Coculture with intraocular lens material-activated macrophages induces an inflammatory phenotype in...

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published online 2 October 2014J Biomater ApplRobert Pintwala, Cameron Postnikoff, Sara Molladavoodi and Maud Gorbet

lens epithelial cellsCoculture with intraocular lens material-activated macrophages induces an inflammatory phenotype in

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Soft Tissues and Materials

Coculture with intraocular lensmaterial-activated macrophages inducesan inflammatory phenotype in lensepithelial cells

Robert Pintwala1, Cameron Postnikoff1, Sara Molladavoodi2 and Maud Gorbet1

Abstract

Cataracts are the leading cause of blindness worldwide, requiring surgical implantation of an intraocular lens. Despite

evidence of leukocyte ingress into the postoperative lens, few studies have investigated the leukocyte response to

intraocular lens materials. A novel coculture model was developed to examine macrophage activation by hydrophilic

acrylic (poly(2-hydroxyethyl methacrylate)) and hydrophobic acrylic (polymethylmethacrylate) commercial intraocular

lens. The human monocytic cell line THP-1 was differentiated into macrophages and cocultured with human lens

epithelial cell line (HLE-B3) with or without an intraocular lens for one, two, four, or six days. Using flow cytometry

and confocal microscopy, expression of the macrophage activation marker CD54 (intercellular adhesion molecule-1) and

production of reactive oxygen species via the fluorogenic probe 20,70-dichlorodihydrofluorescein diacetate were exam-

ined in macrophages. a-Smooth muscle actin, a transdifferentiation marker, was characterized in lens epithelial cells. The

poly(2-hydroxyethyl methacrylate) intraocular lens prevented adhesion but induced significant macrophage activation

(p< 0.03) versus control (no intraocular lens), while the polymethylmethacrylate intraocular lens enabled adhesion and

multinucleated fusion, but induced no significant activation. Coculture with either intraocular lens increased reactive

oxygen species production in macrophages after one day (p< 0.03) and increased expression of a-smooth muscle actin

in HLE B-3 after six days, although only poly(2-hydroxyethyl methacrylate) induced a significant difference versus control

(p< 0.01). Our results imply that—contrary to prior uveal biocompatibility understanding—macrophage adherence is

not necessary for a strong inflammatory response to an intraocular lens, with hydrophilic surfaces inducing higher

activation than hydrophobic surfaces. These findings provide a new method of inquiry into uveal biocompatibility, spe-

cifically through the quantification of cell-surface markers of leukocyte activation.

Keywords

Lens epithelial cells, macrophages, intraocular lens biomaterials, biocompatibility, coculture in vitro model, flow

cytometry, confocal microscopy

Introduction

The crystalline lens is the structure of the eye respon-sible for accommodation, or the ability of the eye tofocus on objects at varying distances. In humans, com-promise of the naturally clear lens can cause a partial orcomplete loss of vision. One form of compromise is thedevelopment of cataracts, or a clouding of the lens. Atpresent, the only treatment for cataracts is the surgicalremoval of the lens and its substitution with an artificialimplant known as an intraocular lens (IOL), commonlymade from foldable hydrophobic acrylic, hydrophilicacrylic, and silicone materials but originally made

from rigid polymethylmethacrylate (PMMA). In somecases, the trauma of surgery and subsequent implant-ation can induce the formation of secondary cataracts.1

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DOI: 10.1177/0885328214552711

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1Faculty of Engineering, Department of Systems Design Engineering,

University of Waterloo, Waterloo, Ontario, Canada2Faculty of Engineering, Department of Mechanical Engineering,

University of Waterloo, Waterloo, Ontario, Canada

Corresponding author:

Maud Gorbet, Systems Design Engineering, University of Waterloo, 200

University Avenue West, Waterloo, Ontario, N2L 3G1 Canada.

Email: [email protected]

at UNIV OF WESTERN ONTARIO on October 14, 2014jba.sagepub.comDownloaded from



Posterior capsular opacification (PCO) is one form ofsecondary cataract and the most frequent complicationof cataract surgery, occurring in 38.5% of patients after3 years.2 Despite decades of effort to improve IOL bio-compatibility and haptic design, PCO remains animportant issue in ophthalmology.3

The physiological lens is comprised of three mainparts: the lens capsule, a type-IV collagen-rich base-ment membrane, the lens epithelium, a monolayer oflens epithelial cells on the anterior surface of the cap-sule, and the lens fibers, transparent, enucleated cellsfilling the volume of the lens. During cataract extrac-tion, the surgeon attempts to remove the epithelium inits entirety. Oftentimes, however, islets of lens epithelialcells survive and experience rapid regrowth and migra-tion on the anterior surface that may continue onto thepreviously cell-free posterior capsular surface, causingthe scattering of light.1,4 Some lens epithelial cellsundergo transdifferentiation into myofibroblast cellsvia the epithelial–mesenchymal transition, leading tothe pathological expression of the contractile proteina-smooth muscle actin (a-SMA), and a loss of expres-sion of type-IV collagen and the intercellular cadherinE-cadherin.5 These myofibroblasts overproduce pro-teins of the extracellular matrix (ECM) such as fibro-nectin, type-I and type-II collagen, and tenascin.5 As aresult of this proliferative and fibrotic response, the lenscapsule wrinkles, causing the substantial loss of visionobserved during PCO.4 Meta-studies of IOL biocom-patibility in vivo have noted a lower incidence rate ofPCO in patients with hydrophobic IOLs compared tohydrophilic IOLs of the same haptic design.6–9 Thiseffect of IOL surface chemistry is believed to be definedby lens epithelial cell adhesion, and therefore migratoryand proliferative potential.10

The primary chemical mediator of PCO has beendemonstrated both in vitro and in vivo to be the cyto-kine transforming growth factor beta (TGF-b).11–18

