Mechanisms of lymphocyte adhesion to cultured human corneal epithelial cells.

PURPOSE: The authors studied the adhesion mechanisms between peripheral blood lymphocytes (PBL) and cultured human corneal epithelial (HCE) cells to investigate the lymphocyte interaction with corneal epithelial cells in the corneal immune response. METHODS: First, the authors examined the expression of intercellular adhesion molecule (ICAM)-1 or lymphocyte function-associated antigen (LFA)-3 on the normal human corneal epithelium and cultured HCE cells by an immunostaining technique and flow cytometry. Effects of inflammatory cytokines such as interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha, on ICAM-1 or LFA-3 expression on cultured HCE cells were also examined. Second, the authors performed an adhesion assay with 51Cr-labeled monocyte-depleted PBL from normal, healthy volunteers and cultured HCE cells, with and without treatment of IFN-gamma or TNF-alpha in 96-well-plates for 1 hour at 37 degrees C in 5% CO2. After unbound PBL were removed, the radioactivity of the sample in each well was counted with a scintillation counter. In addition, the authors evaluated the blocking effects of monoclonal antibodies (mAbs) on the adhesion of PBL to the cultured HCE cells. RESULTS: ICAM-1 expression was not detected in the normal human corneal epithelium. However, the expression of ICAM-1 was detected on the cultured HCE cells with Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum. In addition, both IFN-gamma and TNF-alpha increased ICAM-1 expression on the cultured HCE cells dramatically. LFA-3 expression was detected in all cell layers of the normal human corneal epithelia. Neither IFN-gamma nor TNF-alpha had any effect on LFA-3 expression on the cultured HCE cells. The PBL adhesion to the HCE cells with and without treatment of IFN-gamma or TNF-alpha was blocked dominantly by anti-ICAM-1 or anti-LFA-1 alpha mAb. Anti-LFA-3 mAb also blocked the PBL adhesion but had less blocking effect than anti-ICAM-1 or anti-LFA-1 alpha mAb. Anti-very late activation antigen beta, or anti-human leukocyte antigen (HLA)-class I or HLA-class II mAb had no effect on the PBL adhesion to the HCE cells. The adhesion percentile of the PBL applied to the HCE cells pretreated with IFN-gamma or TNF-alpha showed a dose-response curve dependent on the concentration of these cytokines. CONCLUSIONS: The results in the present study demonstrate that (i) adhesion of lymphocytes to HCE cells could be mediated by the LFA-1-ICAM-1 pathway and/or the CD2-LFA-3 pathway; (ii) the LFA-1-ICAM-1 pathway could be crucial in lymphocyte adhesion to HCE cells; (iii) IFN-gamma or TNF-alpha exerts an enhancing effect not only on the ICAM-1 expression on HCE cells but also on the adhesion of lymphocytes to HCE cells.

Characterization of immortalized rabbit corneal epithelial cells with SV40 large T antigen.

The authors developed a line of immortalized rabbit corneal epithelial cells by transfecting a primary culture of New Zealand White (NZW) rabbit corneal epithelial cells with Simian virus 40 (SV40) T antigen gene using a calcium phosphate precipitation method. The transfected cells, which survived more than 40 passages and 400 population doublings, showed some ability to form colonies in soft agar, and had doubling times and plating efficiencies not significantly different from those of untransfected cells. The epithelial nature of the transfected cells was confirmed by immunofluorescence staining with the antibody to 64 kD keratin (AE5), a specific marker for corneal type epithelial cells. Immunofluorescence microscopic examination revealed that the transfected cells expressed major basement membrane components of epithelial cells, including collagen types IV and VII and laminin. Electron microscopic studies demonstrated the presence of microvilli and intercellular in cultured transfected cells. These results indicate that the transfected cells retain some of the normal phenotypic characteristics of corneal epithelial cells and may therefore be used to study the corneal epithelium in vitro.

Human leukocyte antigen-class II-positive human corneal epithelial cells activate allogeneic T cells.

