Towards a closed eye model of the pre-ocular tear layer.

Although the tear film has been extensively studied as it exists in the open eye state, until recently very little was known as to what happens to the tear film on eye closure. Recent studies have shown that eye closure results in a profound change in the composition, origins, turnover and physiological functions of the tear film. These changes include a shift from an inducible, neurologically controlled, lacrimal secretion containing among other proteins primarily lysozyme, lactoferrin and tear specific lipocalin, to a much slower, constitutive-type of secretion, composed almost exclusively of sIgA. This change is accompanied by the build-up of sialoglycoproteins of epithelial and goblet cell origin, the build-up and activation of complement and the build-up of serum proteins. In addition, various cytokines and proinflammatory mediators accumulate, including some which are potent inducers of angiogenesis and leukochemotaxis. The closed eye also exhibits the recruitment and activation of massive numbers of PMN cells. This results in a stagnant, closed eye layer, which is extremely rich in reactive complement products, PMN cell proteases including protease-3, elastase, capthepsin G, MMP-9 and urokinase. We have postulated that this shift represents a fundamental change in host-defense strategies from a passive-barrier defense to an active immune, inflammatory, phagocyte-mediated process and that this shift is necessitated in order to protect the cornea from entrapped microorganisms. Studies have shown that autologous cell damage is avoided in closed eye tear fluid, by the accumulation of several modulators of complement activation, which shift activation towards opsonization of entrapped microorganisms and the build-up of a wide array of antiproteases. Some of the latter are likely to arise from the ocular surface tissues. Corneal neovascularization may be avoided in part by the build-up of alpha2-macroglobulin and the conversion of plasminogen to angiostatin. It is highly probable that other bioactive protein fragments are produced in the closed eye, which contribute to homeostasis. Areas of future study are indicated.

Diurnal tear cycle: evidence for a nocturnal inflammatory constitutive tear fluid.

To investigate the tear film in the closed eye, microliter tear samples were collected without overt reflex stimulation throughout the diurnal cycle, with closed eye samples recovered immediately upon eye opening. Samples were subjected to agarose, polyacrylamide, and two-dimensional electrophoresis, coupled with immunofixation, immunoblot, and lectin blot assays. Major protein constituents were densitometrically and immunologically quantified. Results revealed a distinct progression in composition from reflex to open to closed eye tear samples. Total protein increased from 6.0 to 9.0 to 18.0 mg/ml, secretory IgA increased from less than 0.23 to 0.85 to 8.40 mg/ml, and serum albumin increased from 0.02 to 0.06 to 1.10 mg/ml. In contrast, concentrations of the major reflex tear components (lysozyme, lactoferrin, and tear specific prealbumin) remained essentially static. Immunoblot assay for complement C3 and C3c revealed that eye closure was associated with C3 activation. Results indicate that: (1) the reflex and closed eye tear layers represent opposite extremes in composition and likely origins, with open eye tear film suggesting an intermediate origin; (2) reflex tears are derived from a neurologically inducible lacrimal or accessory gland secretion composed almost exclusively of lysozyme, lactoferrin, tear specific prealbumin, and a minor mixed alpha to beta globulin fraction; (3) upon eye closure, reflex secretion ceases or greatly diminishes, with ongoing slower flow maintained by a constitutive secretion composed almost exclusively of secretory IgA; (4) the closed eye environment induces a subclinical inflammation, accounting in part for the marked rise in albumin concentration. This increase, coupled with that of secretory IgA, may play a critical role in protecting the closed eye environment from pathogens. However, this may render the closed eye environment particularly vulnerable to inflammatory and immune-mediated pathological processes, such as those seen with extended wear soft contact lenses.

Specificity and biological activity of the protein deposited on the hydrogel surface. Relationship of polymer structure to biofilm formation.