Additionally, members of the matrix metalloproteinase(MMP) family of zinc-dependent endoproteinases—specifically MMP-2 and MMP-9—have been shownto play a regulatory role in the formation of PCO.5

Interestingly, these three mediators are secretory prod-ucts of macrophages, who play a crucial role in regulat-ing inflammatory processes and the wound-healingresponse to biomaterials.19–21 Macrophages are alsoimportant cellular mediators of fibrotic diseases else-where in the body.22,23 In the eye, macrophages havebeen observed on the surface of IOLs explanted fromhuman patients.24–27 In an animal model of PCO,depletion of macrophages reduced the number of lensepithelial cells in the center of the posterior capsule,28

suggesting that macrophages may play a role in lensepithelial cell mobility during PCO. Despite this evi-dence, few studies have investigated the leukocyte

biocompatibility of IOL materials (often referred toas uveal biocompatibility) and its potential contribu-tion to PCO development. To date, uveal biocompati-bility has been measured by counting the numberof adherent leukocytes on the surface of an IOL,9,29,30

the hypothesis being that a higher number of adher-ent cells indicates a less biocompatible material.However, there is limited information on the potentialfor IOL materials to induce an inflammatory responsein leukocytes, and how this response is related toadhesion.

Macrophages are highly responsive to their tissueenvironment and may exhibit a combination of hostdefence, wound-healing, and immune regulationinflammatory phenotypes.31 The exact nature of macro-phage activation can be characterized by examining theexpression of cell-surface proteins involved in theforeign-body response, such as CD14, CD36, andCD54, as well as the production of reactive oxygen spe-cies (ROS) and reactive nitrogen species (RNS), both ofwhich are used to attack and degrade macrophage tar-gets.32 Intercellular adhesion molecule-1 (ICAM-1),also known as CD54, is upregulated on macrophagesin response to foreign-body stimuli in vitro andin vivo.33–37 CD36, a multi-ligand scavenger receptor,is downregulated in macrophages by the anti-inflamma-tory, wound-healing cytokine TGF-b and upregulatedby a selective number of pro-inflammatory cytokinesand is therefore useful for examining the wound-healing response of macrophages.38,39 CD14 is apattern recognition receptor for bacterial and viralpathogens and can be used to specifically examine thecontribution of microbial contamination to the overallhost-defence response.40

In this study, we hypothesized that material-activated macrophages may secrete pro-inflammatorycytokines and other mediators to induce a PCO-likephenotype in lens epithelial cells. Two biocompatibilityoutcomes were investigated: whether common IOL bio-materials could activate macrophages and whether thisactivation could induce an inflammatory phenotype inlens epithelial cells. Cell activation was characterizedusing flow cytometry and confocal microscopy.

Methods and materials

Cell culture

A novel in vitro coculture model of the postoperativelens environment was developed. The human acutemonocytic leukemia cell line THP-1 (ATCC,Manassas, VA) was selected as the macrophage surro-gate for its confirmed ability to produce macrophage-secretory products.41 The SV-40 virus immortalizedhuman lens epithelial cell line HLE B-3 (ATCC) was

2 Journal of Biomaterials Applications 0(0)




used as the lens epithelial surrogate due to its wide-spread use in in vitro PCO studies.42–44

THP-1 monocytes were cultured in Roswell ParkMemorial Institute (RPMI) 1640 cell culture medium(Invitrogen, Oakville, ON) supplemented with 10%fetal bovine serum (FBS; VWR, Mississauga, ON) and0.9% penicillin/streptomycin (Invitrogen, Oakville,ON) and maintained in suspension at 5� 105 cells/mlat 37�C and 5% CO2. THP-1 monocytes were differen-tiated into macrophages using the protein kinase C(PKC) activator phorbol 12-myristate 13-acetate(PMA; Sigma–Aldrich, Oakville, ON), following anoptimized protocol combining that of Park et al.45

and Daigneault et al.46 THP-1 monocytes were seededin 6-well tissue-culture polystyrene (TCPS) plates at aconcentration of 5� 105 cells/well and treated with5 nM PMA for 72 h. THP-1 cells were inspected after24 and 48 h for adherence and viability. At 72 h, mediawas replaced without PMA, and the adherent THP-1cells were allowed to rest for 48 h. Media was replacedonce again and the THP-1 macrophages were allowed torest for a further 48 h. To prepare for coculture, maturedifferentiated macrophages were washed twice withroom-temperature phosphate-buffered saline (PBS) for5min and disassociated by incubation with TrypLETM

Express (Invitrogen, Oakville, ON) for 20min at 37�C.Cells were centrifuged in RPMI 1640 media with 10%FBS, resuspended in fresh media, and transferred to thecoculture plate.

HLE B-3 were cultured in Dulbecco’s ModifiedEagle Medium (DMEM; Invitrogen, Oakville, ON)supplemented with 10% FBS and 0.9% penicillin/streptomycin and maintained at 37.0�C in 5% CO2.To prepare for coculture, cells were serum starved for24 h.12 Cells were dissociated from a flask with TrypLEExpress and then centrifuged in serum-free DMEM.

To begin coculture, 24-well polyethylene terephthal-ate (PET) 1-mm membrane hanging cell-culture inserts(EMD Millipore, Billerica, MA) were inverted in a 6-well plate, and 5� 104 HLE B-3 cells were seeded ontothe superior surface. Inserts were incubated overnightto allow for strong cellular adherence, and thenreturned to their normal orientation in serum-freemedia in a 24-well plate (EMD Millipore) until thestart of the coculture.

