RESEARCH PAPER

α1-antitrypsin and C-reactive protein levels in tear fluid after continuouscontact lens wear

Clin Exp Optom 2014; 97: 66–71 DOI:10.1111/cxo.12093

Aaron Barrett* ABO NCLE MLSDerek Gnehm* MLSJordan Jones* MLSBarbara C Trask† PhD* Medical Laboratory Sciences Department and† Zoology Department, Weber State University,Ogden, Utah, USAE-mail: btrask@weber.edu

Background: Corneal inflammation has long been associated with contact lens wear and theuse of extended-wear lenses enhances the risk of corneal injury. Elucidation of the molecularmediators of contact lens-associated inflammation has the potential to provide injury-identifying markers early in the inflammatory process, as well as determine potentialtherapeutic targets.Methods: This cross-over study investigated a potential correlation between overnightcontact lens wear and the concentrations of two markers of inflammation, α1-antitrypsinand C-reactive protein, in tear fluid. To obtain baseline measurements, 17 subjects adaptedto wearing silicone hydrogel contact lenses wore their prescribed eye glasses for one week,after which tears were collected and ocular health assessed by a licensed optometrist.Subjects then returned to wearing their prescribed silicone hydrogel lenses continuouslyfor one week. A second tear sample was collected and ocular inflammation was againassessed. Enzyme-linked immunosorbent assays were performed on all tear samples for bothα1-antitrypsin and C-reactive protein.Results: α1-antitrypsin was significantly (p = 0.01) elevated after continuous contact lenswear, with increases above baseline concentrations averaging 2.48-fold. Optometric assess-ment of inflammation loosely correlated with levels of this inflammatory marker. C-reactiveprotein was detected in the tears of subjects at both times and levels were also slightly elevatedafter extended lens wear, but not significantly (p > 0.5) and not consistently in all subjects.Conclusion: The results of this study suggest that α1-antitrypsin in tear fluid may be usefulas an early marker of contact lens-associated ocular irritation and inflammation. Thepresence of C-reactive protein in the tears of contact lens wearers is a novel finding which,while not correlative with either α1-antitrypsin concentrations or clinically observableinflammation, may warrant further study.

Submitted: 14 November 2012Revised: 28 January 2013Accepted for publication: 5 February 2013

Key words: acute phase proteins, contact lenses, continuous wear, inflammation, tears

Silicone hydrogel soft contact lenses areapproved and commonly prescribed forovernight wear. They are preferred by manyof the 36 million contact lens wearers inthe United States for their reported com-fort, convenience and ease of use.1–4 It isestimated that over three million peoplein the US now wear silicone hydrogel lensesfor cosmetic purposes.1 Retail sales of theselenses are on the rise, having accounted for29 per cent of new soft lens fittings in 2005and roughly 67 per cent in 2011.1,5 In addi-tion to those for whom silicone hydrogellenses have been prescribed, many daily-wear soft contact lens wearers opt to use theirlenses during sleep, disregarding manufac-turer and clinician recommendations.5

Regardless of the type of lens, overnightwear has been associated with an increased

risk of corneal infection and injury. Forexample, relative to daily use soft contactlenses, the risk of developing ulcerative kera-titis, one of the more severe complicationsassociated with overnight lens wear, hasbeen reported to be elevated approximatelyfour- to 15-fold.6,7 In one study, over 20 percent of all reported cases of microbial kera-titis were associated with contact lens wear.8

Similarly, corneal infiltrative events are eighttimes more likely for patients who sleep intheir lenses.9

As the site of contact lens-induced injury,the human cornea is comprised of a super-ficial, avascular stratified squamous epithe-lium that is supported by an underlyingcollagen-rich stroma containing fibro-blastic keratinocytes. Following injury to thecorneal epithelium, the corneal stroma

exhibits the typical hallmarks of inflamma-tion, including pain, redness and exudativeleukocytic infiltration.10