The in-situ lens-bound protein layer (LBPL) was characterized on hydrogels of varying water content and ionic-binding capacity. The LBPL proved to be critically dependent on the ionic binding capacity of a given hydrogel. On nonionic polymers the LBPL invariably was thin and largely insoluble. Histochemical staining allowed the detection of all major types of tear proteins. Amino acid analysis revealed a variable composition. Extractable protein proved devoid of active lysozyme. Electrophoresis of pooled samples revealed a variable mixture of acidic, neutral, and basic bands. To what extent variability is dependent on tear film composition and lens structure awaits use of more sensitive analytic procedures. On anionic hydroxyethylmethacrylate copolymer lenses, the LBPL proved radically different. Here the LBPL invariably was much thicker and composed primarily of loosely bound protein. Electrophoresis and enzymatic analysis revealed a homogenous layer consisting primarily of lysozyme much of which retains enzymatic activity. The amino acid analysis of the insoluble protein suggests a similar composition. Specificity of deposition can be attributed to ionic affinity. Conformational integrity can be attributed partly to the unique stability of lysozyme. Electrophoresis of a pooled anionic lens extract revealed an unknown, highly mobile, basic protein. This presumably represents the selective accumulation of a highly basic trace or transient constituent of the tear film. The specificity and biological activity of the LBPL on the anionic lens may modify hydrogel biocompatibility affecting risk of spoilage, microbial colonization, and propensity to trigger an inflammatory and immune response.

Complement and complement regulatory proteins in human tears.

PURPOSE: The complement system is part of the innate defense system of the body, and it contributes to inflammatory conditions. The current study examined tears for the presence of complement components, the activity of the components, and the presence of regulatory components. METHODS: The significance of a functional complement system in tears was examined in four ways. First, the presence and concentration of complement components in tear samples (open-eye, closed-eye, and reflex tears) was examined by sandwich enzyme-linked immunosorbent assay. Second, the presence of an active pathway in each tear type was established by supplementation of complement-deficient sera. Third, Western blotting of tear samples was used to determine whether complement components were activated in tears. Fourth, the presence of regulatory components was examined by enzyme-linked immunosorbent assay and by the inhibition of the ability of tears to supplement deficient sera. RESULTS: Components C1q, C3, factor B, C4, C5, and C9 were detected in closed-eye tears. Only C3, factor B, and C4 were detected in open-eye and reflex tears. Tears were able to supplement complement-deficient sera, indicating that the components were in an active state. Complement components C3, factor B, C4, and C9 were activated in closed-eye tears. The regulatory protein decay-accelerating factor was found only in closed-eye tears. Lactoferrin, another regulatory protein present in all tear types, was shown to inhibit complement-mediated red blood cell lysis, although the inhibition by closed-eye tear lactoferrin was reduced compared to that isolated from other tear types. CONCLUSIONS: This study has demonstrated that the complement system in tears was functionally active and that the concentration of all components was increased greatly in closed-eye tears. In spite of the presence of regulatory proteins, proteins of the complement cascade in tears were shown to be activated.

Host-defense mechanism of the ocular surfaces.

The defense of the ocular surfaces presents an unique challenge in that not only must integrity be maintained against microbial, inflammatory and physical assault, but it must be done while minimizing the risk of loss of corneal transparency. This puts severe limitations on the degree to which scarring or neovascularization can occur in the cornea secondary to any infectious, inflammatory, immunological or wound healing process. Moreover, this defense system must be equally effective under two extremes of conditions: those found in the open eye and the closed eye environments. It is our contention that these constraints have resulted in the evolution of a highly complex fail-safe defense system that utilizes distinctly different strategies in open and closed eye conditions. The extraordinary effectiveness of this system is evidenced by the fact that despite continued exposure to a microbe rich environment, the external ocular surfaces maintain a very low microbial titer and are highly resistant to breaching by all but a few pathogens. It is the intent of this review to provide a working model of this defense system as it operates under both open and closed eye conditions, to provide evidence in support of this model as well as highlight some of the many areas of uncertainty.

Diurnal variations in angiostatin in human tear fluid: a possible role in prevention of corneal neovascularization.

