Secretory Phospholipase A₂ Deposition on Contact Lenses and Its Effect on Bacterial Adhesion

Abstract

purpose. Secretory phospholipase A₂ (sPLA₂) is a potent antibacterial enzyme in tears and has been found to kill Staphylococcus aureus rapidly in vitro. The purpose was to determine whether sPLA₂ deposition is associated with contact lens (CL) type, if sPLA₂ remains active on CLs, and if this has an effect on bacterial adhesion.

methods. Ionic (etafilcon A) and nonionic (Polymacon) high-water, soft CLs were used. CLs were worn for 6 hours (daily wear, n = 39) or 6 nights on an extended-wear schedule (n = 25). Tears were collected from patients and worn contact lenses were removed and protein and active enzymes extracted for estimation of their levels. The number of S. aureus adhering to sPLA₂-soaked CLs in vitro was also quantified.

results. There was no significant difference in the concentration of sPLA₂ in tears between groups of daily CL wearers. Significantly less sPLA₂ was recovered from Polymacon CLs for both daily and extended wear compared with etafilcon A CLs (daily wear: 3 vs. 5 ng/lens; extended wear: 3 vs. 6 ng/lens; P < 0.05). sPLA₂ activity correlated with protein amounts from lenses. Relatively less active sPLA₂ was recovered from Polymacon contact lenses. sPLA₂ reduced adhesion of Staphylococcus to contact lenses in vitro.

conclusions. Etafilcon A CLs absorb more active sPLA₂ than Polymacon CLs, which increases with length of CL wear. The sequestering of sPLA₂ onto CLs did not affect amounts of the enzyme in tears. sPLA₂ adsorbed to a CL can reduce the viable Staphylococcus adhering to the CL, which may protect the eye from colonization by this pathogen.

The leading cause of microbial keratitis in non–contact-lens wearers and the most commonly isolated Gram-positive bacteria from contact-lens–related infections is Staphylococcus. ¹ Staphylococcus aureus has been associated with contact-lens–induced peripheral ulceration. ² The first line of defense against these conditions is the nonspecific antibacterial properties of tears. There are many tear components with antibacterial properties, including lysozyme, ^{3 4} lactoferrin, ^{3 4} lipocalin, ^{5 6 7} vitronectin, ⁸ immunoglobulins, ^{3 4 9 10} and complement. ¹¹  

Secretory phospholipase A₂ (sPLA₂) has been found to be present in both rabbit ¹² and human tears. ^{13 14 15 16 17} Cells in the main and accessory lacrimal glands secrete this enzyme. ¹⁴ sPLA₂ cleaves fatty acids from phospholipids at the sn-2 position. With Gram-positive bacteria, membrane damage is due to the cleavage of fatty acids from phospholipids and the large number of cationic residues on the surface of the enzyme. ¹⁸ This enzyme is extremely active against S. aureus in vitro ^{19 20 21} and is currently thought to be the most potent antistaphylococcal factor in tears. ¹⁷ The anti-staphylococcal activity of sPLA₂ is enhanced in the presence of agents that permeabilize the bacterial cell wall, such as lysostaphin. ²² In addition, sPLA₂ has shown activity against the Gram-negative bacteria Escherichia coli and Listeria monocytogenes. ²³ The Gram-negative antibacterial activity of sPLA₂ is increased in the presence of bactericidal/permeability-increasing protein. ²⁴ Apart from its antibacterial function, sPLA₂ is proinflammatory and acts on a range of cells. sPLA₂ activates arachidonic acid metabolism and induces cyclooxygenase-2, leading to the release of prostaglandins and leukotrienes. ²⁵ sPLA₂ is also responsible for cytokine production, such as IL-6 and TNF-α. ²⁶ Intolerant contact lens wearers have been found to have elevated concentrations of this enzyme in their tears. ²⁷  