Because of the prevalence of contact lens-associated corneal injury and subsequentinflammation, understanding the processesby which corneal injury occurs is paramountto its prevention. Currently, the combina-tion of poor lens hygiene and the resultantbacterial contamination of lenses, often byPseudomonas sp. is presumed to be one of theprimary causes of corneal injury associatedwith contact lens wear.7,10,11 At least one studysuggests that contact lens-associated cornealinjury may not be as tightly linked with poorlens hygiene as might be expected, if bacte-rial contamination were its only cause.12 Inaddition to understanding the mechanismsby which lens-associated injuries occur, it is

C L I N I C A L A N D E X P E R I M E N T A L

OPTOMETRY

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equally as important to investigate the physi-ologic responses to these injuries. Under-standing these responses may both providemeans of early detection and lead to theidentification of physiologic targets for phar-maceutical intervention.

Some potential targets may have alreadybeen identified. Although several solublefactors associated with inflammation havebeen recognised, some of which are oftenused clinically to assess acute injury, theirpresence in tears has received relativelylittle attention. Among these inflammatorymarkers are cytokines, anti-bacterial enzy-mes and the acute phase proteins, includingα1-antitrypsin and C-reactive protein.

Along with mucins produced by conjunc-tival goblet cells, soluble tear proteins (manyof which are produced by secretory cellsof the lacrimal glands) function to lubricatethe anterior surface of the eye, to maintainthe health of corneal epithelial cells and toprotect the cornea against injury. The totalprotein concentration of normal tear fluidhas been estimated to be approximately7.5 mg/ml, which is about 10 per cent that ofblood serum.13 Using highly sensitive massspectrometric techniques, as many as 491proteins have been identified in the tears ofhealthy subjects. Of the 68 secreted proteinsidentified with this method, 20 are involvedin the defense response or have known anti-microbial function.14 The most abundant ofthese is the anti-bacterial enzyme lysozyme,which comprises roughly 30 per cent of totaltear protein.13

The protein composition of normal tearfluid changes in response to extendedcontact lens wear.13,15 While not identified byde Souza, Godoy and Mann14 other proteinsthat modulate the inflammatory response,including various interleukins and othercytokines, can be produced by the lacrimalgland and conjunctival epithelium, as wellas by corneal epithelial cells and stromalkeratinocytes in response to infection,disease and/or injury.16,17

To assess whether acute phase proteins intear fluid might be correlated with cornealinflammation, we performed a cross-overstudy in which clinical assessments ofcorneal inflammation before and after con-tinuous contact lens wear were compared tothe tear fluid concentrations of two solublemarkers of inflammation, α1-antitrypsinand C-reactive protein. α1-antitrypsin wasselected because, as a normal tear fluid com-ponent, it has previously been describedto increase in certain inflammatory condi-

tions.18,19 Although not identified as a tearfluid component prior to this study,C-reactive protein is routinely used as amarker of inflammation severity in manydisorders, including rheumatoid arthritis,inflammatory bowel disease and myocardialinfarction.20–22

METHODS

SubjectsSeventeen (10 male) non-astigmatic adultsubjects adapted to silicone hydrogelcontact lens wear were selected for thisstudy. Ten subjects (seven male) wereadapted to lenses of lotrafilcon B (threeto Air Optix and seven to O2 Optix, CIBAVision, Novartis AG, Duluth, GA, USA) andseven (three male) to senofilcon A lensmaterial (Acuvue Oasys, Vistakon, Johnsonand Johnson, Jacksonville, FL, USA). Meantypical lens wear of all subjects was 5.8 daysper week (median = 7) for an averageof 16.7 hours per day (median = 14). Threesubjects were adapted to continuous wearand three subjects had never worn lensesovernight until participation in this study.Of the remaining subjects, usual overnightwear was, on average, 9.7 days per month(median = 4). All subjects (mean age 21.8years, median of 21 and range 18 to 35 years)were free of current eye disease as well as per-sonal and family history of eye disease. Allwere otherwise healthy and non-smoking.Informed consent and institutional reviewboard approval were obtained, in accord-ance with both the Association for Researchin Vision and Ophthalmology and theHelsinki convention.