IOL preparation

Commercially available, surgical-grade PMMA andpoly(2-hydroxyethyl methacrylate) (pHEMA) IOLswere purchased from Freedom Ophthalmic(Mississauga, ON) in both square and round-edgeddesigns. To prepare for culture, the IOLs were incu-bated in RPMI 1640 with 10% FBS for 2 h to allowfor protein adsorption.47

Coculture

The coculture system diagram is shown in Figure 1.Prepared IOLs were transferred into fresh wells of a24-well Millipore plate in media containing 10% FBS.Disassociated mature THP-1 macrophages were thenseeded at 1� 105 cells/well. Plates were incubated for2 h to allow for macrophage adherence. Hanging insertswith adherent HLE cells were then introduced to thewells containing one of the following: macrophages andan IOL (treatment), macrophages and no IOL (con-trol), an IOL and no macrophages, or no macrophagesand no IOL. Coculture plates were incubated for eitherone, two, four, or six days before analysis. Media wasreplaced every two days.

Immunolabeling reagents

Fluorescein isothiocyanate (FITC)-conjugated mousemonoclonal antibody against CD36 (multiligand scav-enger receptor), R-phycoerythrin-cytochrome 5 (PE-Cy5)-conjugated monoclonal antibody against CD45(human leukocyte common antigen), R-phycoerythrin(PE)-conjugated monoclonal antibody against CD54(intercellular adhesion molecule 1 or ICAM1), PE-conjugated mouse monoclonal antibody against CD14(pattern recognition receptor), Alexa Fluor� 647(AF647)-conjugated mouse monoclonal antibodyagainst E-cadherin, and PE-conjugated polyclonal anti-body against rabbit IgG produced in donkey were pur-chased from Becton Dickinson Pharmingen(San Diego, CA, USA). Monoclonal rabbit antibodyagainst fibronectin, and FITC-conjugated mousemonoclonal antibody against a-SMA were purchasedfrom Sigma–Aldrich. All other chemicals were of ana-lytical reagent grade.

Flow cytometry

Upon completion of the coculture, inserts with adher-ent HLE cells were removed from the coculture system.

Figure 1. Coculture system diagram. Inserts are inverted and

lens cells are seeded on the superior surface before being

returned to their normal orientation in a well-containing adher-

ent macrophages and an IOL. Note: IOL: intraocular lens.

Pintwala et al. 3




Wells containing macrophages were incubated at 37�Cwith TrypLE Express for 45min. THP-1 cells were thencentrifuged in media containing 10% FBS and resus-pended in 100 ml fresh media with serum. Aliquots of50 ml were incubated in saturating concentrations ofconjugated fluorescent monoclonal antibodies for 1 hat room temperature in the dark. Leukocyte popula-tions were identified by adding anti-CD45 antibody toall samples. To assess macrophage activation, sampleswere incubated with anti-CD54 or anti-CD14 antibody,as well as anti-CD36 antibody. After incubation, sam-ples were diluted with HEPES-Tyrode’s buffer (HTB:137mM NaCl, 2.7mM KCl, 5mM MgCl2, 3.5mMHEPES, 1 g/L glucose, and 2 g/L bovine serum albumin(BSA), PH 7.4) and fixed with paraformaldehyde (finalconcentration of 1% w/v). Samples were read on athree-color FACSCalibur flow cytometer (BectonDickinson).

To assess intracellular ROS/RNS production,macrophages were cultured with or without an IOLfor 24 h. Macrophages on the IOL surface were disso-ciated separately from those on the TCPS surface.After centrifugation, samples were incubated inserum-free media containing 100 mM of 20,70-dichloro-dihydrofluorescein diacetate (DCFH-DA) (Invitrogen)for 30min at 37�C in the dark. Cells were read imme-diately on a three-color FACSCalibur flow cytometer.Samples were analyzed to completion. Cell counts foradherent macrophage on each IOL were performed inCellQuest Pro (BD) by distinguishing single cells fromsubcellular debris on the basis of size and granularity,estimated by forward scatter and side scatter,respectively.

Inserts with adherent HLE cells were submerged in0.25% Trypsin–ethylenediaminetetraacetic acid(EDTA) (Invitrogen) and incubated at 37�C for 2 h ona shaker at 250 rpm. Once detached, cells were resus-pended in DMEMwith 10% FBS before centrifugation.Excess media was removed. Cells were resuspended andthen fixed in paraformaldehyde at a final concentrationof 4% w/v for 15min, at room temperature in the dark.Samples were washed, and cells were resuspended andpermeabalized with 0.5% Triton-X (Sigma–Aldrich) for5min, at room temperature in the dark. Sampleswere then centrifuged and resuspended in PBS contain-ing 1% w/v fatty-acid free BSA (EMD Millipore).Samples were separated into three aliquots and stainedwith antibodies against either a-SMA (FITC conju-gated), E-cadherin (AF647 conjugated), or fibronectinovernight, at 4�C in the dark. The following morning,samples containing anti-fibronectin were labeled withPE anti-rabbit IgG secondary antibody for 1 h, atroom temperature in the dark. Samples were read imme-diately on a three-color FACSCalibur flowcytometer (Becton Dickinson).

Confocal microscopy

Confocal microscopy was used to confirm the pheno-type of the lens epithelial cells, as well as to determinemacrophage adherence to each IOL material. To serveas a positive control, some HLE cells were culturedwithout macrophages and treated with 5 ng/ml ofTGF-b2 (Sigma–Aldrich) for two days prior to ima-ging. To serve as a negative control, HLE cells werecultured without macrophages for six days and notexposed to any other treatment.

AdherentHLE cells on the insert were washed twicewith room-temperature PBS for 5min. Cells were fixedin paraformaldehyde at a final concentration of 4% w/vfor 15min, washed once with PBS, and then permea-balized with 0.5% Triton-X in PBS for 5min in thedark.42 Cells were washed with PBS, and then stainedovernight in 1% BSA in PBS with antibodies fora-SMA, fibronectin, or E-cadherin at 4�C in the dark.For the samples labeled with anti-fibronectin, second-ary antibody was added after washing with PBS and thesamples were incubated for 1 h at room temperature inthe dark. Samples were washed with PBS at room tem-perature and then imaged by an inverted laser scanningconfocal microscope (LSM 510 Meta, Carl Zeiss,Germany) using an argon laser (488 nm excitation)and HeNe laser (543 nm and 633 nm excitation).