PURPOSE: Although overnight eye closure is known to result in hypoxia and release of potent angiogenic factors, even prolonged eye closure does not result in corneal neovascularization. This suggests that the closed eye tear film may contain factors that can impede neovascularization. Closed eye tear fluid is known to contain proteases capable of converting plasminogen/plasmin to angiostatin and other angiostatin-like A-chain fragments which are potent inhibitors of angiogenesis. This study was designed to characterize open and closed eye tear fluid for the presence of these entities. METHODS: Open and closed eye tears were collected by microcapillaries from normal individuals. Tears were centrifuged and the supernatants analyzed by SDS-PAGE and western blotting. Membranes were probed with antibodies specific for the A-chain of plasmin and plasminogen and with antibodies specific for conformational domains on the smaller N terminal kringles 1-->4 and kringles 1-->3 fragments which are known angiogenesis inhibitors. Supernatants were also analyzed after fractionation by HPLC and binding to lysine sepharose 4B. The isolated fragments were identified based on size, lysine-binding capabilities, antigenic properties and by comparison with standards. RESULTS: Open eye tear fluid from all normal individuals contained low levels of plasminogen, but no detectable antigens consistent with free A-chain or angiostatins. Tears collected after overnight eye closure contained significant amounts of plasminogen, A-chain antigen and various A-chain fragments including kringles 1-->4 and kringles 1-->3 and most likely free kringle 5, all known to have anti-angiogenesis properties. These were often present in concentrations likely to be physiologically significant. In samples collected from an atopic subject, the concentration of angiostatins in CTF increased markedly during active phases of the disease reaching levels of several ng/microl. In these instances and in similar samples obtained from other atopic individuals experiencing active reactions, angiostatin was often detectable in basal-type tear fluid. CONCLUSION: A-chain fragments, which can inhibit angiogenesis, are often present at physiologically significant levels in human tear fluid collected after overnight eye closure. These fragments may play a role in preventing neovascularization in the hypoxic closed eye environment and may well be up regulated during inflammatory reactions.

Identification, origins and the diurnal role of the principal serine protease inhibitors in human tear fluid.

PURPOSE: Previous work identified polymorphonuclear leukocyte (PMN) elastase as the major caseinolytic entity in tears collected after overnight eye closure. This study was designed to identify the principal serine protease inhibitors (serpins) in tears and to determine their function in the regulation of PMN cell proteases on eye closure. METHODS: Reflex and closed eye tear samples were collected by microcapillary tube and centrifuged. After reflex and closed eye supernatants (R and C) were fractionated by HPLC, samples were subjected to casein zymography and reverse zymography. Western blots were utilized to screen tears and HPLC fractions for elastase, cathepsin G and proteinase-3 and to obtain semi-quantitative data on alpha 1-protease inhibitor (alp1), alpha 1-antichymotrypsin (alpha 1-Achy), secretory leukocyte protease inhibitor (SLPI), elafin and alpha 2-macroglobulin (alpha 2-M) as well as associated complexes and products. To confirm specificity of reactivity, samples were immunoprecipitated for a given protease or serpin and screened for the coprecipitation of interacting species. RESULTS: Although R fluid contains no caseinolytic activity, it contains low levels of serpin-like activity principally in the form of SLPI (5-10 ng/microliter). Lesser amounts of alpha 2-M, alpha 1-Achy and alp1 (approximately < 1-3 ng/microliter) are also evident. C fluid is associated with very high levels of PMN cell proteases along with a approximately 5-20-fold increase in the concentrations of all of the above inhibitors. Trace levels of elafin were also detected. The concentrations of rapid reacting inhibitors exceeded that of proteases, with SLPI, alpha 1-Achy and alp1 being the principal functional entities. In atypical samples, complexes of elastase and alpha 2-M were also encountered. CONCLUSIONS: SLPI, a known antimicrobial agent and an elastase and cathepsin G inhibitor, is the principal serpin in R fluid. C fluid is associated with a marked increase in the concentrations of an array of rapid reacting serpins capable of inhibiting all known PMN cell serine proteases. In the normal closed eye, the concentration of rapid reacting inhibitors always exceeds that of proteases with C fluid also containing a functional reserve of the slow reacting inhibitor alpha 2-M.

