activating antibody (Crowston JG, et al. IOVS 2001;43:ARVO Abstract 4734). ^{16 17 18} In this study of the regulation of Fas-dependent apoptosis in LECs, we determined the role of ECM in regulating survival factor production and the role of survival factors in regulating Fas sensitivity. 

Methods

Ethical Review

Ethical approval for the experimentation was granted by the Moorfields Eye Hospital Research Ethical Committee. Capsulotomy specimens for primary LEC culture were obtained after informed consent was obtained in line with the Declaration of Helsinki. 

Determination of LEC Sensitivity to Fas Ligation

LEC sensitivity to Fas ligation was measured in a standardized protocol. HLE-B3 cells ¹⁹ or anterior capsulotomy specimens were transferred to serum-free minimum essential medium (MEM) and incubated for 48 hours at 37°C in 5% CO₂ with 500 ng/mL of either control or Fas-stimulating IgM (CH11; Upstate Biotechnology, Lake Placid, NY). Lens capsule specimens were fixed with 1% glutaraldehyde for 30 minutes at room temperature, washed with PBS, and incubated with 10 μm Hoechst 33342 for 1 minute before they were flatmounted and examined using a fluorescence microscope (DM1L; Leica, Wetzlar, Germany). For cells cultured in TC wells, Hoechst was added directly to the wells, and the cells were examined in situ. Condensed brightly staining aggregations of nuclear material mark apoptotic cells out clearly from the larger, less densely stained nuclei of living cells after Hoechst staining (Fig. 1) . Sensitivity to Fas ligation was quantified by recording percentage counts of apoptotic nuclei in random fields at ×20 magnification, counting >300 cells/specimen. Statistical significance of the results was evaluated by unpaired Student’s t-test. 

To obtain evidence of the activation of members of the caspase family of proteases cleavage of the caspase substrate, poly(ADP-ribose) polymerase (PARP) was measured. After incubation with control or Fas-stimulating antibody, as described earlier, HLE-B3 cells were solubilized in nonreducing sample buffer, subjected to SDS-PAGE, and Western blotted with rabbit anti-PARP antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Bands were detected using horseradish-peroxidase (HRP)–conjugated secondary antibodies (DakoCytomation, High Wycombe, UK) and chemiluminescence reagent (ECL; Pierce Biotechnology, Rockford, IL). Bands were visualized with image-analysis software (Intelligent Dark Box II; Fugi, Tokyo, Japan; and Image Reader LAS-1000 software; Fuji). Semiquantitative analysis was performed with NIH Image (available by ftp at zippy.nimh.nih.gov/ or at http://rsb.info.nih.gov/nih-image; developed by Wayne Rasband, National Institutes of Health, Bethesda, MD). 

Fas Sensitivity in Primary LECs and an LEC Line on Various Adhesion Substrates

Apoptotic responses to CH11 and control IgM were compared by the protocol used in primary LECs cultured as free-floating lens capsule specimens. Anterior capsule specimens were collected as waste tissue after capsulorrhexis in routine cataract surgery. Donors had age-related cataracts but no significant copathology. Each specimen was divided in half at the time of collection for immediate immersion in either test or control culture medium. 

The role of lens capsular extracellular matrix in regulating sensitivity to apoptosis was then investigated by culturing the human LEC line HLE-B3 on TC plastic, porcine lens capsule, and laminin- or collagen IV-coated TC plastic. Porcine eyes (Fresh Tissue Supplies, Ltd., West Sussex, UK) were washed thoroughly and transferred into an iodine solution for 5 minutes. From each eye, the posterior chamber was removed and the lens excised with surgical scissors under a dissection microscope. The anterior capsule was cut circumferentially along the lens equator, and TC inserts (6 mm diameter; Transwell-COL; Costar, Cambridge, MA) were preprepared by removing the original membrane and coating the bottom edge with cyanoacrylate glue. The support was then gently glued onto the central anterior lens capsule. After a complete and tight seal was established between lens capsule and the TC insert support, the remainder of the lens was gently teased away from the capsule. The inserts were then washed thoroughly with PBS in the presence of 0.5 mg/mL streptomycin and 500 U/mL penicillin (Sigma-Aldrich, Poole, UK) and left for 2 to 4 days in a sterile environment at 37°C, to ensure that capsules were not contaminated. Before use, the capsule inserts were washed thoroughly with serum-free MEM. Wells of 24-well TC plates were coated by incubation at 4°C for 15 hours with 50 μg/mL of mouse collagen IV (BD Biosciences, Oxford, UK) in 0.25% acetic acid or laminin (Chemicon, Harrow, UK) in PBS. Unmodified TC wells, porcine lens capsule preparations and coated wells were washed with serum-free MEM, seeded with HLE-B3 cells at 2 × 10⁴ cells/well, and cultured for 3 to 4 days with 20% FCS before treatment with antibody. 