PURPOSE: To achieve a better understanding of the mechanism of corneal immune diseases, including corneal allograft rejection, the authors examined the potential of human corneal epithelial (HCE) cells to activate allogeneic T lymphocytes. METHODS: The mixed lymphocyte-HCE cell reaction (MLCER) was performed as follows: HCE cells from primary cultures, with or without treatment with interferon-gamma (IFN-gamma), were treated with mitomycin C and then mixed with peripheral blood lymphocytes (PBL) from normal volunteers. Triplicate cultures were incubated for 7 days. Interleukin-1-alpha (IL-1-alpha) was added to some cultures to examine its effect on MLCER: The lymphocyte responses were measured by 3H-thymidine uptake for the last 18 hours of incubation in MLCER: RESULTS: IFN-gamma-treated, HLA-class-II-bearing HCE cells stimulated allogenic lymphocytes, whereas IFN-gamma nontreated, class-II-negative HCE cells did not. The stimulation by IFN-gamma-treated HCE cells was blocked by anti-HLA class II monoclonal antibody. In addition, exogenous IL-1-alpha reduced the lymphocyte response in MLCER: This effort was inhibited by indomethacin. CONCLUSIONS: This study demonstrates that HLA-class-II-bearing HCE cells can activate allogeneic PBL by a major histocompatibility complex class II-dependent mechanism. In addition, HCE cells may regulate immune reactions, probably through prostaglandin production caused by IL-1.

Surgical results of penetrating keratoplasty in essential iris atrophy.

BACKGROUND: Results of penetrating keratoplasty in iridocorneal endothelial syndrome have been considered favorable based on past studies; however, documented results in eyes specifically with essential iris atrophy are lacking. METHODS: A retrospective study was performed to evaluate all patients at the University of Pittsburgh with essential iris atrophy who had undergone penetrating keratoplasty for corneal decompensation over 21 years (1971-1992). RESULTS: Penetrating keratoplasty had been performed on six eyes with essential iris atrophy for corneal decompensation. All eyes postoperatively had evidence of persistent anterior uveitis resistant to corticosteroid treatment with one or more episodes of graft reaction. Five of the six eyes (83.3%) ultimately went on to graft failure. Two of the six eyes (33.3%) rejected grafts on two separate occasions. CONCLUSIONS: Penetrating keratoplasty in essential iris atrophy was frequently associated with chronic anterior uveitis and immunologic graft failure.

Clinical and subclinical ophthalmic findings with retinol deficiency.

BACKGROUND: Patients at risk for retinol deficiency in developed countries include those with hepatic dysfunction and malabsorption states. Symptoms of retinol deficiency may go unrecognized or unreported. METHODS: The authors describe 15 patients with hepatic dysfunction, two of whom had procedures that would predispose to malabsorption and were ophthalmologically symptomatic of retinol depletion. The other 13 patients were ophthalmologically asymptomatic liver transplant candidates examined prospectively for subclinical evidence of retinol deficiency. Combined laboratory analysis, Schirmer's testing, conjunctival impression cytology, and electroretinography were performed. RESULTS: Twelve of 15 patients had serum retinol levels below the lower limit of normal. Aqueous tear production was reduced in 7 of 14 patients. Abnormal conjunctival morphology was noted in 6 of 12 patients. Electroretinograms were abnormal in the two patients who were visually symptomatic and in seven of nine patients who were ophthalmologically asymptomatic. CONCLUSION: Subclinical, physiologically significant retinol deficiency may be a frequent and unrecognized problem among patients with hepatic dysfunction.

Basement membrane synthesis by human corneal epithelial cells in vitro.