The presence of a contact lens in the tear film may change the dynamics and structure of the tear film. Findings regarding tear proteins and their alteration by contact lens wear are varied. Lysozyme, albumin, and lactoferrin have been found to remain unchanged in extend-wear contact lens users. ²⁸ However, amounts of fibronectin increase ²⁹ in tears of patients on an extended lens wear schedule. Stapleton et al. ³⁰ found that overnight contact lens wear significantly increases concentrations of total protein, sIgA, and complement proteins in comparison to tears from baseline and daily contact lens wear patients. Proteins from the tear film deposit on contact lenses. Deposits analyzed from contact lenses vary according to contact lens material and charge. ³¹ These deposits are thought to be detrimental to contact lens wear and ocular health. Once deposited onto a lens, the proteins may alter and change their bactericidal properties. For example, lactoferrin deposited onto a contact lens actually promotes Gram-negative bacterial adhesion, once adherent, the bacteria are killed by this protein. ³²  

The purpose of our investigation was to determine whether concentrations of sPLA₂ in tears are affected by contact lens wear, whether sPLA₂ deposits onto contact lenses and, if so, whether the concentration of absorbed sPLA₂ is dependent on contact lens type. In addition, we examined the effect of absorbed sPLA₂ on the adhesion of S. aureus to contact lenses. 

Methods

SPLA₂ Concentrations from Contact Lenses and Tears

Study procedures were subject to review by the Committee on Experimental Procedures Involving Human Subjects at The University of New South Wales and were performed in accordance with the guidelines of the Declaration of Helsinki. Thirty-nine subjects were enlisted in the 6-hour contact lens wear study and wore ionic (etafilcon A) and nonionic (Polymacon), high-water contact lenses contralaterally. Tears (approximately 5 μL) from each eye were collected into a 10-μL glass capillary tube (n = 39) before contact lens wear and before contact lens removal. Twenty-five subjects were enlisted in an extended-wear schedule and wore ionic (etafilcon A) and nonionic (Polymacon), high-water contact lenses contralaterally. 

Worn contact lenses were removed, and protein and active enzyme were extracted for estimations of their levels. Lenses were extracted in 5 mL of an extraction buffer comprising 4 M urea (BDH Laboratory Supplies, Poole, UK), 0.1% sodium dodecyl sulfate (Sigma-Aldrich, St. Louis, MO), 1 mM dithiothreitol (Sigma-Aldrich), 0.1 M Tris (pH 7.5; Sigma-Aldrich) at 95°C for 1.5 hours. Extracts were buffer exchanged into phosphate-buffered saline (PBS; pH 7.4) and concentrated in concentrator tubes (Centricon 10; Millipore Corp., Bedford, MA) to a final volume of 100 μL. 

Concentration of sPLA₂ in contact lens extracts and tears were measured by sandwich ELISA specific for sPLA₂ (Cayman Chemical Co., Ann Arbor, MI). The standard used for this assay was sPLA₂ from human synovial fluid and the sensitivity of the assay was 15.6 pg/mL. Catalytic activity of sPLA₂ from samples was measured by using the 1,2-dithio analogue of diheptanoylphosphatidylcholine as a substrate for sPLA₂ (Cayman Chemical Co.). sPLA₂ catalytic activity was also measured before and after extraction conditions using the activity assay to determine the effect of the extraction process on sPLA₂. 

Bacterial Adhesion to sPLA₂-Soaked Contact Lenses

S. aureus (strain Saur 31) isolated from a patient with a contact-lens–related peripheral ulcer was used. Bacteria were grown overnight in tryptone soya broth (Oxoid, Basingstoke, UK), washed, and resuspended in PBS supplemented with CaCl₂ (0.07 M) to approximately 1 × 10⁸ cfu/mL. Etafilcon A and Polymacon contact lenses were soaked in 20 ng/mL sPLA₂ II in PBS (Sigma-Aldrich) overnight at 35°C. The amount of sPLA₂ used was determined as follows. It is estimated that approximately 10% to 20% of initial concentration in a protein soak is retained on the lens, therefore we soaked contact lenses in a stock solution of 20 ng/mL to have a final amount of approximately 2 to 4 ng/lens (worn contact lens concentrations extracted from lenses in this study) of sPLA₂. sPLA₂-soaked lenses were then incubated in 1 mL of the bacterial suspension for 10 minutes at 35°C. After incubation, contact lenses with adherent bacteria were washed three times in PBS to remove any bacteria associated with, but not adherent to, the contact lens. Lenses were then homogenized in PBS with a dispersing tool (Ultra-Tarrax T-8; IKA Labortechnik, Rawang, Malaysia), serially diluted in PBS, plated on nutrient agar, and incubated overnight at 35°C, to enumerate viable bacteria. 

