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Intracorneal Instillation of Latex Beads InducesMacrophage-Dependent Protection againstAcanthamoeba Keratitis

Daniel W. Clarke, Hassan Alizadeh, and Jerry Y. Niederkorn

PURPOSE. Instillation of sterile 1.0 M latex beads into thecentral corneal epithelium renders Chinese hamsters resistantto corneal infection with Acanthamoeba castellanii. By con-trast, activation of the adaptive immune response by subcuta-neous immunization with A. castellanii antigens fails to pro-tect against Acanthamoeba keratitis. This study wasundertaken to examine the mechanisms that mediate latexbead-induced resistance to Acanthamoeba keratitis.

METHODS. In vitro experiments examined the effect of latexbead treatment on the capacity ofA. castellaniitrophozoites toadhere to and kill corneal epithelial cells. In vivo administrationof antineutrophil antiserum was used to evaluate the role ofneutrophils in latex-beadinduced protection against Acan-thamoeba keratitis. Liposomes containing the macrophagi-cidal drug clodronate were used to deplete conjunctival mac-rophages and determine the role of macrophages in the latex-beadinduced resistance.

RESULTS. Latex bead treatment did not affect adherence oftrophozoites to the corneal epithelium or protect corneal ep-ithelial or stromal cells from trophozoite-mediated cytolysis invitro. Neutrophil depletion did not abrogate the latex beadsprotective effect. Latex bead treatment induced a significantinfiltration of macrophages into the corneas that peaked at day4 of infection. Moreover, depletion of conjunctival macro-phages with the macrophagicidal drug clodronate eliminatedthe latex beads protective effect.

CONCLUSIONS. The results indicate that intracorneal injection oflatex beads induces a remarkable resistance to Acanthamoebakeratitis that is largely, if not entirely, mediated by macro-phages. These results underscore the importance of the innateimmune apparatus in the resistance to Acanthamoebakeratitis. (Invest Ophthalmol Vis Sci. 2006;47:49174925)DOI:10.1167/iovs.06-0266

The Acanthamoeba spp. is a ubiquitous, free-living proto-zoan that has been isolated from a variety of habitatsincluding public water supplies, soil, and air.1,2 Although

Acanthamoeba spp. can cause granulomatous amebic menin-

goencephalitis (GAE) and cutaneous acanthamebiasis, theymost commonly infect the eye to produce Acanthamoebakeratitis. Acanthamoeba keratitis is caused by at least sevenspecies of pathogenic amoebae and is a potentially blindingcorneal infection if left untreated.1,3 However, prompt antimi-crobial treatment often controls infection and preserves vision.The pathogenic cascade of Acanthamoeba keratitis beginswith trophozoite adherence to mannose glycoproteins on thecorneal epithelium via a 136-kDa mannose-binding protein(MBP) on the trophozoite membrane.4 Exposure to mannoseinduces Acanthamoeba trophozoites to release a 133-kDa pro-tein, termed mannose-induced protein (MIP-133), that is highlycytolytic to corneal epithelial cells in vitro.5After the pathogenbinds to the corneal epithelium, the pathogenesis of Acan-thamoeba keratitis proceeds, with trophozoite-mediated de-struction of the corneal epithelium and penetration of theunderlying Bowmans membrane. Subsequently, Acan-thamoeba trophozoites elaborate a variety of proteases thatfacilitate destruction of the corneal stroma.6 The pathogenesisof Acanthamoeba keratitis abruptly stops before invasion ofthe corneal endothelium and entry into the anterior chamberof the eye, as Acanthamoeba spp. rarely progresses beyondthe corneal endothelium to produce intraocular infections.7

Environmental exposure to Acanthamoeba spp. is com-mon, as viable Acanthamoeba trophozoites have been isolatedfrom nasopharyngeal and oral specimens collected fromasymptomatic individuals.2 Moreover, peripheral blood lym-phocytes from 50% of healthy individuals demonstrated T-cellproliferative responses to Acanthamoeba antigens, suggesting

that environmental exposure to this protozoan stimulates theadaptive immune apparatus.8 At least one component of theadaptive immune apparatus, anti-Acanthamoeba secretory IgAantibody, protects against disease by preventing trophozoiteadhesion to the corneal epithelium by augmenting neutrophil-mediated lysis of trophozoites, and by inhibiting MIP-133mediated lysis of the corneal epithelium and stroma.9 MucosalIgA antibodies to Acanthamoeba antigens and MIP-133 are theonly effective component of the adaptive immune responsethat seem to be capable of controlling Acanthamoeba keratitisand are only effective if induced before corneal infections areinitiated in experimental animals.1018 The adaptive immuneresponse also appears to be ineffective in controlling Acan-thamoeba keratitis in humans, as acute infections occur in the

presence of IgG serum antibodies specific for Acanthamoebaantigens in patients with Acanthamoeba keratitis, includingthose with recrudescent infections.10 However, the adaptiverecrudescence can occur in hosts not possessing anti-Acan-thamoeba IgA antibodies, indicating that the adaptive immuneapparatus is not effective at preventing reinfection.