IOLs were removed from the coculture model andfixed in 4% paraformaldehyde for 1 h. To confirm thepresence of adherent macrophages on the IOLs, sam-ples were blocked with 1% BSA in PBS and then incu-bated with saturating concentrations of fluorescentlylabeled antibodies against CD36, CD54 or CD14, andCD45 overnight at 4�C in the dark. The followingmorning, samples were rinsed in PBS and imaged byan inverted LSM (LSM 510 Meta, Carl Zeiss,Germany) using an argon laser (488 nm excitation)and HeNe laser (543 nm and 633 nm excitation).

Statistical analysis

Flow cytometry fluorescence values are presented as themean� the standard error of the mean (SEM).Fluorescence values and fluorescence ratios were sub-jected to a multivariate analysis of variance using ageneral linear model, including terms for length of incu-bation, lens material, order of cell-culture plate, andwhen applicable, the presence or lack of macrophagesin the coculture. Pairwise comparisons between mater-ials, incubation times, and material–time combinationswere performed using the Bonferoni criterion inMinitab 16.2.3 (State College, PA, USA). A p valueof less than 0.05 was required for significance. Thenumber of experiments was always equal to or greaterthan 3.





Results

Macrophages increase a-SMA expression in lensepithelial cells

The novel coculture model was employed to examinethe possible inflammatory effect of macrophages onlens epithelial cells. As illustrated in Figure 2, after asix-day incubation, both the presence of macrophagesand an IOL material were required to significantlyaffect the expression of the myofibroblast proteina-SMA in lens epithelial cells. In the lens cell monocul-ture, the presence of an IOL alone had no effect ona-SMA expression (p> 0.59); note that in our in vitromodel, the lens cells are not in physical contact with thelenses. In the absence of an IOL, the presence of macro-phages in the coculture model induced a small but sig-nificant increase in a-SMA expression (p< 0.031).Coculture with pHEMA IOLs induced a significantupregulation of a-SMA in lens epithelial cells whencompared to both lens cell monoculture withpHEMA IOLs (p< 0.033) and coculture with no IOL(p< 0.007). Similarly, coculture with PMMA IOLsinduced a significant upregulation of a-SMA whencompared to the lens cell monoculture with PMMAIOLs (p< 0.02), but this upregulation was not statistic-ally significant when compared to coculture with noIOL (p< 0.08). Neither two nor four days of coculture

provided the required length of time to detect differ-ences in the expression of a-SMA in lens cells (seeTable 1).

As shown in Table 1, no significant difference infibronectin expression in cocultured lens cells wasinduced by the presence of an IOL material.However, fibronectin expression significantly decreasedbetween two and four days of coculture (p< 0.0008)with a PMMA IOL, and between four and six daysof coculture with no IOL (p< 0.003). Similarly, no sig-nificant difference in E-cadherin expression wasinduced by the presence of an IOL material.However, E-cadherin expression was significantly upre-gulated (p< 0.03) after six days of coculture with apHEMA IOL compared to E-cadherin expression atboth two days and four days.

Differences in a-SMA expression observed in flowcytometry were confirmed by confocal microscopy.For comparison purposes, treatment with 5 ng/ml ofTGF-b2 for two days was included as a positive control,and lens cell monoculture included as a negative con-trol. As illustrated in Figure 3, high levels of a-SMAexpression (shown in green) were observed forthe TGF-b2, coculture with pHEMA IOLs, and cocul-ture with PMMA IOLs groups (Figure 3(a)–(c),respectively). Coculture in the absence of an IOLresulted in a reduced expression of a-SMA (Figure3d) and lens cell monoculture (Figure 3e) showed the

Table 1. a-SMA, fibronectin, and E-cadherin expression on

macrophages cultured for two, four, or six days with a PMMA

lens, pHEMA lens, or no lens. Protein expression was measured

by flow cytometry and is reported as the geometric mean of

arbitrary fluorescence units (AFU)� the standard error of

measurement (SEM).

IOL material

Incubation

time Fibronectin E-cadherin

No material 2 days 33.66� 8.14 13.29� 1.43

4 days 22.19� 4.85 12.20� 2.58

6 days 17.56� 6.36* 13.73� 1.37

PMMA 2 days 34.75� 4.59 12.68� 1.11

4 days 22.27� 3.93þ 13.11� 1.88

6 days 21.42� 6.74þ 13.83� 1.03

pHEMA 2 days 28.92� 4.43 11.85� 1.02

4 days 22.91� 4.48 12.78� 2.22

6 days 20.93� 6.87 15.41� 1.36z

Note: a-SMA: a-smooth muscle actin; PMMA: polymethylmethacrylate;

pHEMA: poly(2-hydroxyethyl methacrylate); IOL: intraocular lens.

n¼ 7 at 2 days, and n¼ 4 at both 4 days and 6 days.

*Significantly different from corresponding value at 2 days (p< 0.003).

þSignificantly different from corresponding value at 2 days (p< 0.0008).

zSignificantly different from corresponding value at 2 days (p< 0.00001)

and 4 days (p< 0.03).

100

80

60

40

20

0No macrophages Macrophages

Culture condition

No Material

PMMA IOL

pHEMA IOL

α-S

MA

Exp

ress

ion

(A

FU

)

*

*

***

Figure 2. Coculture with macrophages increases expression of

a-SMA in HLE B-3 cells versus cells in a monoculture. All cells

were cultured for six days. a-SMA expression was measured by

flow cytometry and is reported as the mean of arbitrary fluor-

escence units (AFU). *Indicates significant difference from

respective a-SMA expression observed in lens cells in mono-

culture (no macrophages) (p< 0.032); **significant difference

(p< 0.01). n¼ 4 for all treatments and controls. Note: PMMA:

polymethylmethacrylate; pHEMA: poly(2-hydroxyethyl

methacrylate).