PURPOSE: Collagen gels may prove to be potential carriers for transplantation of cultured corneal epithelial cells. The purpose of this study was to evaluate the suitability of collagen gels in comparison with corneal stromal blocks as the substrate to support the growth of human corneal epithelial cells in culture and the synthesis and deposition of the basement membrane components by these cells. METHODS: Corneal epithelial sheets, freed from the culture dishes using Dispase II (Boehringer Mannheim, Indianapolis, IN), were cultured on corneal stromal blocks. Deposition of laminin, type IV collagen, type VII collagen, and perlecan (heparan sulfate proteoglycan) were evaluated immunohistochemically after 4 days, 7 days, 2 weeks, and 3 weeks. Human limbal explant cultures were established on collagen gels prepared from bovine type I collagen with or without addition of cultured human corneal fibroblasts. After 1, 2, 3, and 4 weeks, the deposition of the basement membrane components was evaluated immunohistochemically. RESULTS: Corneal epithelial cells, cultured on corneal stromal blocks as well as on collagen gels with or without fibroblasts, deposited laminin, type IV collagen, perlecan, and type VII collagen at the interface of the cells and the substrates. However, different substrates differentially influenced the temporal pattern of the deposition of various basement membrane components. On the stromal blocks, deposition of laminin, type IV collagen, and perlecan by the epithelial cells was evident at 1 week. Type VII collagen was detected at 2 weeks. On the collagen gels with fibroblasts, deposition of laminin, type IV collagen and perlecan was detectable at 1 week. In the epithelial cultures on the collagen gels without fibroblasts, only perlecan was detectable at 1 week. At 2 weeks, all of the basement membrane components, including type VII collagen were detectable on the collagen gels, either with or without fibroblasts. CONCLUSION: Human corneal epithelium cultured on collagen gels or on corneal stromal blocks can synthesize and deposit basement membrane components, including laminin, type IV collagen, type VII collagen, and perlecan within 2 weeks in culture. Therefore, collagen gels may serve as potential carriers for human corneal epithelial transplantation.

Conjunctival epithelial cell hypermitosis and goblet cell hyperplasia in atopic keratoconjunctivitis.

Atopic diseases that include eczema (atopic dermatitis), asthma, and seasonal and perennial rhinoconjunctivitis are common manifestations of abnormal immediate hypersensitivity. Ocular involvement, such as atopic keratoconjunctivitis, characteristically includes conjunctival and corneal inflammation, and in a severe form, conjunctival scarring, symblepharon, corneal epitheliopathy, and visual loss. To examine the conjunctival cellular abnormalities in atopic keratoconjunctivitis, we studied the in vivo differentiation and tissue-culture growth characteristics of conjunctiva from normal subjects and patients with severe atopic keratoconjunctivitis. We examined conjunctival biopsy specimens to determine epithelial mitotic rate and goblet cell frequency, and we studied conjunctival explants to determine the latent period for fibroblast outgrowth and fibroblast doubling time. The mitotic rate for atopic keratoconjunctivitis, 6.7% +/- 2.1% (11 patients), was statistically significantly greater than for normal subjects, 2.0% +/- 0.63% (seven subjects) (P = .05). Also the goblet cell frequency for atopic keratoconjunctivitis, 14.6% +/- 3.4% (11 patients), was statistically significantly greater than for normal subjects, 4.8% +/- 0.92% (seven subjects) (P = .02). The latent period for fibroblast outgrowth and the fibroblast doubling time for atopic keratoconjunctivitis were not statistically significantly different from normal control subjects. Therefore, atopic keratoconjunctivitis was associated with conjunctival epithelial hypermitosis, goblet cell hyperplasia, and normal fibroblast tissue-culture growth. These characteristics may be useful in the diagnosis of atopic keratoconjunctivitis. We previously studied another disease characterized by chronic conjunctival inflammation and scarring, cicatricial pemphigoid, which also demonstrated conjunctival epithelial hypermitosis, but in contrast there was near absence of goblet cells, and the fibroblasts were hyperproliferative. These differences may be used to distinguish atopic keratoconjunctivitis from cicatricial pemphigoid.

Transforming growth factor-beta stimulates collagen and fibronectin synthesis by human corneal stromal fibroblasts in vitro.

The effects of transforming growth factor-beta (TGF beta) and epidermal growth factor (EGF) on the synthesis of collagen and fibronectin, and on the proliferation of human corneal stromal fibroblasts in vitro, were evaluated. Human corneal stromal fibroblasts in culture were incubated for 48 hours with TGF beta or EGF in the absence of serum. Collagen and fibronectin in the culture media were measured by a collagenase-digestion assay and a competitive ELISA, respectively. The effects of the growth factors on proliferation were assessed by 3H-thymidine incorporation. Collagen synthesis was dose-dependently stimulated by TGF beta; at a concentration of 1 ng/ml of TGF beta, a 120% increase in collagen synthesis was seen over that of controls (p < 0.01). EGF, at a concentration of 10 ng/ml, induced a 40% increase in collagen synthesis over that of controls (p < 0.01). The maximum stimulation by TGF beta was greater than that by EGF (p < 0.05). Fibronectin synthesis was stimulated by TGF beta and EGF in a dose-dependent manner; 230% (p < 0.001) and 210% (p < 0.01) increases in fibronectin synthesis were caused by 10 ng/ml TGF beta and EGF, respectively. TGF beta and EGF dose-dependently stimulated 3H-thymidine incorporation. The maximum increases in 3H-thymidine incorporation reached 180% (p < 0.001) and 190% (p < 0.001) over that in controls, at 10 ng/ml concentrations of TGF beta and EGF, respectively. In conclusion, both TGF beta and EGF are potent stimulants of collagen and fibronectin synthesis and proliferation. Therefore, these two growth factors may be effective alternatives or additional choices for the treatment of corneal ulcer.