Fas Expression in HLE-B3 Cells Cultured on Different Substrates

To determine whether relative protection from Fas-stimulated apoptosis on lens capsule and collagen IV-coated TC plastic might be mediated by changes in Fas expression, lens capsule specimens and HLE-B3 cells cultured on TC plastic or collagen IV were solubilized in reducing sample buffer, subjected to SDS-PAGE, and Western blotted with goat anti-Fas antibody (Santa Cruz Biotechnology) and mouse anti-tubulin antibody (Zymed, Cambridge, UK) as a loading control. 

Fas Sensitivity of Primary LECs in Explant Cultures

Anterior capsulotomy specimens were pinned flat in 12-well plates and cultured for 3 to 4 weeks in MEM containing 20% FCS at 37°C in 5% CO₂. During this period, LECs proliferated and migrated from the capsule to form a pericapsular halo. In some specimens, capsules were removed by removing the pins and the lens capsule with attached LECs, and all specimens were cultured in serum-free medium for 48 hours before addition of control or anti-Fas antibody, as described earlier. Apoptosis was determined, after 48 hours’ incubation, by Hoechst staining as described earlier. 

Effect of Conditioned Medium Transferred from HLE-B3 Cultures on Unmodified and Collagen IV-Coated TC Plastic

To determine whether secreted soluble factors might protect LECs from Fas-dependent apoptosis, conditioned medium was collected from HLE-B3 cells after 48 hours’ culture in serum-free conditions on either unmodified or collagen IV-coated TC plastic. After low-speed centrifugation to remove any cells in suspension, media samples were subjected to high-speed centrifugation (100,000g for 30 minutes at 4°C) to remove debris. Separate HLE-B3 cell cultures on either unmodified or collagen IV-coated TC plastic were then treated with test (from HLE-B3 cells on collagen IV) or control (from HLE-B3 cells on unmodified TC plastic) conditioned media and cultured for 48 hours with either Fas-stimulating or control IgM, with determination of Fas sensitivity as described earlier. 

Effect of Cell Density on Protection from Fas-Stimulated Apoptosis

Reasoning that cell density in culture should not affect protection mediated directly by the adhesion substrate, but would, by relative dilution in less-dense cultures, modulate protection by secreted soluble factors, we examined the effect of seeding density on Fas sensitivity. HLE-B3 cells were seeded at high (2 × 10⁴ cells/well), medium (6.7 × 10³ cells/well), or low (2.2 × 10³ cells/well) density on unmodified or collagen-coated TC plastic, and sensitivity to Fas-stimulated apoptosis was determined after 48 hours’ serum-free culture with either Fas-stimulating or control IgM as described earlier. 

Results

Sensitivity of Primary Human LECs in Their Natural Adhesion Environment to Fas-Stimulated Apoptosis

In serum-free medium Fas-stimulating antibody (CH11) did not induce significant amounts of apoptosis in primary LECs, compared with control IgM (Fig. 1) , with <15% of cells apoptotic after treatment with either control or Fas-stimulating antibody after 48 hours (Fig. 2) . 