Pintwala et al. 5




weakest staining for a-SMA. Regardless of incubationconditions expression of E-cadherin (shown in red) wassimilar, with significant background staining on allPET substrates.

Macrophages upregulate CD54 expression inresponse to pHEMA lenses

Macrophage activation induced by the presence of IOLmaterials was characterized over six days. Upregulatedcell-surface CD54 receptor expression was observed attwo days, four days, and six days of incubation. Asshown in Figure 4, after two days of incubation, macro-phages cultured with a pHEMA lens significantly upre-gulated CD54 expression relative to cells cultured withno lens (p< 0.03), but not compared to the expressionon cells cultured with PMMA lens (p< 0.117). CD54expression decreased significantly after two days(p< 0.0001) and remained approximately constantbetween four and six days incubation (Figure 4). Thepresence of a PMMA lens did not result in a significantupregulation of CD54 when compared to control(macrophages with no IOL, p¼ 1.0).

As for the expression of the lipopolysaccharidereceptor CD14, the presence of an IOL materialinduced no changes in expression (Table 2). However,CD14 expression was upregulated at four days com-pared to two days (p> 0.05) and subsequently down-regulated at six days relative to both two days and fourdays (p< 0.02).

Cell-surface expression of CD45, the human leuko-cyte common antigen, and CD36, the thrombospondinreceptor, were not affected by the presence of an IOLmaterial (Table 2). However, for all samples, expressionof both receptors on macrophages appeared to decreaseover time: CD45 expression was significantly decreased

Figure 3. HLE B-3 cells imaged at 40� zoom using a confocal

laser scanning microscope. Scale bar represents 50 mm. Left

(green): stained for a-SMA. Right (red): stained for E-cadherin. (a)

Monoculture with 5 ng/ml of TGF-b2 for two days; positive

control. (b) Coculture with pHEMA IOL for six days. (c)

Coculture with PMMA IOL for six days. (d) Coculture with no

IOL for six days. (e) Monoculture with no IOL for six days;

negative control. Images are representative of triplicate of each

treatment category.

200

150

100

50

02 days 4 days 6 days

*

CD

54 E

xpre

ssio

n (

AF

U)

Incubation Time

No MaterialPMMA IOL

pHEMA IOL

Figure 4. CD54 cell-surface receptor expression on macro-

phages cultured for two, four, or six days with a PMMA lens,

pHEMA lens, or no lens. CD54 expression was measured by flow

cytometry and is reported as the mean of arbitrary fluorescence

units (AFU). *Indicates a significant difference of p< 0.03. n¼ 7 at

two days and n¼ 4 at both four and six days. Note: PMMA:

polymethylmethacrylate; pHEMA: poly(2-hydroxyethyl

methacrylate).





at six days when compared to both two and four days(p< 0.01), while significantly decreased expression ofCD36 was observed at four and six days when com-pared to two days (p< 0.0001).

pHEMA surfaces prevent strong macrophageadhesion

The relationship between CD54 upregulation andmacrophage adhesion to IOLmaterials was investigatedusing confocal microscopy. After two days of incuba-tion, the surface of the pHEMA IOL was devoidof macrophages, as determined by immunostainingfor CD45. Instead, the surface was covered inCD45þ/CD54þ microparticles of two forms: a sharp,jagged appearance that stained strongly for CD54 (* inFigure 5a), and a granular appearance that stained lessstrongly for CD54 (z in Figure 5b). Also found infre-quently on the surface were larger, cell-sized impressionsstaining positively for CD54 and CD45 (þ in Figure 5b).These impressions lacked the three-dimensionalgeometry of macrophages and instead appeared as athin film on the IOL surface. A pHEMA lens culturedwithout macrophages and treated with the same stainingprotocol served as a negative control for the binding ofCD45 andCD54 (Figure 5c), confirming specific bindingof the fluorescent antibodies.

In contrast, hundreds of CD45þ macrophages butvery few microparticles were found on the hydrophobicsurface of the PMMA IOL after two days of incubationtime. These cells stained very strongly for CD54, and

Figure 5. The surface of a pHEMA IOL observed on a confocal

laser scanning microscope with a 40� objective. Scale bar rep-

resents 50mm. Images (a) and (b) show a lens cultured with

macrophages for two days and then stained with anti-CD54

(shown in yellow) prior to imaging. Both images depict different

sections of the same IOL, and are representative of three sam-

ples. Image (c) depicts a lens cultured without macrophages,

stained for CD54 and CD45, included as a negative control.

Table 2. CD45, CD36, and CD14 cell-surface receptor

expression on macrophages cultured for 2, 4, or 6 days with a

PMMA lens, pHEMA lens, or no lens. Receptor expression was

measured by flow cytometry and is reported as the geometric

mean of arbitrary fluorescence units (AFU)� the standard error

of measurement.

IOL material

Incubation

time CD45 CD36 CD14

No material 2 days 57.2� 13.2 25.8� 5.93 17.3� 5.01

4 days 56.7� 12.1 16.3� 8.46* 23.6� 5.54

6 days 37.9� 9.10*þ 17.1� 6.60* 15.0� 3.91þ

PMMA 2 days 57.7� 6.29 25.5� 3.88 22.0� 4.69

4 days 53.9� 9.29 16.4� 5.40* 20.5� 2.37

6 days 38.4� 5.93*þ 14.0� 8.23* 14.7� 1.96þ

pHEMA 2 days 54.0� 6.35 23.4� 4.24 19.2� 3.88

4 days 63.9� 8.46 18.5� 6.21* 25.6� 3.84

6 days 40.3� 6.27*þ 14.8� 6.14* 17.4� 2.50þ

Note: PMMA: polymethylmethacrylate; pHEMA: poly(2-hydroxyethyl

methacrylate); IOL: intraocular lens.

n¼ 7 at 2 days, and n¼ 4 at both 4 days and 6 days.