Graft failure in keratoepithelioplasty.

We report three patients undergoing keratoepithelioplasty using donor limbal tissue. All three had significant vascularization of the recipient bed and all three had inflammatory reactions with apparent partial replacement of the donor epithelium with host epithelium. Limbal transplantation, which is likely to include significant numbers of donor Langerhans' cells, may well increase the risk of graft failure in keratoepithelioplasty.

Corneal epithelial cell attachment with endogenous laminin and fibronectin.

PURPOSE: To evaluate the role of endogenously produced laminin and fibronectin as well as the effect of exogenous laminin and fibronectin in the attachment of human corneal epithelial cells in vitro. METHODS: Primary cultured human corneal epithelial cells labeled with 3H-thymidine were seeded onto plates coated with laminin or fibronectin, or onto uncoated bacteriologic plates. Attachment of cells was measured in the presence or absence of antisera against laminin or fibronectin, by counting radioactivity. RESULTS: Human corneal epithelial cells attached to plates coated with human laminin or human fibronectin in a dose-dependent manner, with 69% and 50% of cells attached to the wells coated with 40 micrograms/ml of laminin and fibronectin, respectively (P < 0.001). The percentage of attachment to uncoated bacteriologic plates increased from 1.2% at 45 min of incubation to 6.7% at 90 min, 22.2% at 3 hr, and 40.1% at 6 hr of incubation. Cycloheximide, a protein synthesis inhibitor, completely inhibited cell attachment. Rabbit antiserum against human fibronectin reduced cell attachment to the uncoated plates to 67% of the control value (P < 0.01), whereas rabbit antiserum against human laminin decreased the attachment to 52% of the control (P < 0.01). A combination of these two antisera reduced cell attachment to 46% of the control (P < 0.01). CONCLUSIONS: Endogenous laminin and fibronectin as well as exogenous laminin and fibronectin play significant roles in the attachment of human corneal epithelial cells in culture.

Trigeminal ganglion neurons affect corneal epithelial phenotype. Influence on type VII collagen expression in vitro.

PURPOSE: To examine whether trigeminal ganglion (TG) neurons in tissue culture influence expression of Type VII collagen (a major component of the anchoring fibrils involved in the attachment of the epithelium to the underlying stroma) by cultured corneal epithelial cells. METHODS: A two-chambered coculture system was used. Fetal rabbit TG neurons were cultured into a central chamber on collagen- or laminin-coated tissue culture dishes. After good neurite outgrowth (average 7 days), rabbit corneal epithelial explants were placed into the outer chamber. Once neurite-epithelial cell interaction occurred, the cultures were immunostained for Type VII collagen. Direct coculture of TG neurons onto confluent passaged rabbit corneal epithelium also was studied. RESULTS: Neurites in contact with the epithelial cells in the outer chamber formed branching complexes, but staining for Type VII collagen was negative. In cocultures of TG neurons onto confluent passaged rabbit corneal epithelium, there was extensive neurite branching on and around the epithelial cells within a week. Scattered epithelial cells, many in clusters, were found to express Type VII collagen, as determined by immunofluorescence staining. CONCLUSIONS: Based on the finding from this study that TG neurons influence production of Type VII collagen by rabbit corneal epithelium in vitro, it is likely that TG neurons influence corneal epithelial phenotypic characteristics that are critical to the maintenance of healthy epithelium in vivo.

Regulation of HLA class II antigen expression on cultured corneal epithelium by interferon-gamma.