Statistics

A two-tailed unpaired Student’s t-test was used to determine significant differences between amounts of sPLA₂ on contact lens types and between wearing schedules. A two-tailed t-test was also used for analysis of bacterial adhesion results. 

Results

There was no significant difference in tear concentrations of sPLA₂ from etafilcon A or Polymacon daily contact lens wearers. Tear concentrations of sPLA₂ from baseline (before contact lens wear) and post-Polymacon contact lens wear were approximately 19 ± 8 μg/mL. Slightly less sPLA₂ was detected in tears after etafilcon A contact lens wear (16 ± 10 μg/mL), but this was not significantly different from the other groups (P = 0.259). 

sPLA₂ was found to absorb to both types of contact lenses. Concentrations of sPLA₂ extracted from etafilcon A daily-wear contact lenses were approximately 5 ng/lens. The concentrations of sPLA₂ extracted from Polymacon daily-wear contact lenses was significantly less at approximately 3 ng/lens, P < 0.05 (Fig. 1A) , which corresponds to 51% less sPLA₂ recovered from etafilcon A lenses. Extended-wear etafilcon A lenses had an sPLA₂ concentration of approximately 6 ng/lens. Extended-wear Polymacon lenses had significantly lower concentrations of sPLA₂ (3 ng/lens; P < 0.05; Fig. 1A ), corresponding to 38% less than etafilcon A lenses. No significant differences were observed between concentrations of sPLA₂ extracted from extended or daily wear for etafilcon A or Polymacon contact lenses. 

Catalytic activity of the enzyme correlated with the amounts of protein extracted from contact lenses (Fig. 1B) . The catalytic activity of sPLA₂ was significantly greater on etafilcon A contact lenses than on Polymacon contact lenses for both extended- and daily-wear contact lens wear schedules (P ≤ 0.013; Fig. 1B ). Polymacon lenses had 94% and 96% less active sPLA₂ than etafilcon A daily- and extended-wear lenses, respectively. Control experiments to determine loss of activity with the extraction method demonstrated that 40% of the activity of commercially bought sPLA₂ was retained after boiling in extraction buffer. 

When incubated with sPLA₂-coated contact lenses, the count of viable S. aureus was reduced on both contact lens types (Fig. 2) . The number of viable bacteria was reduced on etafilcon A contact lenses soaked with sPLA₂ by approximately 44% compared with uncoated control etafilcon A lenses, but the number was not reduced on Polymacon contact lenses. However, differences between sPLA₂-soaked and control lenses were not significant. The trend in activity correlated with that of sPLA₂ concentrations detected on the two types of contact lenses (i.e., more sPLA₂ was recovered from etafilcon A lenses with a corresponding lower load of viable bacterial on these lenses). 

Discussion

This is the first report to show that sPLA₂ deposits on contact lenses from the tear film in both daily- and extended-wear contact lens schedules. We have also demonstrated that the lens material type influenced the amount of enzyme deposited. Etafilcon A contact lenses absorbed more sPLA₂ than Polymacon contact lenses, and the amount did not increase significantly from daily- to extended-wear contact lenses. Although we did not compare contact lens wearers to non–contact-lens wearers, Aho et al. ¹⁵ found that long-term soft contact lens wearers had lower concentrations of sPLA₂ in their tears than did non–contact-lens wearers. We found that there was a trend for less sPLA₂ to be recovered from tears after etafilcon A lens wear, suggesting that the contact lens could be sequestering sPLA₂ from the tears. Diurnal variations in sPLA₂ concentrations do exist and are at their highest from 12 PM until 4 PM. ³³ As post–daily-wear tears were collected during this period, the effects of any contact lens sPLA₂ depletion may have been masked. 