Tear fluid collectionTo obtain baseline levels of inflammatoryproteins in tear fluid, study subjects wereasked to wear only their prescribed eye-glasses for a period of one week. No reportsof eye irritation were made during thisperiod. To obtain test levels, study subjectswere then asked to wear their prescribedsilicone hydrogel contact lenses continu-ously for a period of one week. Preserva-tive-free lubricating eye drops (SystanePreservative-Free, Alcon, Novartis, Irvine,CA, USA) were provided with direction touse as needed. Some subjects did indicateexperiencing dry eyes and irritation relatedto contact lens wear during this period butno other injuries or irritation unrelated to

lens wear were reported. At both times, tearfluid was collected by placing 8.0 mm polyu-rethane minisponges (Pele Tim, VOCOAmerica, Briarcliff Manor, NY, USA) in botheyes as previously described for a maximumof 10 minutes or until sponges were visiblysaturated.23 Sponges were carefully removedso as not to induce corneal injury. Saturatedsponges were placed in filtered micro-centrifuge tubes and subjected to centrifu-gation at 16,000 g for 10 minutes. Fluid fromboth eyes of each subject was pooled, creat-ing total volumes for each subject rangingfrom 40 to 110 ml. Tear samples were storedat -80°C until diluted for assay.

Clinical corneal assessmentPrior to and following continuous contactlens wear, a licensed optometrist examinedeach participant’s eyes and recorded anyclinically observable ocular inflammation ona scale from zero to 3 (zero = no inflam-mation, 1 = mild, 2 = moderate, 3 = severe).Parameters used to score ocular inflamma-tion were as follows:1. some injection of the conjunctiva; no

corneal infiltrates or limbal oedema2. conjunctival injection and limbal

oedema (less than 50 per cent or 180degrees of the limbus); early cornealinfiltrates isolated to the limbus

3. conjunctival injection, limbal oedema(greater than 50 per cent or 180 degreesof the limbus); epithelial oedema andcentral corneal infiltrates.

ELISAs for α1-AT and CRPCommercially-available enzyme-linked im-munosorbent assay (ELISA) kits for α1-AT(GenWay Biotech, San Diego, CA, USA)and C-reactive protein (Helica Biosystems,Santa Ana, CA, USA) were used according tomanufacturers’ protocols to measure tearfluid concentrations of these acute phaseproteins. Lower limits of detection forthese assays were 7.8 ng/ml and 0.2 ng/ml,respectively. Tear fluid samples were diluted1 : 3 serially down to 1 : 243 in the diluentsolution supplied (α1-antitrypsin) or exclu-sively diluted 1 : 6 in phosphate-bufferedsaline, pH 7.4, containing one per centbovine serum albumin (both from SigmaChemical Co, St. Louis, MO, USA) prior toassay. Samples were assayed in duplicate.Only 10 of the 17 subjects (five male; fourwearing lotrafilcon and six wearing seno-filcon lenses) provided sufficient tear fluidsuch that C-reactive protein concentrations

α1-AT and CRP with continuous lens wear Barrett, Gnehm, Jones and Trask

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could be assayed. An automatedspectrophotometer (Versa Max, MolecularDevices, Sunnyvale, CA, USA) was used toquantify analyte levels. Absorbance datafrom all dilutions within the linear range of astandard curve were averaged to calculatetear fluid concentrations of α1-antitrypsin.

Statistical analysisPaired Student’s t-tests were used to assessstatistical significance when comparing base-line to test inflammatory protein concentra-tions; p-values of ≤ 0.05 were consideredsignificant. Data are shown as mean valuesand standard error of the mean (SEM).

RESULTS

Baseline levels of α1-antitrypsin (mean =11.07 mg/ml) became significantly elevated(p = 0.01) after continuous lens wear in 16 of17 subjects, with an average elevation of9.5 mg/ml (Figure 1). While two individualsexperienced only small increases (less thansix per cent), α1-antitrypsin levels rose inmost subjects, with elevations rangingbetween 14 and 103.5 per cent (that is,1.14- to more than 10-fold; mean 2.62-foldincrease for those subjects experiencing anincrease). Seven of these 14 subjects experi-enced greater than two-fold increases in tearfluid α1-antitrypsin. Of these, all but onedisplayed clinically observable increasesin inflammation as well (Table 1). When

lotrafilcon B and senofilcon A lens wearerswere assessed independently, no significantdifferences were detected (not shown)between them in either baseline or continu-ous wear α1-antitrypsin levels or in clinicallyobserved inflammation.