Elements of the innate immune system are crucial in theresolution of Acanthamoeba keratitis in experimental ani-mals.18,19 Neutrophils and activated macrophages are capableof killing trophozoites and cysts in vitro. 14,2022 Moreover,depletion of macrophages with the macrophagicidal drug clo-dronate results in a chronic, more severe form of Acan-thamoeba keratitis.18 Similarly, depletion of neutrophils by in

From the Department of Ophthalmology and Microbiology, Uni-versity of Texas Southwestern Medical Center, Dallas, Texas.

Supported by National Eye Institute Grants EY09756 and R24

EY016664, Grant-In-Aid of Research from the National Academy ofSciences, administered by Sigma Xi, The Scientific Research Society(DWC) and an unrestricted grant from Research to Prevent Blindness,Inc.

Submitted for publication March 13, 2006; revised April 26, 2006;accepted August 22, 2006.

Disclosure: D.W. Clarke , None; H. Alizadeh, None; J.Y. Nieder-korn, None

The publication costs of this article were defrayed in part by pagecharge payment. This article must therefore be marked advertise-ment in accordance with 18 U.S.C. 1734 solely to indicate this fact.

Corresponding author: Jerry Y. Niederkorn, Department of Oph-thalmology, UT Southwestern Medical Center, 5323 Harry Hines Bou-levard, Dallas, TX 75390-9057; [email protected].

Investigative Ophthalmology & Visual Science, November 2006, Vol. 47, No. 11

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vivo treatment with antineutrophil antiserum exacerbates dis-ease in Chinese hamsters.19 Recent results suggest that neutro-phils are also important in preventing Acanthamoeba keratitisfrom progressing to intraocular infection.7

Previous studies have shown that intracorneal instillation ofsterile latex beads induces an increased number of Langerhanscells (LCs) in the central cornea.23 In other infectious diseasesof the cornea, the induction of increased numbers of LCs intothe central cornea exacerbates keratitis by promoting a morerobust delayed-type hypersensitivity (DTH) response.24,25

However, induction of LC migration into the central corneabefore infection as a means of promoting DTH responses to

Acanthamoeba antigens did not exacerbate Acanthamoebakeratitis as anticipated, but instead resulted in a robust resis-tance to infection and a mitigation of corneal inflammation.23

The mechanism by which corneal latex bead treatment pro-tects against Acanthamoeba keratitis remains unknown.

The adaptive immune apparatus is not usually effectiveagainst Acanthamoeba keratitis, as neither T-cellmediatednor humoral immunity protects against disease.11 Moreover,the latex beads protective effect is observed within 4 days ofexposure to Acanthamoeba trophozoites. Thus, if the immuneapparatus is involved in the protective effect, it is likely to be

the innate immune apparatus and not the adaptive immuneapparatus that mediates protection.

This study explored the immune and nonimmune mecha-nisms that could be responsible for the latex beads protectiveeffect. One hypothesis proposed that latex beads provide pro-tection against infection by preventing the adhesion of tropho-zoites to the corneal epithelium. We further hypothesized thatlatex beads could protect against disease by inhibiting tropho-zoite-mediated cytolysis of corneal cells, including the cornealepithelium and the corneal stroma. A final hypothesis pro-posed that latex beads provide protection by recruiting and/oractivating cells of the innate immune system, including neutro-phils and macrophages, which can kill trophozoites.

METHODSAnimals

Chinese hamsters were purchased from Cytogen Research and Devel-

opment, Inc. (West Roxbury, MA) and used at 4 to 6 weeks of age. All

corneas were examined before experimentation to exclude animals

with preexisting corneal defects. Animals were handled in accordance

with the ARVO Statement for the Use of Animals in Ophthalmic and

Vision Research.

Amoebae and Cell Lines

Acanthamoeba castellanii (ATCC 30868), originally isolated from a

human cornea, was obtained from the American Type Culture Collec-

tion (Manassas, VA). Trophozoites were grown as axenic cultures in

peptone-yeast-glucose (PYG) medium at 35C, as described previ-ously.23,26

Chinese hamster corneal (HCORN) epithelial cells were immortal-

ized with human papillomavirus E6 and E7 genes, as previously de-

scribed27 and cultured in complete Eagles minimum essential medium

(EMEM; BioWhittaker, Walkersville, MD) containing 10 mM HEPES

buffer solution; 1% nonessential amino acid solution; 1% L-glutamine

(BioWhittaker); 1% penicillin, streptomycin, and amphotericin B (Fun-

gizone; BioWhittaker); 1% sodium pyruvate (BioWhittaker); and 10%

fetal calf serum (FCS; HyClone Laboratories, Logan, UT).

Normal human keratocytes (NHKs) were a generous gift from

James Jester (University of California at Irvine). Cells were cultured in

Dulbeccos modified Eagles medium (DMEM; BioWhittaker) contain-

ing 10 mM HEPES buffer solution; 1% nonessential amino acid solution;

1% L-glutamine; 1% penicillin, streptomycin, and amphotericin B; 1%

sodium pyruvate; and 10% fetal calf serum.