Sensitivity of the Human LEC Line, HLE-B3, on TC Plastic to Fas-Stimulated Apoptosis

After serum-free culture of the human LE cell line, HLE-B3, on TC plastic in the presence of anti-Fas antibody, 55% of nuclei were apoptotic versus 22% with control IgM (Figs. 1 2) . Apoptosis was confirmed by demonstrating that incubation with anti-Fas antibody caused increased cleavage of PARP, compared with incubation with control IgM (Fig. 1) . PARP is a 112-kDa substrate that, on caspase activation, is cleaved to an 85-kDa form. Semiquantitative analysis showed that the intensity of the 85-kDa cleaved form of PARP was increased approximately sixfold after incubation with anti-Fas antibody, compared with the IgM-treated control. The apoptosis that occurred in the presence of control IgM was due to the effects of serum withdrawal, rather than the presence of the IgM. Similar levels of apoptosis were observed in the presence or absence of control IgM in serum-free medium (results not shown). Because of the high level of apoptosis due to serum withdrawal, we determined whether Fas-dependent apoptosis could be measured in the presence of serum. Although the presence of only 2% serum reduced the level of apoptosis in the presence of IgM from 22% to 10% it also reduced the levels of apoptosis in the presence of Fas-activating antibody to a level that was not significantly different from that in control cells (Fig. 2) . It was necessary, therefore, to perform all the subsequent experiments in the absence of serum. Although a significant amount of apoptosis was induced by serum withdrawal, there remained an additional highly significant degree of apoptosis induced by Fas-stimulating antibody. 

Effect of Adhesion to Lens Capsule and Collagen IV and Laminin on Fas-Stimulated Apoptosis

Similar levels (25%) of apoptosis were observed in the presence of control IgM in HLE-B3 cells cultured on porcine lens capsule as that observed in cells cultured on TC plastic. However, culture on porcine lens capsule appeared to protect from Fas-stimulated apoptosis, so that there was no significant difference in levels of apoptosis between control and anti-Fas-treated cells (Fig. 2) . 

Culture on collagen IV, but not on laminin, reduced Fas-stimulated apoptosis to levels similar to that observed after culture on lens capsule but had no significant effect on levels of apoptosis in the presence of control IgM (Fig. 3) . 

Expression of Fas after Culture on Collagen IV

HLE-B3 cells still expressed Fas after culture on collagen IV or laminin for 5 days (Fig. 4) . Semiquantitative analysis of Western blot analysis indicated that Fas expression was reduced by culture on collagen IV (by up to 50%) or laminin (∼25%), compared with culture on untreated TC plastic. 

Effect of the Presence of the Original Explant on Fas Sensitivity of Primary Human LECs in Explant Culture after Migration onto TC Plastic

Anterior capsulotomy specimens were pinned flat in culture plates, and the LECs were allowed to proliferate and migrate from the explant to form a pericapsular halo. Fas-stimulating antibody did not induce significant amounts of apoptosis in the pericapsular halo compared with control IgM, unless the explant was removed before addition of antibody. Under these conditions levels of apoptosis were significantly greater in the presence of Fas-stimulating antibody than with control IgM (Fig. 5) . 

Transfer of Relative Protection from Fas-Stimulated Apoptosis to HLE-B3 Cells on Unmodified TC Plastic by Conditioned Medium Derived from HLE-B3 Cells Cultured on Collagen IV

Conditioned medium from cells cultured on collagen IV provided relative protection against Fas-dependent apoptosis in cells cultured on TC plastic (Fig. 6) . A slightly higher level of protection was observed in cells cultured directly on collagen IV (Fig. 6) . A small, but significant, reduction in Fas-dependent apoptosis was also observed in conditioned media derived from HLE-B3 cells cultured on TC plastic (Fig. 6) . These results suggest that adhesion to collagen IV stimulates production of one or more soluble secreted factors that provide protection from Fas-stimulated apoptosis. 

Correlation of Reduced Cell Density with Reduced Protection from Fas-Mediated Apoptosis for HLE-B3 Cells Grown on Collagen IV

As expected, at high density, HLE-B3 cells were protected from Fas-dependent apoptosis by culture on collagen IV, whereas HLE-B3 cells cultured at the same density on TC plastic were highly Fas sensitive (Fig. 7) . Protection from Fas-dependent apoptosis was considerably reduced when HLE-B3 cells were cultured on collagen IV at low density (Fig. 7) , suggesting that protective factors released by HLE-B3 cells cultured on collagen IV were insufficiently concentrated in low-density cultures to confer protection against Fas-dependent apoptosis. Cells cultured at medium density exhibited Fas sensitivity when cultured on collagen IV intermediate between that shown at high and low density. No equivalent increase in apoptosis rates were observed with reduced seeding density in LECs on untreated TC plastic. 