Diurnal variations in tear glycoproteins: evidence for an epithelial origin for the major non-reducible > or = 450 kDa sialoglycoprotein(s).

PURPOSE: To characterize the nature and origin of changes in tear glycoproteins accompanying eye closure. METHODS: Reflex (R) and overnight closed (C) eye tears collected by capillary tubes were centrifuged with the resulting R pellets (primarily desquamated epithelial cells) and C pellets (primarily PMN and some epithelial cells) extracted in acidic PBS. Extracts and supernatants were separated by size-exclusion HPLC and/or SDS-PAGE. Gels were stained or blotted and immune- or lectin-probed. An HPLC glycoprotein fraction of > or = 450 kDa isolated from all four sources was characterized before and after partial deglycosylation, using antibodies specific to known mucin and carbohydrate epitopes. Immunofluorescence microscopy was carried out on human conjunctiva, using as probe a MAb to salivary mucin specific for a sialyl Lea epitope, which was found to cross-react specifically with the major non-reducible high molecular weight sialoglycoproteins (SGs) in tears. These SGs were immunoprecipitated and blot-probed along with tissue extracts. RESULTS: R fluid contained minor amounts of numerous glycoproteins, including probably several of inducible lacrimal secretory origin. Results confirmed sIgA as the principal source of the intense reducible glycoprotein bands common to C fluid. Smaller amounts of free secretory component and serum glycoproteins were also visualized. The HPLC fraction (> or = 450 kDa) consisted of four major non-reducible glycoproteins. In R fluid, this fraction (< 1% total protein) consisted primarily of two entities: a 450-500 kDa SG and a larger asialoglycoprotein. The SG accounts for as much as 85% of the total protein in the R pellet extract. C fluid was associated with a selective increase in SGs and a shift in distribution to two SGs > 500 kDa. All SGs exhibited a common antigenicity reacting specifically with the MAb for the sialyl Lea epitope. SGs indistinguishable in size and antigenicity were recovered in epithelial extracts. Immunofluorescence microscopy revealed that reactivity was localized to the epithelial plasma membrane, increasing in intensity from basal to apical cells. Although these SGs exhibited some properties in common with MUC1, immunological and other data suggest a unique SG. CONCLUSIONS: Tear glycoproteins are derived from four principal sources. In R fluid, an inducible lacrimal secretion predominates. In C fluid, a constitutive sIgA secretion predominates, augmented by a serum exudate and SGs derived at least in part from the epithelium. In R fluid and pellet extracts, the SGs consist primarily of a 450-500 kDa species that is most probably derived from the plasma membrane. Larger antigenically related SGs are prevalent in C fluid.

Polymorphonuclear leukocyte cells and elastase in tears.