The monocyte-macrophage cell line originally derived in BALB/c

mice, RAW 264.7 cells (TIB 71), were obtained from ATCC. Cells were

cultured in complete RPMI-1640 (BioWhittaker) containing 10 mM

HEPES buffer solution; 1% nonessential amino acids solution; 2 mM

L-glutamine; 1 mM sodium pyruvate solution; 1% penicillin, streptomy-

cin, and amphotericin B; and 10% fetal calf serum.

BALB/c corneal epithelial cells were established as described pre-

viously.28 Briefly, corneal cells were isolated from corneal explants and

immortalized with the human papillomavirus E6 and E7 genes.29 Cells

were cultured in complete RPMI-1640 (BioWhittaker) containing 10

mM HEPES buffer solution; 1% nonessential amino acid solution; 2 mM

L-glutamine; 1 mM sodium pyruvate solution; 1% penicillin, streptomy-

cin, and amphotericin B; and 10% fetal calf serum.

Intracorneal Instillation of Latex Beads

Latex beads (1 m; Polysciences, Inc., Warrington, PA) were deposited

into shallow corneal epithelial incisions as a reproducible method to

induce centripetal LC migration to the center of the corneal epithelium

of the Chinese hamster.23 Briefly, hamsters were deeply anesthetized

as described previously.23 Next, 10 L of sterile 1 m polystyrene

beads suspended in phosphate-buffered saline (PBS) were deposited

into shallow incisions in the center of the corneal epithelium 10 days

before infection. Hamsters were challenged 10 days after latex bead

instillation, as this time point had been shown to produce optimal

resistance to corneal infection with Acanthamoeba trophozoites.23

Adherence Assay

The ability of Acanthamoeba trophozoites to adhere to latex-bead

treated corneas was examined using a modification of the assay in van

Klink et al.23 Briefly, hamsters were killed 10 days after latex bead

treatment, and the eyes were enucleated. The corneal epithelium was

abraded immediately before enucleation in all groups. Normal,

abraded, but otherwise untreated eyes served as the control. Eyes were

sterilized in 10% iodine in water for 1 minute and rinsed with saline.

Subsequently, the eyes were placed into 200-L pipette tips (eye

cups), cornea side up. The eyes became wedged in the eye cups such

that the corneal surface served as the bottom of the cup. Acan-thamoeba castellanii trophozoites were metabolically labeled, as de-

scribed previously.27 Briefly, trophozoites (5 106/mL PYG) were

cultured with 100 Ci of [35S] methionine (ICN Biomedicals, Inc.,

Irvine, CA) overnight at 35C. Radiolabeled trophozoites were washed

three times in PYG and added to each eye cup at 10 5 trophozoites/eye.

The control cultures of trophozoites were incubated in the presence of

100 mM mannose on untreated corneas. Adherence of trophozoites to

corneas was determined after 2 hours of incubation at 35C. The eye

cups were washed three times with PYG to remove unbound amoe-

bae. The eyes were then transferred to scintillation vials containing 2

mL of scintillation cocktail (Ready-Gel; BD Biosciences, San Diego, CA),

and counts were measured in a liquid scintillation counter (model

LS3801; Beckman, Fullerton, CA). The results are expressed as the

percentage adherence, with 100% corresponding to the number of

trophozoites that bound to untreated corneas.

Assay for Cytopathic Effects

The cytopathic effect (CPE) of trophozoites on corneal cells was

determined with a previously described photometric assay.5 Briefly,

latex beads were added to confluent monolayers of HCORN or NHK

cells to obtain a concentration of 10 beads/cell. Latex beads were not

added to control wells. Trophozoites (5 103, 5 104, or 5 105)

were then added to the confluent monolayers and incubated for 48

hours at 37C. Wells were stained with a manual staining system

(Hema 3; Fisher Scientific, Pittsburgh, PA) and the optical density (OD)

was read at 590 nm in a microplate reader (Molecular Devices, Menlo

Park, CA). The OD of trophozoites alone was subtracted from the OD

of experimental wells to determine the OD of live cells. The results are

expressed as the percentage of cells alive.

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In Vivo Corneal Infections

Acanthamoeba keratitis was induced in Chinese hamsters as described

previously.23 Briefly, approximately 25% of the cornea was abraded

with a sterile cotton applicator, and then amoeba-laden contact lenses

were placed on the center of the cornea. The contact lenses were

removed 4 days after infection, and the corneas were visually in-

spected for severity of disease. Visual inspection results were recorded

daily during the times indicated, and infections were scored based on

the degree of corneal infiltration, corneal neovascularization, and cor-neal ulceration.19 Each pathologic criterion was scored 0 to 5 based on

the degree of corneal involvement19: 0, no disease; 1, 10% of the

cornea involved; 2, 10% to 25% involved; 3, 25% to 50% involved; 4,

50% to 75% involved; and 5, 75% to 100% involved. The scores for all

three criteria were lumped together, and the average combined score

was determined for each time point and was expressed as the clinical

severity of keratitis (%). In Chinese hamsters, Acanthamoeba keratitis

resolves in approximately 3 weeks.