Discussion

These results demonstrate that LECs express Fas, but do not undergo apoptosis in response to Fas ligation when adherent to lens capsule. Adhesion to collagen IV but not laminin produced relative protection from Fas-stimulated apoptosis similar to that observed for adhesion to lens capsule. This effect did not appear to be mediated by changes in Fas expression. Transfer of protection via conditioned media and the weakened protection observed in cells seeded at low densities suggest that cells adherent to collagen IV secrete one or more soluble survival factors that provide relative protection from Fas-stimulated apoptosis. 

Our finding that primary human LECs cultured as free-floating anterior capsulotomy specimens were not sensitive to Fas-stimulated apoptosis differs from the previously published work of Nishi et al., ¹⁸ who found that apoptosis is induced in 80% of primary LECs after culture for 24 hours in the presence of Fas-activating antibody. We observed no significant apoptotic effect at either 24 or 48 hours. This difference occurred despite the use of multiple batches of the same Fas-stimulating antibody (CH11) as that used by Nishi et al. which induced apoptosis in >95% of Jurkatt T cells within 24 hours of treatment in control testing (data not shown). The difference also cannot be explained by the presence of serum in the studies of Nishi et al., versus the serum-free conditions used in the present study. Our demonstration that serum protects HLE-B3 cells from Fas-dependent apoptosis suggests that serum contains survival factors and would therefore be expected to protect against rather than promote apoptosis. Indeed, in pilot experiments we could not demonstrate significant Fas-dependent apoptosis on anterior capsulotomy specimens in either the presence or absence of serum. Hueber et al., ¹⁷ found that both Fas ligand and anti-Fas antibody could induce apoptosis in primary porcine LECs, but in that study the LECs were not adherent to the lens capsule. 

Capsule-mediated protection does not appear to be restricted to Fas. Tholozan et al. (IOVS 2002;43:ARVO E-Abstract 4649) have recently noted that LECs adherent to the lens capsule are highly resistant to staurosporine-induced apoptosis. This protection appears to be cell specific, as lens capsule did not protect other non-LEC cell lines. 

Relative sensitivity of HLE-B3 cells to Fas-stimulated apoptosis has been reported. ¹⁶ The sensitivity of primary LECs and HLE-B3 cells was not complete under any conditions and never approached the >95% levels observed in Jurkat T cells in our experimentation. Despite variation of both culture duration and the dose of Fas-stimulating IgM in pilot studies, we never achieved >60% apoptosis in HLE-B3 cells adherent to TC plastic. This result suggests that we must understand the regulation of downstream modulators of Fas sensitivity better before death surfaces (lens or capsule ring surfaces modified with polyethylene glycol–tethered Fas ligand) will offer any scope for clinical application in either prevention of visual loss or preservation of capsular tissue compliance after cataract surgery. 

In common with other groups, we have found primary culture on substrates other than lens capsule unreliable for human LECs. Cultures were relatively slow to grow, exhibited considerable interspecimen variation, and were difficult to passage successfully. We, therefore, used the HLE-B3 LE cell line to investigate the role of extracellular matrix in the regulation of Fas sensitivity in more detail. We have demonstrated that HLE-B3 cells cultured on lens capsule mimic the Fas resistance of primary LECs in contact with lens capsule and that primary LECs in explant culture become at least partially Fas-sensitive in the absence of the donor capsule and attached cells. Taken together, these data suggest that HLE-B3 cells mimic the responses of primary human LECs to Fas stimulation with reasonable accuracy. However, HLE-B3 cells exhibited a greater apoptotic response to serum deprivation than primary human LECs, even on lens capsules, suggesting the presence of protective mechanisms for primary human LECs on capsules which were not reproduced in this model. 

As with other basement membranes, the major components of lens capsule are laminin, collagen IV, entactin-1/nidogen 1, and heparan sulfate proteoglycans. ²⁰ Our surface modification studies were restricted to laminin and collagen IV, and so we cannot exclude the possibility that capsular adhesion signals other than those present on collagen IV may be important regulators of apoptosis; however,the finding of protection levels for collagen IV similar to those observed for HLE-B3 cells cultured on lens capsules, and the absence of any protective effect for HLE-B3 cells cultured on laminin, suggests that signaling by collagen IV-binding integrins (e.g., α1β1, α2β1, α3β1, α5β3) may play a role in the regulation of Fas sensitivity in LECs. 