PURPOSE: To characterize the effects that mode of sampling and overnight eye closure have on the nature of caseinolytic activity recovered in tear fluid. METHODS: Reflex, open and closed (R, O and C) eye tear fluids were collected by microcapillary tubes or from the inferior formix by Schirmer strip. Microcapillary collected samples were centrifuged and recovered cells cytochemically characterized and probed by immunofluorescence microscopy, or alternatively extracted in acidic PBS. Tear supernatants, pellets and Schirmer strip extracts were subjected to casein zymography or SDS-PAGE and immunoprobed for plasmin/plasminogen. To identify caseinolytic activity, samples were immunoprecipitated with antibodies to plasmin/plasminogen or to elastase, and the immunoprecipitated materials were subjected to zymographic analysis. RESULTS: Immunoblot assays revealed R and O samples contained low levels of plasminogen (approximately 1.1 micrograms/ml) and only trace levels of plasmin (< 0.1 ng/ml). Insufficient levels of caseinolytic activity were present to allow zymographic detection. Cytochemical analysis revealed that R and O pellets consisted almost exclusively of desquamated epithelium. Immunoblot analysis revealed that C fluid was associated with an increase in plasminogen and its partial conversion to plasmin (approximately 3.2 ng/ microliter), high molecular weight covalent complexes and degradative products. Zymographic analysis disclosed much greater caseinolytic activity than could be attributed to plasmin or its cleavage products. This consisted primarily of three bands (30-26 kDa) which were identified as polymorphonuclear leukocyte (PMN) cell elastase based on size and antigenicity. This is derived from PMNs recovered from the C pellet. Elastase could also be recovered from Schirmer strips from 90% of donors, provided that the strips were extracted in sample loading buffer. The activity was restricted to the portion of the strip that had been in contact with the ocular tissue. CONCLUSIONS: The main source of caseinolytic activity in C fluid is elastase. This arises from PMNs that undergo recruitment, activation and degranulation in the C environment. In contrast, the elastase recovered in Schirmer strip extracts is derived from intact PMNs that adhere to the strip during sample collection. This would suggest that PMN cells undergo a low level of recruitment into the open eye environment.

Temporal sequence of changes in tear film composition during sleep.

Overnight eye closure induces a shift in the nature and composition of the tear film, from a dynamic reflex tear-rich to a stagnant secretory IgA-rich layer. This is accompanied by the induction of a state of sub-clinical inflammation, as evidenced by increases in albumin levels, plasminogen activation, conversion of complement C3 to C3c, and the recruitment of polymorphonuclear (PMN) cells into the tear film. To determine the time course and functional relationship between these potentially interdependent processes, tear samples were collected from ten non-contact lens wearers after 1, 2, 3 and 5 hours of sleep. A subgroup of 6 subjects also self-collected tear samples after 8 hours of sleep. Tear samples were analysed for albumin by quantitative immunofixation assay, secretory IgA (sIgA) by radial immunodiffusion assay, plasmin-like activity using a chromogenic substrate, and complement C3 to C3c conversion by immunoblot assay. Epithelial and PMN cells in the precorneal tear film were recovered from corneal washings from the same subjects after 1, 3, 5 and 8 hours of sleep, and quantified. Results revealed that, unlike epithelial cells which exhibited a slow progressive accumulation as a function of the period of sleep, PMN cell concentration exhibited a lag phase, with recruitment occurring after between 3 and 5 hours of eye closure. This was preceded by plasminogen activation, increases in albumin and sIgA levels, and complement C3 to C3c conversion, all of which occurred within 1 to 3 hours after eye closure. Plasmin-like activity appeared to plateau after 3 hours and then decreased.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of eye closure on protein and complement deposition on Group IV hydrogel contact lenses: relationship to tear flow dynamics.