Production of Anti-Chinese HamsterNeutrophil Antiserum

Anti-Chinese hamster neutrophil antiserum was produced and ab-

sorbed as described previously.19 Antiserum was absorbed for 2 hours

at 4C with hamster spleen cells to remove antibodies against Chinesehamster histocompatibility and lymphoid antigens. The absorbed se-

rum was then centrifuged at 1700g for 10 minutes, and the antiserum

was removed and tested for cytotoxicity to neutrophils in vivo and in

vitro. To test for cytotoxicity in vivo, Chinese hamsters were injected

with 1 mL of anti-Chinese hamster antiserum or normal rabbit serum

IP. Blood was collected from tail veins at 0, 2, and 4 hours after

injection and streaked on a slide. Slides were stained with the manual

staining system (Hema 3; Fisher Scientific) for histologic examination.

Neutrophils and lymphocytes were counted in 10 random fields per

slide. The results are expressed as mean number of cells per five

high-power fields.

The anti-neutrophil antibody was also examined with an in vitro

cytotoxicity assay, as described previously.19

Chinese hamsters were injected intraperitoneally with 0.5 mL of

absorbed serum twice daily for 7 days beginning at day 3 after infec-tion.

Preparation of Clodronate Liposomes

Multilamellar liposomes were prepared as described previously.18,30

Clodronate liposomes were tested for in vitro toxicity against macro-

phages before use. Clodronate liposomes selectively deplete macro-

phages, and are not toxic to other phagocytic cells.30,31

Clodronate Liposome Treatment

Clodronate-containing liposomes (50 L) were administered via sub-

conjunctival injection on days 9, 7, 5, and 3 before infection with

Acanthamoeba-laden lenses.18 Clodronate liposomes were injected

into four quadrants of the eye, encircling the entire conjunctiva. Van

Klink et al.23 showed that injection of PBS-containing liposomes does

not deplete resident macrophages or alter the severity of disease.

Quantification of NO Synthesis

Macrophage activation was quantified by measuring nitric oxide (NO)

production.32 RAW cells were plated at 5 104 cells/well in a 24-well

plate and allowed to grow to confluence. As a positive control for

activation, the indicated wells were stimulated with 2 g/mL lipopoly-

saccharide (LPS; Sigma-Aldrich) and 100 ng/mL IFN-(BD-PharMingen,

San Diego, CA), as described previously.20 To determine whether latex

beads induce macrophages to produce NO, 1.0-m latex beads were

added to the indicated wells to obtain concentrations of 100, 1000, or

5000 beads/cell. After 24 and 48 hours of incubation at 37C, 100 L

of supernatant was removed and assayed for nitrite, a stable reaction

product of NO. Supernatants were then incubated with 100 L of

Griess reagent (Sigma-Aldrich) in a 96-well flat-bottomed plate for 10

minutes, and the OD was read at 590 nm in a microplate reader as

described previously.21 To determine NO concentration, a standard

curve of NO production was generated by incubating serial dilutions of

sodium nitrite (Sigma-Aldrich) with Griess reagent.32 Results are ex-

pressed in micromolar of NO.

To determine whether latex beads induce corneal epithelial cells to

produce a factor that activates macrophages, BALB/c corneal epithelial

cells were grown to confluence in a 24-well plate. Latex beads (1.0

m) were added to the indicated wells to obtain concentrations of 10,

100, and 1000 beads/cell. After 24 hours of incubation at 37C, the

supernatant was collected and added to RAW cells in a 24-well plate.

As a positive control for activation, the indicated wells of RAW cells

were stimulated with 2 g/mL LPS (Sigma-Aldrich) and 100 ng/mL

IFN- (BD-PharMingen). After 24 and 48 hours, supernatant was re-

moved and assayed for NO activity as just described. The same method

was used to determine whether latex bead treatment induces BALB/c

corneal keratocytes to produce a factor that activates macrophages.

Acid Phosphatase Staining

To determine whether there were more macrophages present in latex-

beadtreated, Acanthamoeba-infected corneas than in untreated, in-

fected corneas, eyes were processed and stained for macrophage-

endogenous acid phosphatase activity, as described previously.18,24,33

Of importance, neutrophils and other leukocytes that may be present

in the cornea do not stain positively with acid phosphatase.18,24,33

Thus, the acid phosphatase stain can be used to specifically stain

macrophages in the cornea.2325 Briefly, Chinese hamster corneas

were infected with A. castellanii, with or without prior latex bead

treatment, as described earlier. Infected eyes of bead-treated and un-

treated Chinese hamsters (n 5/group) were enucleated 3, 4, and 5

days after infection, sectioned, and processed for acid phosphatase

staining.34 Frozen spleen sections were used as a positive control for

acid phosphatase staining. Acid-phosphatasepositive cells were

counted via light microscopy by three masked observers.

Statistics

Statistical analyses of all data except clinical scores were performed by

using unpaired Students t-tests. Clinical severity scores were analyzed

by the Mann-Whitney test.