The observation that primary LECs on TC plastic were protected from Fas-dependent apoptosis by the presence of neighboring LECs adherent to the lens capsule provided the first indication that LECs cultured on lens capsule may produce soluble factors protecting against Fas-dependent apoptosis. The protective effect of conditioned media from HLE-B3 cells cultured on collagen IV when transferred to HLE-B3 cells cultured on TC plastic confirms this hypothesis. 

The protective effect of transferred conditioned media was not as great as that observed for HLE-B3 cells cultured directly on collagen IV. This result could be because soluble survival factors produced by cells cultured on collagen IV are labile. Alternatively, more than one mechanism could operate to protect LECs cultured on collagen IV from Fas-dependent apoptosis. In addition to alterations in cytokine expression, integrin signaling may influence proapoptotic and antiapoptotic gene expression directly. For endothelial cells, Fas-dependent apoptosis induced by detachment from Descemet’s membrane is mediated partly through upregulation of Fas. ¹⁵ There was some reduction in Fas expression after culture of HLE-B3 cells on collagen IV, which could contribute to protection against Fas-dependent apoptosis, but the observation that seeding at low cell densities tends to negate the protective effect of collagen IV on Fas sensitivity would suggest that cytokine dependent survival signaling predominates. Cytokine-independent mechanisms would not normally be affected by cell density, whereas cytokine dilution in conditions of reduced cell density would be expected to downregulate any cytokine-dependent effect. 

LECs are known to secrete soluble factors necessary for their own survival. Culture of primary rat LECs at high density on TC plastic allowed their survival for many weeks in serum-free medium, and conditioned medium from high-density LECs promoted the survival of LECs seeded at low density. ¹² We observed that conditioned medium from LECs cultured on collagen IV had no effect on survival of LECs in serum-free medium (in the absence of anti-Fas antibody), suggesting that the survival factors produced by LECs cultured on collagen IV may be different from those whose presence was identified by Ishizaki et al. ¹²  

We have not identified which survival factor(s) protect LECs from Fas-dependent apoptosis. Several growth factors/cytokines have been shown to modulate LEC proliferation and/or survival, including epidermal growth factor (EGF), ²¹ FGF, ²² platelet-derived growth factor (PDGF), ²³ transferrin, ²⁴ hepatocyte growth factor (HGF), ²⁵ and lens epithelium–derived growth factor (LEDGF), ²⁶ but their effect on Fas-dependent apoptosis and the effect of culture on collagen IV on their production remain to be determined. 

We have demonstrated that primary human LECs attached to lens capsule are resistant to Fas-stimulated apoptosis. This resistance remains for the halo of LECs growing out onto TC plastic from pinned human lens capsule specimens, but is lost if the capsule specimens are removed. In this study of LECs we linked the ECM with the production of soluble survival factors and showed that interaction of LECs with collagen IV promotes the production of soluble survival factors which protect LECs from Fas-dependent apoptosis. Visual degradation after cataract surgery results from PCO and the loss of natural accommodation. The scarring response to cataract surgery responsible for both these adverse consequences is mediated exclusively by LECs. A greater understanding of the regulation of LEC Fas sensitivity and further elucidation of the mechanism of collagen IV-mediated protection could hold the key to a new dimension of pseudophakic visual performance. 

 Supported by the Special Trustees of Moorfields Eye Hospital, Fight for Sight, and the Wellcome Trust.

 Submitted for publication January 25, 2005; revised April 13, 2005; accepted April 25, 2005.

 Disclosure: C.E. Futter, None; J.G. Crowston, None; B.D.S. Allan, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

Fas sensitivity of primary LECs and HLE-B3 LECs. (A) Primary LECs cultured as free-floating anterior capsulotomy specimens or HLE-B3 cells cultured on TC plastic were incubated with either control IgM or anti-Fas IgM for 48 hours at 37°C. Apoptotic cells were identified by Hoechst staining. Nuclei of apoptotic cells are condensed and stain very brightly with Hoechst (arrowheads). Bar, 32 μm. (B) HLE-B3 cells cultured on TC plastic were incubated with either control IgM or anti-Fas IgM for 24 hours at 37°C and Western blotted with anti-PARP antibody. Cells treated with anti-Fas IgM showed decreased levels of intact PARP and increased levels of the cleaved 85-kDa fragment.