PURPOSE: This study was designed to determine the effect of overnight eye closure on the rate and composition of protein deposition on high water content ionic matrix soft contact lenses (Group IV SCLs) and to extrapolate from this data information on the probable change in the rate of reflex-type tear secretion associated with eye closure. METHODS: Group IV SCLs were temporally sampled after equivalent periods of wear under closed eye (C) or open eye (O) conditions. Lenses were rinsed in saline and the majority of the tightly bound protein extracted at 90 degrees C in 40% urea, containing 1% SDS, 1 mM DTT, 100 mM Tris-HCl (pH 8.00). Residual protein was determined by Coomassie staining of the extracted lenses and densitometric analysis. Extracted protein was quantitated and separated by SDS-PAGE. Gels were either stained with Coomassie blue or reversibly stained with imidazole-zinc and blotted. Blots were PAS stained, or lectin and antibody probed for glycoproteins, secretory IgA (sIgA), IgG, lysozyme and complement C3. Laboratory simulated deposition studies were carried out on unworn lenses exposed to HPLC purified lysozyme. RESULTS: The protein in the saline rinse, to a large degree mirrored the composition of tear fluid in which the lens had been residing (O or C). This would suggest that the saline wash consists of residual tear fluid and loosely adherent protein. In contrast, the urea extracts were highly homogeneous consisting primarily of lysozyme and to lesser extent lysozyme dimer. This supports the contention that the Group IV SCL functions in the eye much as cationic exchange resin selectively absorbing lysozyme. C extracts also proved relatively enriched in trace amounts of sIgA, IgG and complement C3 and its breakdown products. High levels of C3 and C3 breakdown products were specifically recovered only in the C worn lens extracts from a subject experiencing unilateral contact lens associated corneal infiltrates from the affected eye. In all subjects, markedly less protein (lysozyme) was recovered in urea extracts of lenses exposed to 7-8 h of closed eye as compared to open eye wear (0.20 +/- .08 versus 0.79 +/- .15 mg/lens (n = 6)). Temporal studies further revealed that deposition was linearly related to duration of wear during the initial phase of conditioning film formation giving rise to rate constants for lysozyme deposition of 2.2 +/- 0.29 (n = 5) and 0.20 +/- 0.06 microgram/min (n = 4) under open and closed eye conditions respectively. With further wear, deposition eventually reached a steady state. Under laboratory conditions, lysozyme was much rapidly and quantitatively removed from solution in a manner following a hyperbolic plot. This suggests that during the initial phase of deposition the rate of deposition is limited by the capacity of the tear fluid to deliver lysozyme to the lens surface under these two extremes of conditions. CONCLUSIONS: Eye closure profoundly affects the rate of lysozyme deposition on Group IV hydrogels and the composition of minor biofilm constituents in a manner that could affect biocompatibility. Findings support the contention that eye closure results in a > 90% reduction in the rate of reflex-type tear secretion.

Vitronectin: Possible contribution to the closed-eye external host-defense mechanism.

Eye closure causes a shift in the preocular tear film, from a reflex tear-rich layer which is in dynamic equilibrium to a secretory IgA-rich layer which is stagnant in nature. This is accompanied by complement conversion and plasminogen activation, followed by polymorphonuclear (PMN) cell recruitment. It is suggested that this shift to a secretory IgA and PMN cell-rich layer serves to protect the ocular surfaces from trapped microorganisms. The mechanisms whereby autologous damage is avoided are uncertain. In other tissues, vitronectin (VN) may be an inhibitor of complement and plasmin induced autolytic damage and a potentiator of microbial phagocytosis. Its presence in the external ocular environment is unknown. To screen for VN, normal human reflex (R), open-eye (O) and closed-eye (C) tear samples were collected, separated by SDS PAGE, and immunoblot probed. Detection was carried out using monoclonal antibodies (MAbs) raised against human VN, coupled to an avidin biotin conjugate-horseradish peroxidase amplification system. Quantitative analysis was carried out using a sandwich ELISA assay. Bovine corneas were sectioned and immunohistochemically stained with MAbs to bovine VN. Results revealed that in going from R to O to C tear fluid there is a marked progressive increase in VN (0.08, 0.75, 3.65 µ/ml). This is accompanied by a shift from the intact 75kDa molecule to the 65kDa breakdown product which is still biologically active, with further degradation occasionally encountered. Immunohistochemical staining of bovine cornea revealed that VN is localized in the corneal epithelium and stromal keratocytes. These findings are compatible with either a local or serological origin for VN, and support the contention that VN may be a component in the external closed-eye host-defense system.

The effect of utilization on health care costs.

This report analyzes the effect of utilization on health care costs. Two important aspects of utilization are examined: (1) the type of obstetric delivery utilized; (2) the extent of utilization of hospital facilities. A retrospective review of the type of delivery employed by five different physicians to deliver 494 primiparous patients revealed cesarean section rates varying from 16% to 40%. Average charges incurred by 100 patients who had undergone uncomplicated abdominal hysterectomies varied by as much as 26%. A simple formula to project cost is included.