The effect of recombinant human interferon gamma (IFN-gamma) on the induction of HLA class II (HLA-DR, -DP, -DQ) antigen expression on human corneal epithelial (HCE) cells was examined in different stages of culture. Primary cultures were established with limbal explants without endothelium. HCE cells in Stage 1 and Stage 2, with cells negative and positive for the 64K corneal keratin (the marker for advanced corneal epithelial differentiation), respectively, were prepared. HCE cells in both stages were treated with IFN-gamma at a concentration of 0 to 1000 U/ml for two to six days and were stained by the avidin-biotin peroxidase complex method. Class II antigens were not detected on HCE cells in either stage without IFN-gamma treatment. IFN-gamma induced three class II antigens on HCE cells in both stages in a dose- and time-dependent manner but at different levels for each antigen (DR greater than DP greater than DQ). In addition, DQ expression was related to cell differentiation, with DQ extremely rare at Stage 1 and more frequent at Stage 2 (5% vs. 20%). These findings indicate that the induction of class II antigens on HCE cells may be regulated by IFN-gamma independently for each of the antigens and that DQ induction may depend upon the differentiation of HCE cells in culture.

Biosynthesis of stromal matrix proteoglycans and basement membrane components by human corneal fibroblasts.

The proteoglycans produced by intact human corneas and corneal cells in culture were compared by characterizing the biosynthetically radiolabeled proteoglycans and by using antibodies to detect their core proteins. Organ cultures of corneas primarily produce a keratan sulfate proteoglycan (KSPG) and a chondroitin and dermatan sulfate proteoglycan (decorin). Immunostaining with antibodies specific for the core proteins of KSPG and decorin showed that these proteoglycans are localized to the corneal stroma. The stroma also contained trace amounts of matrix that stained with antibodies to basement membrane heparan sulfate proteoglycan (perlecan) and laminin. Corneal fibroblasts in culture produced decorin, but the synthesis of KSPG appeared to be blocked at the level of core protein synthesis. Corneal fibroblasts in culture, however, produced perlecan in greater amounts than they did in organ cultures, and they synthesized both perlecan and laminin in greater amounts than did corneal epithelial cells in culture. These results indicate that the synthesis of proteoglycans by human corneal fibroblasts in culture is altered, resulting in increased production of basement membrane-associated proteoglycans and decreased synthesis of corneal stroma-associated proteoglycans.

Differentiation in cultured limbal epithelium as defined by keratin expression.

The authors investigated differentiation of cultured corneal and limbal epithelial cells by immunochemically evaluating the changes in the profiles of keratins recognized by two monoclonal antibodies: AE5, which recognizes K3, and AE1, which recognizes a group of acidic keratins including K16, which is present in the hyperproliferative cells. After 1 and 2 weeks in culture, the human epithelial cells did not react with AE5 but did react strongly with AE1. At 3 weeks, only suprabasal cells exhibited a moderate reactivity with AE5, whereas AE1 binding was seen in all of the cells. After 5 to 6 weeks in culture, all of the cells reacted moderately with AE5 and AE1. Treatment of 2-week-old limbally derived cultures with mitomycin C (mitosis inhibitor) did not inhibit subsequent K3 expression. Thus, K3 expression was associated with maturation or a later stage of differentiation that did not require an additional cell division. Unlike human epithelial cells, rabbit suprabasal epithelial cells expressed K3 (reactivity to AE5) after only ten days in culture. The epithelium derived from central human cornea lost K3 by 1 week in tissue culture but expressed keratin(s) recognized by AE1. Even after 4-6 weeks, cells derived from the central cornea did not become confluent and did not react with AE5. Thus, limbally derived human and rabbit epithelial cells undergo chronological changes in K3 expression similar to that seen in rabbit epithelial cells derived from central cornea. However, cultured human limbal epithelial cells take a significantly longer time to express K3 (a phenotypic characteristic of differentiated corneal epithelium) than do rabbit epithelial cells.

Increased aggrecan (cartilage proteoglycan) production in the sclera of myopic chicks.