Figure 2.

 

Quantitation of Fas sensitivity of primary LECs and HLE-B3 LECs cultured on TC plastic and lens capsule. Primary LECs cultured as free-floating anterior capsulotomy specimens or HLE-B3 cells cultured on TC plastic or porcine lens capsule were incubated with either control IgM or anti-Fas IgM in the presence or absence of 2% FCS for 48 hours at 37°C. Apoptotic cells were identified by Hoechst staining. Results are expressed as the mean ± SEM of 6 to 12 observations. Anti-Fas IgM induced significant apoptosis compared with IgM-treated controls in HLE-B3 cultured on TC plastic in serum-free conditions (P < 0.0001).

Figure 3.

 

Effect of culture on collagen IV and laminin on Fas-sensitivity of LECs. HLE-B3 cells cultured on untreated, collagen IV- or laminin-coated wells were incubated with control IgM or anti-Fas IgM (CH11) for 48 hours at 37°C. Apoptotic cells were identified by Hoechst staining. Results are expressed as the mean ± SEM of 6 to 12 observations. Culture on collagen IV significantly reduced Fas-dependent apoptosis (P < 0.0001), but had no effect on apoptosis in the presence of control IgM, whereas culture on laminin had no significant effect on apoptosis in the presence of either control or anti-Fas IgM.

Figure 4.

 

Fas expression in HLE-B3 LECs cultured on different substrates. HLE-B3 cells were cultured on untreated, collagen IV- or laminin-coated wells for 5 days at 37°C. Cells were solubilized, subjected to SDS-PAGE, and Western blotted with anti-Fas antibody and anti-tubulin antibody as a loading control.

Figure 5.

 

Effect of capsule and attached LECs on Fas sensitivity of LECs in the pericapsular halo. (A) Anterior capsulotomy specimens were pinned flat in culture and cells allowed to proliferate and migrate from the capsule (caps) to form a pericapsular halo. The capsule (asterisks, arrows) was either left or removed before culture of the remaining cells in the presence of anti-Fas or control IgM for 48 hours at 37°C. Apoptotic cells were identified by Hoechst staining (arrowheads). (B) There was no significant Fas-dependent apoptosis when the capsule and attached cells were present, but removal of the capsule caused significant levels of apoptosis in the presence of anti-Fas IgM, compared with control IgM (P = 0.0021). Results are expressed as the mean ± SEM of six observations.

Figure 6.

 

Effect of conditioned medium from LECs cultured on collagen IV on Fas sensitivity of LECs cultured on TC plastic. HLE-B3 cells cultured on untreated (plastic) or collagen IV-coated (collagen) wells were incubated with control IgM or anti-Fas IgM in the presence of serum-free medium (SFM) or conditioned medium (CM) from HLE-B3 cells cultured on untreated wells or collagen IV-coated wells. Apoptotic cells were identified by Hoechst staining. Results are expressed as the mean ± SEM of five to six observations. Treatment with conditioned medium from cells cultured on collagen IV provided significant protection against Fas-dependent apoptosis compared to SFM (P < 0.0001). A smaller but significant protection against Fas-dependent apoptosis was provided by conditioned medium from cells cultured on plastic (P = 0.008).

Figure 7.

 

Effect of cell density on Fas sensitivity of LECs cultured on TC plastic and collagen IV. HLE-B3 cells seeded at high, medium, and low densities on untreated or collagen IV-coated wells were incubated with control IgM or anti-Fas IgM in serum-free medium for 48 hours at 37°C. Apoptotic cells were identified by Hoechst staining. Results are expressed as the mean ± SEM of five to six observations. Culture at low density on collagen IV significantly increased sensitivity to Fas-dependent apoptosis compared with culture at high density (P < 0.0001), whereas culture at medium density had an intermediate but significant effect (P = 0.0363).

The authors thank Usha Andley (Washington University, St. Louis, MO) for generously providing HLE-B3 cells.