Baseline C-reactive protein levels (mean =9.15 ng/ml) increased slightly to an averageof 10.95 ng/ml; however, not consistently,as levels increased in only four out of 10samples tested. The observed slight eleva-tion in C-reactive protein was not statisticallysignificant (p = 0.74; Figure 2). Samplesfrom individuals with the most extensivechanges in clinically observable inflamma-tion following continuous wear were unfor-

tunately unable to be assayed for C-reactiveprotein due to insufficient tear fluid col-lected. Although each of the four subjects,who displayed an elevation in tear fluidC-reactive protein also displayed moreadvanced observable inflammation, two outof six subjects, for whom C-reactive proteinlevels did not increase also experiencedobservable exacerbations in corneal in-flammation (data not shown). Similar toα1-antitrypsin, when lotrafilcon B andsenofilcon A lens wearers were assessed inde-pendently with respect to C-reactive proteinconcentration, no significant differencesbetween them were detected in either base-line or continuous wear levels.

Baseline CW

25

20

15

10

5

0

μg/m

l

Figure 1. Tear fluid α1-antitrypsin con-centrations at baseline and following con-tinuous wear. Tear fluid α1-antitrypsinconcentrations significantly increased (p =0.01) following continuous wear (CW) ofsilicone hydrogel contact lenses for oneweek. Graph represents the mean tear fluidconcentration of 17 subjects (±SEM).

Sample No. Fold increasein α1-AT

Graded baselineinflammation

Graded inflammationafter CW

Inflammation Δ

1 10.35 0 1 +12 0.52 1 1 03 2.74 1 2 +14 1.21 0 0 05 1.01 1 2 +16 2.29 1 2 +17 1.23 2 2 08 2.16 0 2 +29 1.53 1 2 +1

10 1.9 0 1 +111 6.25 0 2 +212 1.14 2 2 013 1.05 1 1 014 2.44 0 2 +215 1.93 1 2 +116 1.81 1 2 +117 2.67 2 2 0

x 2.48 0.88 1.65 +0.82

Table 1. Tear fluid α1-antitrypsin (α1-AT) concentrations compared with clinicallyobservable ocular inflammation show a correlation. Differences in tear fluidα1-antitrypsin (α1-AT) (fold increase in continuous wear relative to baseline) for eachsubject are shown in the column on the left. Inflammation was graded by a licensedoptometrist on a 0–3 scale, as described in ‘Methods’ both before (baseline) and aftercontinuous wear (CW) for one week. Differences in observed inflammation after CW(relative to baseline), if present, are indicated in the right column (Inflammation Δ). Sixsubjects (No. 1, 3, 6, 8, 11 and 14) experienced a two-fold or greater increase in tear fluidα1-AT concentration. A single subject (No. 17) experienced an increase in tear fluidα1-AT with no concomitant clinically observable inflammation, while another (No. 5)experienced an increase in clinically observable inflammation with no concomitantelevation in tear fluid α1-AT.

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DISCUSSION

Understanding the molecular mechanismsunderlying contact lens-associated inflam-mation and its resolution is important,as it is a major cause of morbidity in theUnited States. This study sought to correlatethe concentrations of two inflammatoryproteins in tear fluid, α1-antitrypsin andC-reactive protein, with continuous wear ofcontact lenses. Our results are similar tothose reported in previous studies linkingα1-antitrypsin with corneal injury in that wealso observed a significant increase in tearfluid levels of this marker relative to base-line.19 Although α1-antitrypsin is normallypresent in tears at low concentration,18

Prause19 observed an increase in tear fluidα1-antitrypsin in patients with corneal ulcersof variable origin, relative to controls. Whilethe concentration of α1-antitrypsin inpatients with corneal ulcers in the formerstudy was elevated approximately 20-fold,only a two-fold increase was observed in thepresent study following continuous wear;however, this small increase was signifi-cant and is consistent with α1-antitrypsinelevation being indicative of subclinicalinflammation. As expected, the precise com-position of lens material was not found tosignificantly impact α1-antitrypsin concen-trations either before or after continuouswear (p = 0.58), although the mean levels

were slightly higher in subjects followingcontinuous wear of lotrafilcon B (20.58 mg/ml) than in subjects wearing senofilcon Alenses (18.5 mg/ml).