RESULTS

Effect of Latex Beads on Trophozoite Adherenceto the Corneal Epithelium

Previous studies have shown that trophozoite adherence tomannose glycoproteins on the corneal epithelium is a crucialprerequisite for the establishment of infection.3538 The extentof trophozoite binding to the corneal epithelium of various

mammalian species correlates closely with the susceptibility ofeach mammalian species to corneal infection in vivo.36 More-over, reports have shown that free-mannose, but not othersugars, inhibits trophozoite adherence to corneal epithelialcells in vitro.37,39,40 To ascertain the mechanism of the latexbeads protective effect, it was first necessary to determinewhether corneal latex bead treatment inhibits the binding oftrophozoites to the corneal epithelium, thereby preventinginfection. Accordingly, the ability of trophozoites to adhere tolatex-beadtreated corneas was examined in an organ culturesystem. Corneal latex bead treatment 10 days before the ad-herence assay did not alter the adherence of A. castellaniitrophozoites to the corneal epithelium (Fig. 1). By contrast,binding of trophozoites to hamster corneas in the presence of100 mM mannose was reduced by 85% compared with the

untreated control.

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Effect of Latex Bead Treatment on the Cytolysisof Corneal Epithelial and Corneal Stromal Cells

In the pathogenic cascade of Acanthamoeba keratitis, adhe-sion to the corneal epithelium is followed by trophozoite-mediated destruction of the corneal epithelium and penetra-tion of the underlying Bowmans membrane. It is possible thatlatex bead treatment protects the corneal epithelium fromtrophozoite-mediated cytolysis. Therefore, we next examinedthe ability of trophozoites to kill HCORN cells that were pre-treated with latex beads in vitro. The results show that latexbeads (10/cell) did not protect HCORN cells from trophozoite-

mediated killing (Fig. 2A). Trophozoites killed as many as 60%of latex-beadtreated and untreated HCORN cells after 48hours Similar results were obtained with HCORN cells thatwere pretreated with 100 and 1000 beads/cell (data notshown).

After desquamation of the corneal epithelium, the patho-genesis of Acanthamoeba keratitis continues, with penetra-tion and dissolution of the underlying stroma. It is possible thatlatex bead treatment provides resistance against Acan-thamoeba keratitis by protecting the corneal stroma fromtrophozoite-mediated cytolysis. However, latex beads did notprotect normal human corneal stromal cells (NHK) cells fromtrophozoite-mediated killing (Fig. 2B). Similar results were ob-tained with NHK cells that were pretreated with 100 and 1000beads/cell (data not shown).

Role of Neutrophils in the Latex BeadsProtective Effect

Neutrophils are capable of killing Acanthamoeba trophozoitesand cysts in vitro.21,22,41 Moreover, in vivo depletion of neu-trophils by intraperitoneal injection of anti-Chinese hamsterneutrophil antibody results in a more severe infection com-

pared with untreated control cells.19 Therefore, the possibilitythat neutrophils are involved in the latex beads protectiveeffect was explored. To determine the role of neutrophils inthe protective effect, rabbit anti-Chinese hamster neutrophilantiserum was generated to deplete neutrophils in latex-beadtreated animals. First, the efficacy of the rabbit anti-Chinesehamster neutrophil antibody was examined in vivo. Antiseruminjections resulted in approximately an 85% decrease in pe-ripheral blood neutrophils after 2 hours and a 95% decreaseafter 4 hours (Fig. 3). Lymphocyte and monocyte counts werenot significantly decreased. Moreover, anti-Chinese hamsterneutrophil antibody (1:100) lysed 83% of neutrophils in thepresence of complement without significantly depletingsplenocytes in vitro (data not shown). As a control, normalrabbit serum was found to be nontoxic to neutrophils in vivoor in vitro (data not shown). Successful depletion of neutro-phils with anti-neutrophil antiserum resulted in a more severedisease than appeared in the untreated control animals. Asreported previously, latex bead treatment reduced the inci-

FIGURE 1. Effect of latex bead treatment on binding of trophozoites tohamster corneas. Sterile latex beads suspended in PBS were depositedinto shallow incisions in the center of the corneal epithelium ofChinese hamsters. Ten days later, the corneas were abraded immedi-ately before enucleation. Adherence of radiolabeled trophozoites tohamster corneas, with or without prior latex bead treatment, wasexamined after 2 hours of incubation in an organ culture system. As acontrol, the adherence assay was also performed in the presence of100 mM mannose. Data are the mean SD of results in five hamstercorneas. The experiment was performed in triplicate. There was nosignificant difference between latex-beadtreated and normal corneas(P 0.05). Attachment to corneas in the presence of 100 mM mannose

was reduced by 85%. **P 0.01.

FIGURE 2. Effect of latex bead treatment on trophozoite-mediated cytolysis of corneal cells. Latex beads were added to HCORN (A) or NHK (B)cells and incubated for 24 hours at 37C. Trophozoites were then added to the indicated wells and incubated for 48 hours. CPE was assessedspectrophotometrically. Data are the mean SD of triplicate counts. CPE on latex-beadtreated cells was not significantly different from that on

untreated cells. (P 0.05). Trophs, trophozoites; HCORN, Chinese hamster corneal epithelial cells; NHK, normal human keratocytes.


