murine keratocytes function as antigen-presenting cells

0014-2980/01/1111-3318$17.50+.50/0 © WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2001

Murine keratocytes function as antigen-presentingcells

Su K. Seo1,2, Bryan M. Gebhardt2, Ho Y. Lim1, Sang W. Kang1, Shiro Higaki2, Emily D.Varnell2, James M. Hill2, Herbert E. Kaufman2 and Byoung S. Kwon1, 2

1 The Immunomodulation Research Center and Department of Biological Sciences, University ofUlsan, Ulsan, Korea

2 LSU Eye Center, Louisiana State University Health Sciences Center, New Orleans, USA

Keratocytes express MHC class I molecules constitutively, and keratocytes stimulated withIFN- + express MHC class II molecules. Unstimulated keratocytes constitutively expressB7–1 and ICAM-1, as well as low levels of CD40 and 4–1BBL. These findings indicate thatkeratocytes may deliver both antigen-specific and costimulatory signals to CD4+ and CD8+

T cells. To demonstrate that keratocytes expressing B7–1 provide a costimulatory signal toT cells, CD4+ or CD8+ mouse T cells were incubated with anti-CD3 mAb and irradiated kera-tocytes. Enhanced proliferation of both CD4+ and CD8+ T cells occurred, and could be inhib-ited by anti-B7–1 mAb, indicating T cell costimulatory activity by B7–1 on the keratocytes. Todemonstrate that keratocytes can deliver an antigen-specific signal, CD4+ and CD8+ T cellsfrom herpes-infected mice were incubated with HSV-1-infected, irradiated keratocytes. Theresulting T cell proliferation and production of Th1 cytokines (IL-2, IFN- + ) indicated T cellactivation by antigens presented by the infected keratocytes. These results show that kera-tocytes in the corneal stroma of the mouse can function as antigen-presenting cells and,thus, may play a role in immune-mediated stromal inflammation such as herpetic stromalkeratitis.

Key words: Costimulation / Herpetic stromal keratitis / Keratocyte / MHC class II / B7–1

Received 19/4/01Revised 31/7/01Accepted 29/8/01

[I 21936]

Abbreviations: HSK: Herpetic stromal keratitis LC: Lan-gerhans cell

1 Introduction

Viral infections generally induce vigorous immune re-sponses. The immune responses and accompanyinginflammation during viral infection often result in localand systemic injury. Antigen-presenting cells (APC)play an important role in virus-induced inflammatoryresponses by activating T cells. The interaction of APCand T cells involves viral antigen presented by MHCclass I or II and costimulatory molecules, resulting ininflammation and associated by-products [1–3].

Herpes simplex virus type 1 (HSV-1) infection of the cor-nea leads to epithelial cell death and the establishmentof a latent infection in the sensory and autonomic gan-glia, from which the virus reactivates at intervals andcauses recurrent disease [4, 5]. Although the immuneresponse functions to prevent the spread of the virus,ensure the maintenance of latency, and suppress recur-rences, this response can also be pathogenic. Recurrent

viral disease can lead to a chronic immune inflammatoryprocess in the corneal stroma, called herpetic stromalkeratitis (HSK), which can result in blindness [6].

HSK in mice is mediated by CD4+ T lymphocytes. It hasbeen demonstrated that Th1 cells from inflamed corneasinfected with HSV-1 strain RE secrete IL-2, IFN- + , andTNF- § / g [7], and that treatment with neutralizing anti-IFN- + or anti-IL-2 antibodies significantly reduces theincidence and severity of corneal inflammation in thesemice [8]. Hendricks and colleagues reported that IFN- +and IL-2 mediate corneal inflammation by facilitatingPMN infiltration [9] and by maintaining PMN viability inthe cornea [10].

Several studies have demonstrated that corneal Langer-hans cells (LC) act as APC. In HSV-1-infected corneas,LC migrate from the conjunctival epithelium into the cen-tral cornea, where they are involved in the activation ofCD4+ T lymphocytes [11]. Inhibition of B7-1 in infectedcorneas prevents corneal inflammation [12]. However,other APC may also play an important role in the patho-genesis of corneal inflammation.

The keratocyte is a fibroblast-like cell in the cornealstroma. Its function is to produce collagen and other

3318 S. K. Seo et al. Eur. J. Immunol. 2001. 31: 3318–3328

Fig. 1. Expression of MHC class II (I-Ad) molecules on IFN- + -stimulated keratocytes. Cultured keratocytes were treatedwith 500 U/ml of IFN- + for indicated times (A) or indicateddose for 3 days (B). Keratocytes were harvested and thenstained for I-Ad with mAb (filled curve) or isotype-control(open curve). I-Ad expression was assessed by flow cyto-metry. (C) Total RNA was extracted from normal (-) or IFN- + -treated (500 U/ml for 3 days) (+) keratocytes, and reversetranscription-PCR was performed as described in Sect. 4.GAPDH was used as the gene control. Data shown are rep-resentative of four independent experiments. GAPDH, glyc-eraldehyde phosphodehydrogenase. (D) 1. Cultured kerato-cytes were stained with PE-anti-mouse CD45 mAb (LCA,Ly-5). 2. BALB/c splenocytes stained with the same mAbserved as the positive control. Open curve, PE-rat IgG2b;filled curve, PE-anti-mouse CD45 mAb.