In addition, increasing concentrations of sPLA₂ in tears have been associated with intolerance to contact lens wear. ²⁷ No significant differences were found in sPLA₂ tear concentrations of patients before and after contact lens wear, suggesting that these wearers tolerated the contact lenses well. Concentrations of sPLA₂ in baseline tears in our study (19 ± 8 μg/mL) are within the range of those reported by others (55 ± 34 and 34 ± 21 μg/mL). ^{16 34} Higher sPLA₂ concentrations have also been reported (70 ± 35 and 37 ± 3 μg/mL). ^{17 33} Combined findings indicate that concentrations of sPLA₂ vary throughout the population. 

We found that extended wear of contact lenses did not significantly alter amounts of sPLA₂ on contact lenses, suggesting there is a saturation point for sPLA₂ deposition on a contact lens. The amount of sPLA₂ on contact lenses is most likely related to the charge of the lenses. sPLA₂ is known to have an affinity for anionic bacterial membranes, ^{17 35} which is due to ligand interactions and may also be partly due to cationic residues surrounding the hydrophobic rim of the enzyme. ^{18 35} The attraction to anionic surfaces may be one reason why more sPLA₂ is recovered from etafilcon A contact lenses, as these lenses are anionic, whereas Polymacon contact lenses are nonionic. 

The sPLA₂ remained active on the contact lenses. Polymacon lenses had 50% less total but 90% less active sPLA₂ than etafilcon A lenses. For both contact lens types, activity appeared to be 10 times less, which may be a result of the extraction process, as experiments mimicking extraction conditions yielded low amounts of initial sPLA₂ activity. However, the results reported herein imply that contact lenses absorb sPLA₂ and maintain active sPLA₂ on the lens surface which, in turn, may render the contact lens antibacterial. We tested this hypothesis by applying sPLA₂ on unworn etafilcon A and Polymacon contact lenses. We found that etafilcon A had reduced counts of viable S. aureus compared with Polymacon contact lenses. These results demonstrate that increasing sPLA₂ absorbed onto a contact lens may result in a concomitant reduction in bacterial adhesion to the contact lens. 

The sequestering of this antibacterial agent from the tear film onto the contact lens may be protective for the cornea against contact-lens–associated staphylococcal infection. With the knowledge that this enzyme is also antibacterial for Gram-negative bacteria, ^{23 24} these findings may have implications for strategies in material design aimed at the reduction of contact-lens–driven adverse responses. 

 Presented in part at the annual meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Florida, May 2002.

 Supported by the Australian Federal Government through the Co-operative Research Centres Program (Vision CRC).

 Submitted for publication November 13, 2003; revised April 7, and May 28, 2004; accepted June 4, 2004.

 Disclosure: E.B.H. Hume, None; N. Cole, None; A. Parmar, None; M.E. Tan, None; Y. Aliwarga, None; T. Schubert, None; B.A. Holden, None; M.D.P. Willcox, 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.

 

(A) Amount of sPLA₂ extracted from extended-wear (n = 25) and daily- (n = 39) wear contact lenses. Error bars: SD. No significant differences were observed between the daily- and extended-wear modalities. Significantly more sPLA₂ was recovered from etafilcon A lenses in extended (*P = 0.02) and daily (**P = 0.002) wear than from Polymacon lenses of either lens-wear regimen, by unpaired Student’s t-test. (B) Activity of sPLA₂ extracted from daily- and extended-wear contact lenses. Error bars, SD. Significantly more sPLA₂ was recovered from etafilcon A lenses in extended (*P = 0.0039) and daily (**P = 0.013) wear than from Polymacon lenses of either lens wear regimen, by unpaired Student’s t-test.

Figure 2.

 

Effect of S. aureus adhesion to contact lenses with absorbed sPLA₂. Error bars, SEM. Contact lenses were soaked with 20 ng/mL sPLA₂, washed, and incubated with 1 × 10⁸ cfu S. aureus strain 31. The y-axis represents colony-forming units recovered per lens (cfu/lens). No significant differences were observed between groups.