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dence, severity, and duration of disease.23 However, anti-Chi-nese hamster neutrophil antibody failed to exacerbate diseasein latex-beadtreated animals (Fig. 4).

The Role of Macrophages in the Latex BeadsProtective Effect

The role of macrophages in resistance to Acanthamoeba ker-atitis is well-established. In vivo depletion of conjunctival mac-

rophages with the macrophagicidal drug, dichloro-methylenediphosphonate (clodronate), results in a chronic, exacerbatedform of Acanthamoeba keratitis.18 Moreover, macrophagescan kill Acanthamoeba trophozoites and cysts in vitro.20,42Wehypothesized that corneal latex bead treatment induces themigration and/or activation of macrophages that mitigate dis-ease. To ascertain the role of macrophages in the latex beadsprotective effect, clodronate was administered subconjuncti-vally to remove corneal macrophages in latex-beadtreatedanimals. The results of this experiment showed that clodronatetreatment eliminated the protective effect of the beads, exac-erbating disease similar to that in the clodronate alone group(Fig. 5). Furthermore, the latex beadsclodronate group wasnot significantly different from the clodronate-treatmentalonegroup.

We next proposed that latex bead treatment may induce theactivation of macrophages, which could kill trophozoites. Todetermine whether latex bead treatment activates macro-phages, the beads were added to the RAW macrophage cells invitro. Macrophage activation was determined by measuringnitrite generation, a stable reaction product of NO. However,the results showed that latex beads did not increase the pro-

FIGURE 4. The role of neutrophils in the protective effect of latexbeads. Sterile latex beads suspended in PBS were instilled into shallowcorneal epithelial incisions in Chinese hamster corneas 10 days beforeinfection in the indicated groups. Hamsters were injected IP with 0.5mL of either anti-neutrophil antiserum or nave rabbit serum twicedaily for 7 days beginning 3 days before corneal infection. Latex beadtreatment reduced the severity of disease at days 4, 5, 6, and 14 afterinfection compared with that in the untreated (infection alone) group(P 0.05). Anti-neutrophil antiserum treatment increased the severityof disease at days 4, 5, 6, 12, 14, 19, and 21 after infection (P 0.05).

Anti-neutrophil antiserum treatment did not exacerbate disease inlatex-beadtreated corneas at any of the time points examined (P

0.05; n 7/group).

FIGURE 5. The role of macrophages in the latex beads protectiveeffect. Latex beads were administered to hamster corneas 10 daysbefore infection in the indicated groups. Clodronate-containing lipo-somes were administered via subconjunctival injection 9, 7, 5, and 3days before infection with Acanthamoeba-laden lenses. Corneas wereobserved for signs of clinical disease beginning at day 4 after infection.Latex bead treatment reduced the severity of disease at days 4 and 5after infection compared with that in the untreated (infection alone)group (P 0.05). Clodronate treatment increased the severity ofdisease at all times examined in both latex-beadtreated and untreatedhamsters (P 0.05). The clodronate treatment alone group was notsignificantly different from the latex-beadtreated, clodronate-treatedgroup (P 0.05). The results are representative of three separate

experiments (n 7/group).

FIGURE 3. Cytotoxic activity of absorbed anti-Chinese hamster neu-trophil antiserum on neutrophils and lymphocytes in vivo. Chinesehamsters were injected with 1 mL of anti-Chinese hamster neutrophilantiserum or normal rabbit serum IP. Blood was collected from tail

veins at 1, 2, and 4 hours after injection. Data represent the mean SDof 10 counts. **P 0.01.


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duction of NO at 24 or 48 hours (Fig. 6A). Treatment withIFN- and LPS significantly activated macrophages comparedwith the untreated control group. Alternatively, it is possiblethat latex beads induce corneal epithelial cells to produce afactor that activates macrophages. To test this, BALB/c cornealepithelial cells were incubated with latex beads for 24 hours.Subsequently, latex-beadtreated BALB/c epithelial cell super-natant was added to RAW macrophages. Latex-beadtreatedcorneal epithelial cell supernatant did not increase productionof NO at 24 or 48 hours (Fig. 6B).

We next hypothesized that latex bead treatment beforecorneal infection may induce the migration of macrophages tothe central cornea, which kill trophozoites and result in themitigation of corneal disease. Previous work by Hazlett et al.24

demonstrated that intracorneal instillation of latex beads be-fore corneal infection with P. aeruginosa induces the cornealmigration of macrophages. Acid phosphatase staining of cor-neal macrophages in latex-beadtreated, Acanthamoeba-in-fected corneas revealed that latex bead treatment induced asignificant increase in the infiltration of macrophages at day 4of corneal infection (Fig. 7).