*Significantly different from corresponding value at 2 days (p< 0.01).

þSignificantly different from corresponding value at 4 days (p< 0.02).

Pintwala et al. 7




took one of the three appearances. First observed weregiant, circular cells of diameter greater than 100 mm, asshown in Figure 6a. These cells were multinucleatedand resembled foreign-body giant cells (FBGCs) inappearance. Also observed were macrophages withpseudopodia extended, often times joining the bodyof other macrophages, as shown in Figure 6d. Thesecells were typically around 250 mm in length, varyingin width along their length. Lastly were globularmacrophages with sharp cytoplasmic projections thatextended in all directions, as shown in Figure 6(c) and(d). These cells varied in size between 30 mm and 60 mmin diameter, and were the most common adherent cellsobserved on the hydrophobic PMMA IOL surface.

Oxidative species are generated in response toPMMA and pHEMA lenses

ROS and RNS generation have been implicated in thedevelopment of a number of inflammatory disorders.48

ROS/RNS production and overall oxidative stress,

assessed by DCF fluorescence, was measured in macro-phages adhered to the IOL as well as those adhered tothe tissue-culture plate after one day of incubation.This incubation length was chosen as a result of theconfocal study, where the absence of strongly adherentmacrophages on pHEMA IOLs at two days of incuba-tion was observed. As shown in Figure 7, incubation inthe presence of either a PMMA or pHEMA IOLcaused a significant increase in production of ROS/RNS in macrophages adherent to the TCPS surface(p< 0.03) compared to control macrophages (noIOL). Furthermore, macrophages on the PMMA orpHEMA IOL surface exhibited significantly lowerDCF fluorescence when compared to both controlmacrophages (no IOL) (p< 0.004) and macrophagesthat remained on TCPS (p< 0.0001).

To examine the capability of the macrophages toadhere to each IOL material after one day of incuba-tion, adherent macrophage were dissociated andcounted using a flow cytometer. The hydrophilic sur-face of the pHEMA IOL had fewer adherent cells at

Figure 6. The surface of two PMMA IOLs imaged with a confocal laser scanning microscope on a 40� objective. Scale bar rep-

resents 50 mm. Lenses were cultured with macrophages for 48 h and then stained with anti-CD54 (shown in yellow) prior to imaging.

(a) Depicts a multinucleated FBGC. (c) and (d) Depict smaller macrophages with sharp cytoplasmic projections. (b) and (d) Depict

macrophages with elongated pseudopodia. Images are a representative area of at least three different IOLs studied.





one day (1773� 844) than the hydrophobic surface ofthe PMMA IOL (3376� 1367), however this differencedid not reach statistical significance (p< 0.07, n¼ 6).

Discussion

Upon exposure to the PKC activator PMA, the humanmonocytic leukemia cell line THP-1 differentiateinto macrophage-like cells that resemble native mono-cyte-derived macrophages in morphology,49 phagocyticability, cell-surface receptor expression, and cytokine/chemokine production.41,46,50,51 Owing to this, THP-1macrophages have been extensively used to investigatematerial biocompatibility in vitro.52–55 Further workhas optimized THP-1 differentiation protocols toensure sensitivity to weak stimuli and a high phenotypicsimilarity to native macrophages,45,46 combined for thefirst time in the present study. The SV-40 virus immor-talized human lens epithelial cell line HLE B-3 wasselected for use in the coculture model. Although dif-ferences between the proteomes of native lens epithelialcells and HLE B-3 have been investigated, namely theabsence of crystalline expression in HLE B-3,56 the cellline continues to be used extensively to study signalingpathways and mechanisms in PCO,12,15,16,42,43,57 andcapsular biocompatibility with IOL materials.58,59

Our objective was to examine the uveal biocompati-bility of PMMA and pHEMA IOL materials by quan-tifying cellular activation. To measure activation, wechose to examine expression of the cell-surface

adhesion protein CD54 (or ICAM-1) on macrophagesin vitro. CD54 has been shown extensively in the litera-ture to be upregulated on macrophages in response toinflammatory stimuli (see Roebuck et al.,33 Sheikhet al.34). Specifically, Imanaka et al.36 observed astrong correlation in vivo between CD54 upregulationand increased production of TGF-b1, and Koyamaet al.35 reported that CD54 activity functioned as asignal transducer for IL-1b gene transcription in vitro.Following two days exposure to an IOL, we observedsignificant increases in expression of CD54 on macro-phages cultured with a hydrophilic acrylic (pHEMA)IOL compared to cells cultured on TCPS, as well ascells cultured with a hydrophobic acrylic (PMMA)IOL, suggesting that hydrophilic acrylic materialsmay cause significant macrophage activation comparedto hydrophobic acrylic materials. This observation is inagreement with previous work by Anderson andJones60 who reported significant levels of macrophageactivation to hydrophilic polyacrylamide (PAAm) sur-faces compared to hydrophobic poly(styrene-co-benzylN,N-dimethyldithio-carbamate) (BDETDC) surfaces,resulting in significantly increased production of inter-leukin (IL)-10, IL-1b, IL-6, and macrophage inflamma-tory protein (MIP)-1b.60 They concluded that a stronginverse relationship exists between macrophage adhe-sion and macrophage activation. A similar inverse rela-tionship was observed in the present study; macrophagesexposed to a pHEMA IOL showed signs of significantactivation but an inability to strongly adhere to the IOLsurface. Differences in CD54 expression between thehydrophobic PMMA IOL and TCPS were insignificant,corroborating work by Bernatchez et al.61 wherein noCD54 upregulation was observed with peritonealmacrophages cultured on hydrophobic polylactic acidsurfaces versus TCPS. Our results appear to agree withprevious work investigating hydrophobic and hydro-philic materials, further suggesting that differentiatedTHP-1 macrophages represent a good in vitro model ofmacrophage biocompatibility.