While the primary site of tear fluid proteinsynthesis is in the secretory epithelial cellsof the lacrimal gland,24 Prause19 postulatedthat injury-induced increases in vascularpermeability of conjunctival vessels allowedfor selective leakage of α1-antitrypsin fromthe systemic circulation in his corneal ulcerpatients. The source of α1-antitrypsin in tearfluid following continuous wear requiresfurther investigation. Additionally, the func-tion of α1-antitrypsin elevation followingcontinuous wear is also unknown, althoughthis proteinase inhibitor is generally thoughtto regulate the inflammatory process andpromote healing.13,25 Although continuouswear has been associated with increasedincidence of microbial keratitis, the con-centrations of α1-antitrypsin measuredfollowing continuous wear are not consist-ent with severe bacterial infection.8 Whilethe elevations observed in this study couldportend the presence of higher bacterialnumbers, bacterial lens contamination wasnot assessed. Therefore, further investiga-tion is necessary to correlate bacterial num-bers with α1-antitrypsin concentrations.

Clinically observable inflammation wascorrelated with increases in tear fluidα1-antitrypsin; however, not as tightly asexpected, based upon the clear associationsbetween α1-antitrypsin levels and otherinflammatory processes that have beenreported in the literature.22,26,27 It is possiblethat the subject displaying clinically observ-able inflammation without a concomitantincrease in α1-antitrypsin experienced someacute exposure to an ocular irritant immedi-ately prior to clinical assessment, such thatthe elevation was not continuous wear-related. While no such injury or irritationwas reported, a survey intended to reveal anyocular injury or irritation was completed byour subjects at the time of tear collection,rather than at the time of clinical assessment.Completion of the questionnaire at the sametime as both tear collection and clinicalassessment would certainly have negated thispossibility. Conversely, one individual in ourstudy experienced a greater than two-foldincrease in α1-antitrypsin without an associ-ated change in observable inflammation.Although participants were selected for nochronic systemic medical problems, oneexplanation for this observation is thatthis subject was experiencing an acute infec-

tion or some other inflammatory eventthat resulted in increased circulatingα1-antitrypsin. This increased serum con-centration could have been reflected in tearfluid levels of this protein. Tear fluid isknown to contain many proteins that are alsopresent in serum (for example, albumen,immunoglobulin G, transferrin),18 althoughto our knowledge, specific correlationsbetween serum and tear fluid concen-trations of inflammatory proteins havenot been performed. Additional studies inwhich serum α1-antitrypsin levels are mea-sured along with tear fluid concentrationsmay shed light on this discrepancy.

Unlike α1-antitrypsin, C-reactive proteinlevels in tear fluid were not significantlyincreased with continuous wear nor wasthere a discernible association betweenC-reactive protein levels and clinicallyobservable inflammation. Although C-reactive protein has been observed toincrease in other ocular tissues, such asthe retina in association with age-relatedmacular degeneration, to the best ofour knowledge this is the first report ofC-reactive protein in tear fluid.28 Unlikeprevious studies that inventoried tearfluid components using very sensitive tech-niques,14,18,29 we found C-reactive protein tobe present even in our baseline samples.Ham, Jacob and Cole30 also failed to identifyC-reactive protein in an assessment oftear components using matrix-assisted laserdesorption ionisation – time of flight massspectrometry; however, rabbits were thesource of the normal and dry eye samplesused in his study. Although both de Souza,Godoy and Mann14 and Green-Churchand colleagues18 used normal human tearsamples in their proteomic approachesto elucidating tear fluid composition,C-reactive protein was not among theprotein components identified. While it ispossible that C-reactive protein concentra-tions were too low to be detectible in theseinvestigations, other likely explanationsinclude inter-species variation in tearfluid protein components and differencesin method of collection. Alternatively, toexplain the absence of C-reactive protein innormal human tear fluid, it is possible thatadaptation to contact lens wear, such as thatexperienced by our research subjects evenbefore participation in this study, resultsin continual, low-level production of thisacute phase protein. This requires addi-tional investigation through comparison ofC-reactive protein concentrations in the