DISCUSSION

The original rationale for instilling latex beads into the centralcornea was based on the hypothesis that latex beads wouldstimulate the appearance of LCs in the central cornea, which inturn would enhance the development of DTH to Acan-thamoeba antigens, as LCs are potent antigen-presenting cellsand are known to enhance DTH responses to alloantigens oncorneal transplants and infectious agents introduced into thecentral cornea.24,25,4345 The pathophysiology of herpes sim-plex virus (HSV) keratitis and Pseudomonas keratitis is largelymediated by DTH responses to the ocular pathogens and isexacerbated in corneas containing resident LC at the time ofocular infection.24,25 However, as shown here and elsewhere,

intracorneal instillation of sterile latex beads 10 days beforeocular challenge with A. castellaniireduced, rather than exac-erbated, the incidence, severity, and duration of Acan-thamoeba keratitis.23 Although intracorneal instillation of ster-ile latex beads before corneal infection with A. castellaniimitigates disease, it also curiously promotes the developmentof parasite-specific DTH.17 This is in sharp contrast to thecondition in HSV keratitis and Pseudomonas keratitis, inwhich latex bead treatment enhances the DTH responses tothe ocular pathogens and thus contributes to the exacerbationof corneal inflammation.24,25

The latex beads protective effect does not appear to bemediated by the adaptive immune response, as protectiondevelops within 4 days of bead instillation, a time well beforethe peak of the adaptive immune response. Moreover, previousstudies in both pigs and Chinese hamsters showed that elicita-tion of Acanthamoeba-specific DTH and complement-fixingantibodies to Acanthamoeba antigens does not protect against

Acanthamoeba keratitis, indicating that the major compo-nents of the adaptive immune apparatus are not typically ef-fective against Acanthamoeba keratitis.11,46 Thus, it is mostlikely that if the immune system contributes to the latex beadsprotective effect, it is through elements of the innate, not the

adaptive, immune system.Initially, we entertained the hypothesis that latex bead treat-

ment decreases binding of trophozoites to the corneal epithe-lium. Trophozoite adherence to mannose glycoproteins on thecorneal epithelium is a crucial prerequisite for the establish-ment of infection, as described in both the pig and Chinesehamster models of disease.23,47 Previous results have shownthat both corneal abrasion and contact lenses upregulatemannose glycoproteins on the corneal epithelium, resultingin increased binding of trophozoites to the corneal sur-face.48,49 It is possible that latex bead treatment decreasesexpression of mannose glycoproteins, thus reducing trophozo-ite binding to the cornea. However, because latex bead treat-

FIGURE 6. Effect of latex beads on macrophage activation. (A) RAW cells were treated with various concentrations of latex beads and incubatedat 37C. As a measure of macrophage activation, NO production was determined at 24 and 48 hours. IFN-and LPS treatment served as a positivecontrol for macrophage activation. IFN- and LPS treatment resulted in increased production of NO at 24 and 48 hours **P 0.05. Latex beadtreatment did not increase NO production at 24 or 48 hours. (B) BALB/c corneal epithelial cells were treated with various concentrations of latexbeads and incubated at 37C for 24 hours. The supernatant was collected and added to RAW cells in a 24-well plate. NO production was determinedat 24 and 48 hours. BALB/c corneal epithelial supernatant did not increase NO production at 24 or 48 hours. The results are representative of three

experiments. SN, supernatant.


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ment did not decrease the adherence of A. castellanii tropho-zoites to the corneal epithelium, we conclude that latex beadsmost likely provide protection against disease via an alternatemechanism.

After adherence to the corneal epithelium, the pathogeniccascade of Acanthamoeba keratitis continues with trophozo-ite-mediated destruction of the corneal epithelium and under-lying corneal stroma.6 Our second hypothesis proposed thatlatex bead treatment protects corneal epithelial or cornealstromal cells against trophozoite-mediated killing. Evidencesupporting this hypothesis shows that corneal epithelial cellselaborate increased amounts of IL-1 after phagocytosis of latexbeads.50 It is possible that latex beads induce corneal epithelialcells or stromal cells to produce a factor, such as IL-1, thatinhibits trophozoite-mediated cytolysis. However, our resultsshow that latex bead treatment did not protect corneal epithe-lial or stromal cells from trophozoite-mediated cytolysis, sug-gesting that latex bead treatment does not induce corneal cellsto produce a factor that inhibits trophozoite-mediated killing.

In this study, we also considered the possibility that im-mune mechanisms may be responsible for the latex beadsprotective effect. Previous studies have shown that in vivodepletion of neutrophils with anti-Chinese hamster neutrophilantibody results in an earlier onset and more severe Acan-thamoeba infections than occurs in untreated hosts.19 More-over, administration of anti-macrophage inflammatory protein(MIP)-2, a powerful chemotactic factor for neutrophils, exac-

erbates Acanthamoeba keratitis.19

Taken together, this evi-dence indicates that neutrophils are involved in protectionagainst Acanthamoeba keratitis. Therefore, we next hypothe-sized that latex bead treatment induces the migration and/oractivation of neutrophils, which kill trophozoites. Althoughadministration of anti-neutrophil antiserum depleted neutro-phils in vivo, it did not eliminate the latex beads protectiveeffect, suggesting that neutrophils were not involved in theprotective effect against Acanthamoeba keratitis.