The scavenger receptor CD36 plays a crucial role inthe phagocytosis of apoptotic cells,62–64 although littleevidence supports inducible expression by biomaterials.We observed no effect on the expression of CD36 by theinclusion of an IOL, but detected a reduction in CD36expression after two days of culture. Draude andLorenz65 reported that exposure to the wound-healingcytokine TGF-b1 supressed CD36 expression onmacrophages, indicating a possible macrophage pheno-typic shift in the present study. The pattern recognitionreceptor CD14 plays a role in the phagocytosis of bothapoptotic cells and bacterial and viral pathogens,40,66

but is typically used as a marker of monocyte differen-tiation into macrophages.67,68 We observed no effect onthe expression of CD14 by the inclusion of an IOL, but

2.0

1.5

1.0

0.5

0.0

* *

** **

**** *

*

TCPS Surface SurfaceIOL

IOL

IOL

Macrophage Substrate

DC

F f

luo

resc

ence

rat

ioPMMA

pHEMA

Figure 7. Reactive oxygen and nitrogen species production in

macrophages cultured for 24 h with a PMMA or pHEMA IOL.

Macrophage adhered to the IOL were treated separately from

those on the tissue culture plate (TCPS) surrounding the IOL.

Values presented are DCF fluorescence measured by flow cyto-

metry and normalized to control DCF fluorescence (no IOL).

*Indicates (p< 0.03); **indicates (p< 0.004); ***indicates

(p< 0.0001). n¼ 6 for all categories. Note: PMMA: polymethyl-

methacrylate; pHEMA: poly(2-hydroxyethyl methacrylate); IOL:

intraocular lens.

Pintwala et al. 9




identified a decrease in CD14 expression by six days ofmacrophage culture. Bosshart and Heinzelmann69

observed a spontaneous decrease in CD14 expressionin blood-isolated monocytes ex vivo, indicating a quies-cent state capable of rapid reactivation upon lipopoly-saccharide stimulation. Kindlund et al.70 concludedthat CD14 downregulation occurred during the differ-entiation process from monocyte to osteoclast. Alsoobserved in the present study was a reduction inCD45 expression after four days of culture, perhapsindicating a change in macrophage activity to a morequiescent state.71 In their work examining the macro-phage response to biomaterial surface chemistry,Anderson and Jones noted a phenotypic change afterthree days of macrophage culture that resembled a shiftfrom a host-defence phenotype to a wound-healingphenotype.60 This shift to a less inflammatory statemay have been echoed in the present study by thedecrease in expression of CD36, CD14, and CD45observed after four days of macrophage culture.

ROS and RNS are important signaling molecules ina number of inflammatory processes and disorders.48

To analyze the acute inflammatory response of macro-phages to hydrophobic and hydrophilic IOL materials,ROS/RNS production and overall oxidative stress weremeasured using the probe DCFH-DA. Due to the factthat very few cells were found adherent to the pHEMAIOLs at two days of incubation, ROS/RNS generationwas quantified after one day of culture. After exposureto either the hydrophobic PMMA or hydrophilicpHEMA IOL, we observed an increase of ROS/RNSproduction in macrophages on the TCPS substratecompared to macrophages not exposed to an IOL.This finding coincides with but cannot be correlatedwith the increased expression of CD54 after two daysof culture with either IOL material. Iribarren et al.37

noted a similar correlation in vivo between increasedROS generation and CD54 upregulation. However,we observed significantly less ROS/RNS productionin macrophages on the surface of either IOL comparedto macrophages on the TCPS substrate surrounding theIOL, as well as macrophages not exposed to an IOL.The DCFH-DA probe is a membrane-permeablefluorogenic compound that is deacetylated by cytosolicesterases into the membrane-impermeable 20-70-dichlor-odihydrofluorescein (DCFH2).

72 Once trapped insidethe cell, DCFH2 is oxidized by peroxidases, cytochromec, and Fe2þ in the presence of intracellular hydrogenperoxide, producing the fluorescent compound 20-70-dichlorofluorescein (DCF).73 Brubacher and Bols74

showed that the DCFH-DA probe underestimatedROS production in macrophages with low esteraseexpression, or a sequestering of esterase into inaccess-ible cellular compartments, a conclusion made previ-ously by Robinson et al.75 Labow et al.76 showed

previously that monocyte-derived macrophages secretea number of esterases to degrade biomaterial surfaces, aprocess known as frustrated phagocytosis.77 In the pre-sent study, it is likely that the compartmentalizationand secretion of cellular esterases led to an underesti-mation of oxidative stress in macrophages adherent tothe IOL surfaces.