Baseline CW

14

12

10

4

6

8

2

0

ng/m

l

Figure 2. C-reactive protein concentra-tions in tear fluid at baseline and followingcontinuous wear. C-reactive protein con-centrations in tear fluid did not signifi-cantly increase following continuous wear(CW) of silicone hydrogel contact lensesfor one week (p = 0.74). Graph representsthe mean of tear fluid concentration from10 subjects (±SEM).

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tears of non-contact lens wearers and thoseof adapted lens-wearing subjects.

The source of tear fluid C-reactive pro-tein remains in question. C-reactive proteinis generally synthesised by hepatocytes inresponse to increases in interleukin-6 (IL-6).31 IL-6 has been reported to be present innormal non-reflexive tears32 and IL-6 levelsare increased transiently both in night tearsand in response to contact lens wear.33,34

Therefore, it seems likely that the tear fluidof our adapted lens-wearing subjects con-tained IL-6 that could possibly stimulatelocal C-reactive protein production. Whichcells might be responsible for this produc-tion, if it occurs, is speculative. Corneal epi-thelial cells and keratinocytes, as well asconjunctival epithelial cells, are known toproduce numerous pro-inflammatory pro-teins.16,17 It is also possible that, similar towhat has been proposed for α1-antitrypsin,the tear fluid C-reactive protein detected inthis study was derived from serum. Normalserum concentrations of C-reactive protein,which are generally less than 1.0 mg/ml,are comparable to those measured in thisstudy.35

C-reactive protein has been positively asso-ciated with inflammatory processes and haslong been used as a clinical indicator ofacute inflammation.31 C-reactive protein hasbeen postulated to be more than an indica-tor and to contribute to the progression ofinflammatory disease.36–38 Pro-inflammatoryeffects of this acute phase protein includecomplement activation, pathogen opsonisa-tion and leukocyte activation; however, con-centrations required to obtain these effectsare generally at least 10-8 mol/l and often10-7 mol/l, roughly 10- to 100-fold higherthan what was measured in our tear fluidsamples.31 While the concentrations ofC-reactive protein detected in this study areinsufficient to produce any of the aforemen-tioned pro-inflammatory effects, it is possi-ble that tear fluid samples obtained at othertimes during continuous wear might havecontained higher concentrations. C-reactiveprotein concentrations are known to fluctu-ate rapidly in response to inflammation.In addition, variable forms of C-reactiveprotein, including aggregates and peptidefragments, have biologic functions that varyfrom those of the intact protein.39 Althoughthe assay used to measure C-reactive proteinin this study was able to detect all forms ofthe protein, it was unable to discern betweenthem. Therefore, their relative concentra-tions in tear fluid are unknown.

Further investigation into the causes,timing and sources of increased α1-anti-trypsin in tear fluid following continuouswear might enable this acute phase proteinto be used effectively as an indicator of sub-clinical ocular inflammation. While theresults of this study seem to discount tearfluid C-reactive protein as an appropriatemarker of ocular inflammation, additionalstudies assessing the concentration of thisacute phase protein at various timesthroughout a week of continuous contactlens wear and/or assessing C-reactive pro-tein aggregates and proteolytic fragmentsmay yield different results.

ACKNOWLEDGEMENTSThe authors would like to thank Dr MarkTaylor for his expertise in clinically evaluat-ing optic inflammation, the study subjectsfor their participation and Dr Leonard GaryNielsen for his contributions to the studydesign.

This study was funded by the WeberState University Office of UndergraduateResearch and by the George S. & DeloresDore Eccles foundation.

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α1-AT and CRP with continuous lens wear Barrett, Gnehm, Jones and Trask

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