Macrophages are also involved in the innate immune re-sponse to Acanthamoeba keratitis.18,41 Removal of conjuncti-val macrophages with the macrophagicidal drug clodronateexacerbates Acanthamoeba keratitis.18 Thus, we hypothe-sized that intracorneal instillation of sterile latex beads inducesthe migration and/or activation of macrophages, which kill

Acanthamoeba trophozoites. Depletion of macrophages abro-

gated the latex beads protective effect, suggesting that mac-rophages play a role in the protective effect against Acan-thamoeba keratitis. Previous studies have indicated that thepresence of IFN- dramatically increases the capacity of mac-rophages to kill trophozoites.41 Moreover, macrophages pro-duce increased amounts of IL-1 after ingestion of latex beads.51

Thus, it is possible that latex bead treatment activates macro-phages, which mitigate disease. However, latex beads did notdirectly activate macrophages in our study. Previous reportshave suggested that corneal epithelial cells phagocytose latexbeads in vitro and in vivo.50 Moreover, phagocytosis of latexbeads induced the corneal epithelial cells to elaborate in-creased amounts of IL-1.50 Because latex beads did not directlyactivate macrophages, we explored the possibility that latexbeads activate macrophages indirectly, by inducing cornealcells to produce a macrophage-activating factor. However, thesupernatant from bead-treated corneal epithelial cells did notactivate macrophages, indicating that beads do not inducecorneal epithelial cells to produce a factor that activates mac-rophages in vitro.

An increased number of macrophages in latex-beadtreated,Acanthamoeba-infected corneas compared with untreated, in-fected corneas would indicate that latex bead treatment in-duces the chemotaxis of macrophages. Latex bead treatmentmay induce corneal cells to produce a chemoattractant formacrophages, such as MCP-1, macrophage inflammatory pro-tein (MIP)-1, IL-1, or IL-8, all of which are produced by

corneal epithelial cells.5256

Previous results have demon-strated that intracorneal instillation of latex beads before cor-neal infection with P. aeruginosa induces the corneal migra-tion of macrophages.24 Acid phosphatase staining of latex-beadtreated, Acanthamoeba-infected corneas revealed thatlatex beads induced the migration of macrophages at day 4after infection compared with untreated corneas. Thus, it islikely that the latex beads protective effect is mediated by thecorneal migration of macrophages, which kill trophozoites atthe ocular surface.

In summary, our results suggest that macrophages play arole in the latex beads protective effect against Acan-thamoeba keratitis. This is not a surprising result, as previousfindings suggest that cells of the innate immune apparatus,including macrophages and neutrophils, are crucial in the

resolution of Acanthamoeba keratitis and Pseudomonas ker-

FIGURE 7. Effect of corneal latex bead treatment on the migration of macrophages. Chinese hamster corneas were infected with A. castellanii,with or without prior latex bead treatment. Infected eyes of bead-treated and untreated Chinese hamsters were enucleated 3, 4, and 5 days afterinfection and 8-m cryostat sections were tested for endogenous acid phosphatase activity as an indicator of macrophage infiltration. Acid-phosphatasepositive cells (pinkish red) were counted by three masked observers. (A) Control eyes not treated with latex beads and collected onday 4 after infection; (B) latex-beadtreated eyes collected on day 4 after infection; and (C) number of acid-phosphatasepositive macrophages inthe corneas of infected mice. Numbers are representative of the three observers counts in untreated and latex-beadtreated corneas. Acid-phosphatasepositive cells were counted in the entire cornea (conjunctiva to conjunctiva) in four corneal sections per eye in each of three separateexperimental eyes per group. Mean SD; *P 0.05.


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atitis in experimental animals.18,19,57 However, elements of theadaptive immune system can exacerbate the pathologic se-quelae of HSV keratitis and Pseudomonas keratitis.24,25 Intra-corneal instillation of latex beads before ocular challenge with

P. aeruginosa results in a corneal infiltration of macrophagesand LCs and an increased expression of IFN-.24 These obser-vations led Hazlett et al.24to conclude that latex beads promotethe generation of a Th1 immune response to Pseudomonasantigens, which results in more severe keratitis, especially inTh2-prone BALB/c mice. They further proposed that the ex-cessive number of macrophages elicited by latex bead treat-ment contributes to corneal perforation in Pseudomonas ker-atitis.24 This conclusion is in sharp contrast to the present andprevious findings suggesting that macrophages are crucial forthe resolution ofAcanthamoeba keratitis.18 Thus, we favor thehypothesis that latex bead treatment stimulates the centripetalmigration of both LC and macrophages into the infected cor-nea. The infiltrating macrophages appear to be the primarymediators of the latex bead-induced protection through theircapacity to eliminate trophozoites from the ocular surface.

Acknowledgments

The authors thank Christina Stevens for preparation of the clodronateliposomes and Sudha Neelam for technical assistance.

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