Using confocal microscopy, the number and pheno-typic appearance of adherent macrophages on each bio-material surface was investigated in vitro. Ziegelaar andFitton52 previously reported that THP-1 cells showeddecreased adherence to pHEMA surfaces compared toTCPS and noted a relationship between adhesion andcollagenase production, but did not normalize for thenumber of adherent cells. In the present study, fewercells were found adhered to the pHEMA IOLs than thePMMA IOLs despite what appears to be significantmacrophage activation. After one day of incubation,approximately half as many macrophages were countedvia flow cytometry on the pHEMA surface comparedto the PMMA surface. After two days of incubation,the PMMA surface showed signs of FBGC fusion whilethe pHEMA IOL was devoid of cells. Macrophageswere instead found on the TCPS substrate surroundingthe pHEMA IOL for up to six days of culture.Macrophages were found loosely adherent to thepHEMA IOL surface after one day exposure butwere no longer present at two days incubation. Rice78

observed the migration of primary osteoblasts fromtitanium oxide surfaces to exposed TCPS surfacesin vitro during a seven-day period,78 and thus a similarmigration may have occurred. Furthermore, CD45þ/CD54þ impressions of detached cells and microparti-cles were found on the surface of the pHEMA lens attwo days incubation. Macrophage microparticles wereobserved by Cerri et al.79 to upregulate inflammatoryprocesses in human airway endothelial cells. Gauleyand Pisetsky80 observed that toll-like receptor stimula-tion of macrophages lead to microparticle release; aprocess influenced by ROS production. These findingssuggest that the microparticles observed on thepHEMA surface, as well as the changes in ROS/RNSproduction, may mediate the response of lens epithelialcells in the coculture. While many studies have charac-terized macrophage adhesion on biomaterials, there islimited work reporting on the adherence of macrophagemicroparticles on biomaterial surfaces, perhaps owingto the limited use of confocal microscopy for macro-phage markers on implant surfaces. Although thepHEMA lens surface prevented adherence, in the post-operative in vivo environment, the newly exposed col-lagen-IV rich anterior lens capsule may serve as asubstrate for macrophage adherence in patients withpHEMA IOLs. On the other hand, the PMMA lensallowed for strong adherence of macrophages and





enabled fusion into what appeared to be multinucleatedFBGCs. Similar to our in vitro results, Ravalico et al.81

observed significant inflammatory cell adhesion andfusion on PMMA lenses in human patients between 7and 180 days after corrective surgery for cataracts, witha corresponding lack of cellular adhesion and fusion onpHEMA lenses.

During the development of PCO, myofibroblasts inthe lens capsule overexpress the ECM protein fibronec-tin and halt expression of the epithelial adhesion pro-tein E-cadherin.5 Chung et al.42 showed that treatmentwith TGF-b1 induced fibronectin expression in HLE B-3 cells, while Hosler et al.16 showed that treatment withTGF-b2 induced fibronectin gene activation in HLE B-3 and primary cells. Choi et al.12 observed that TGF-b1treatment repressed E-cadherin expression in HLE B-3cells, while a similar study by Yao et al.82 observed thesame finding but in alveolar epithelial cells. In the pre-sent study, no difference in fibronectin or E-cadherinexpression was induced by lens cell and macrophagecoculture with an IOL material. These findings mayindicate that the TGF-b family of cytokines may notparticipate in the interaction between coculturedmacrophages and lens epithelial cells. Indeed, anenzyme-linked immunosorbent assay for TGF-b1 wasperformed on coculture and activated macrophagesupernatants to examine a possible effect, but nonewas found. In light of these findings, it seems probablethat another soluble mediator is responsible for theobserved interaction.

The novel in vitro coculture model presented hereinincorporated lens epithelial cells and macrophages onseparate substrates in the same media environment.Similar coculture models have demonstrated the abilityof macrophages to promote inflammation in hepato-cytes and adipocytes,41,83,84 stimulate bone resorp-tion,85 and direct apoptosis and suppress cell divisionin mesangial cells.86 More relevant to this investigation,Glim et al.87 demonstrated that M2 (wound-healing)macrophages induced the expression of the myofibro-blast protein and secondary-cataract marker a-SMA indermal and gingival fibroblasts via the secretion of pla-telet-derived growth factor-CC.87 After six days ofcoculture with macrophages activated by a pHEMAIOL, lens epithelial cells significantly increased expres-sion of a-SMA relative to coculture with no IOL, asdemonstrated both by flow cytometry and confocalmicroscopy. a-SMA expression has been shown toincrease contractile force in fibroblasts,88 and Loiset al.28 observed a reduction in the number of lens epi-thelial cells in the center of the posterior capsule aftersystemic macrophage depletion, indicating a possiblerole for macrophage activation to IOL biomaterials inthe severity of posterior capsule wrinkling during PCO.Anderson and Jones60 noted that macrophages cultured

on hydrophilic PAAm surfaces produced greater quan-tities of inflammatory cytokines than macrophages cul-tured on hydrophobic BDETDC despite beingsubstantially fewer in number, concluding that totalvolume of cytokine production was a function of thenumber of activated cells and the degree to which theywere activated. In the present study, macrophages cul-tured on a hydrophilic pHEMA surface were signifi-cantly more activated and fewer in number thanmacrophages cultured on a hydrophobic PMMA sur-face. However, both groups induced approximately thesame upregulation of a-SMA in cocultured lens cells,lending further evidence to the hypothesis that the grossforeign-body reaction to a biomaterial is a function ofboth the numbers and degree of macrophageactivation.

Conclusions

Our findings provide a novel insight into uveal biocom-patibility. Previous work exploring IOL biocompatibil-ity have based their conclusions on the number ofleukocytes adherent to a given material, concludingthat hydrophilic surfaces that prevent cellular adhesionare more biocompatible as they prevent leukocytefusion into FBGCs.9,29,30 Our results have shownin vitro that FBGC formation, and indeed macrophageadherence in general, are not necessary for a stronginflammatory response to an IOL. Rather, the inflam-matory response to an IOL appears to be a function ofboth the amount of stimulation and the number ofinflammatory cells stimulated, with a hydrophilicacrylic surface providing a stronger stimulus for macro-phage activation than a hydrophobic acrylic one. Thesefindings provide a new method of inquiry into uvealbiocompatibility, specifically through the quantificationof cell-surface markers of leukocyte activation.

Declaration of conflicting interests

The authors declared no potential conflicts of interest with

respect to the research, authorship, and/or publication of thisarticle.

Funding

20/20: the NSERC Ophthalmic Materials Network and

Ontario Graduate Scholarship have funded this research.

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