A previously characterized chick model of myopia was used to evaluate biochemical changes in the sclera which are associated with ocular enlargement and myopia. Chicks were monocularly occluded for 10 days and the DNA, hydroxyproline, and glycosaminoglycan contents of the sclera were compared between the normal and the myopic eyes. No significant differences could be detected in total DNA or hydroxyproline content. There was, however, a 34% increase in glycosaminoglycans and a 20.7% decrease in cell density within the posterior sclera of myopic eyes. The biosynthesis of scleral proteoglycans was determined by measuring 35SO4 incorporation in the sclera of chicks visually occluded for 5, 10, and 15 days. No differences could be detected in 35SO4 incorporation into the cornea or the anterior sclera. However, 35SO4 incorporation was significantly increased in the posterior sclera of myopic eyes by 64% at Day 5, 39% at Day 10, and 49% at Day 15. When fractionated on Sepharose CL-4B, scleral proteoglycans were resolved into two peaks which were identified by Western blot analysis as aggrecan (cartilage proteoglycan) and decorin. Furthermore, Western blot and dot blot analyses indicated that significantly more aggrecan core protein was present in the sclera of myopic eyes compared with equivalent amounts of sclera from control eyes. These results indicate that increased synthesis and accumulation of aggrecan, which increases the volume of extracellular matrix in the posterior sclera, are responsible for the ocular enlargement observed in this model of myopia.

Regional heterogeneity in human corneal and limbal epithelia: an immunohistochemical evaluation.

The authors studied the distribution of specific keratins within the superior, inferior, medial, and lateral regions of human limbus and cornea to determine whether the limbal epithelium exhibits regional heterogeneity in its microstructure. A corneal epithelial basic keratin (K3), recognized by monoclonal antibody AE5, was immunohistochemically undetectable in the basal layers of the limbus in these four regions, but was seen in all layers in the central cornea. The pattern of immunostaining with another monoclonal antibody, AE1, which recognizes several acidic keratins, was complementary to AE5 staining in that AE1 recognized a similar heterogeneity in the limbal epithelial cells. AE1 immunoreacted with the basal cells of the limbus, but not those of the central corneal epithelium. Limbal characteristics, as defined by AE1-positive and AE5-negative staining, extended deeply into peripheral cornea in the superior and inferior regions, but to a lesser extent in the lateral and medial regions. The broader regions of epithelium with limbal characteristics in the superior and inferior regions raises the possibility that these regions play an important role in corneal epithelial maintenance and wound healing.

Growth of acanthamoeba on human corneal epithelial cells and keratocytes in vitro.

Acanthamoebic keratitis, a potentially devastating infection usually associated with contact lens wear, has been recognized with increasing frequency in recent years. Once the Acanthamoeba organisms gain access to the human cornea, it is not clear which constituents of the corneal milieu provide a substrate for their growth. The growth of Acanthamoeba polyphaga was investigated on cultured monolayers of human corneal epithelial cells, stromal keratocytes, and stromal homogenate suspensions. Growth was determined through organism counts and observation of cytopathic effects on tissue culture dishes. Compared with tissue culture media controls, acanthamoebic growth was supported by cultured epithelial cells and keratocytes but not stromal homogenates. These results suggest that in acanthamoebic keratitis the organisms depend on the cellular components of the cornea as substrates for growth. This in vitro model may also provide further information on the pathogenesis of keratitis and a system for drug sensitivity testing.

Healing of excimer laser ablated monkey corneas. An immunohistochemical evaluation.

The healing response of the cornea following excimer laser anterior keratomileusis (a 4-mm-diameter ablation to a depth of 11, 23, or 46 microns) was analyzed immunohistochemically in adult rhesus monkeys. The ablated surface had reepithelialized and the synthesis of type VII collagen (a major component of anchoring fibrils) was evident by 7 days; the reestablishment of a nearly continuous anchoring fibril zone was evident after 12 weeks. Stromal fibroblasts, activated in response to wounding, expressed a fetal antigen for approximately 6 weeks. Although fibronectin and type VII collagen were present only transiently in the regenerating subepithelial stromal matrix in the subepithelial regions, some other alterations, including the presence of type III collagen, increased levels of keratan sulfates, and discontinuities in the anchoring fibril zone were evident even 18 months after wounding. The depths of the regenerated stroma, which were estimated from the depths of remnant staining for type VII collagen in the stromal matrix, generally, but not always, corresponded to the depths of the calculated ablation.