extracellular matrix proteins. MHC class I molecules areexpressed at low levels by keratocytes in the normal cor-nea but at higher levels in the inflamed cornea [13]. T cellclones isolated from human corneas with HSK can bestimulated by IFN- + -treated, HSV-1-infected keratocytes[14]. Other studies have shown that HSV-1 infectedhuman keratocytes express ICAM [15] and that thesecells are phagocytic [16]. Treatment of human keratocy-tes with TNF- § or IL-1 § can induce IL-8 production [17].IL-8 plays an important role in inflammation through itscapacity to recruit T cells and nonspecific inflammatorycells such as neutrophils into inflammatory sites [18].Although these studies suggest that stromal keratocytesmay function as APC, a role for these cells in the immuneresponse in the cornea has not been identified.

To determine whether stromal keratocytes can functionas APC in the mouse cornea, we used primary culturesof murine keratocytes induced to express MHC class IIor costimulatory molecules to evaluate the response ofCD4+ and CD8+ T lymphocytes in terms of T cell prolifer-ation and cytokine production.

2 Results

2.1 Expression of MHC class II molecules onkeratocytes

IFN- + induces the expression of MHC molecules on pro-fessional APC [1]. To determine if keratocytes can pro-vide antigen-specific signals to T lymphocytes with MHCclass II-peptide complexes, we first examined the ex-pression of MHC class II molecules by cultured kerato-cytes incubated with recombinant murine IFN- + at differ-ent concentrations for varying periods of time. Flowcytometric analysis of these cells showed that MHCclass II expression increased following IFN- + stimulation.Peak expression occurred with stimulation by 500 U/mlof IFN- + for 3 days (Fig. 1A and B). For mRNA detection,the RNA was extracted from unstimulated and stimu-lated keratocytes. MHC class II mRNA was detectedonly when the cells were stimulated with IFN- + (Fig. 1C).These results indicate that keratocytes are capable ofexpressing MHC class II molecules; therefore, they mightbe able to provide antigen-specific signals to CD4+

T cells.

The cultured keratocytes did not stain with anti-CD45,whereas splenocyte cultures clearly showed stainingwith this mAb (Fig. 1D). The cultured keratocytes also didnot stain with mAb to CD11b, CD11c, or F4/80 (data notshown). These results indicate that our cultured kerato-cytes were not contaminated with bone marrow-derivedcells.

Eur. J. Immunol. 2001. 31: 3318–3328 Murine keratocytes as APC 3319

Fig. 2. Expression of the costimulatory molecules B7-1, B7-2, CD40, 4-1BBL, and ICAM-1 by LPS-stimulated keratocy-tes. Cultured keratocytes were stimulated with LPS (15 ? g/ml) for 0–3 days; stained with mAb to B7–1 (A), B7–2 (B),CD40 (C), or ICAM-1 (D); and analyzed by flow cytometry.Experimental, filled curve; isotype control, open curve. (E)Top: B7-1, B7-2, CD40, and 4-1BBL mRNA was detected incultures of normal (-) and LPS-treated (15 ? g/ml for 3 days)(+) keratocytes using RT-PCR. 1,2 = B7–1; 3,4 = B7–2; 5,6 =CD40; 7,8 = 4–1BBL. Bottom: GAPDH was used as the genecontrol. Data shown are representative of four independentexperiments.

2.2 Expression of costimulatory molecules onkeratocytes

To determine whether keratocytes can provide costim-ulatory signals to T lymphocytes, we examined theexpression of the costimulatory ligands B7–1, B7–2,CD40, and 4–1BBL on LPS- or PMA/ionomycin-stimulated keratocytes. B7–1 was highly expressed con-stitutively on unstimulated keratocytes (day 0), as well ason the 1-, 2-, and 3-day stimulated cells (Fig. 2A). Bycontrast, B7–2 was not expressed even after stimulationfor 3 days (Fig. 2B). CD40 was constitutively expressedbut the level was low (Fig. 2C). PMA did not induce CD40(data not shown). 4–1BBL was constitutively expressedat a level similar to that of CD40 (data not shown). Thekeratocytes also expressed ICAM-1 constitutively at ahigh level (Fig. 2D). B7–1, CD40, and 4-1BBL mRNAwere detected on unstimulated keratocytes (Fig. 2F).These results indicate that keratocytes constitutivelyexpress costimulatory ligands, in particular B7–1, CD40,and 4–1BBL; therefore, they can provide costimulatorysignals to T cells.

2.3 Keratocytes provide T cell costimulation

To test whether the keratocytes provide T cells with cos-timulatory signals, purified CD4+ and CD8+ T cells wereincubated with anti-CD3 mAb as the first signal and irra-diated keratocytes as the costimulatory signal. Boththe CD4+ and CD8+ T cells showed high proliferativeresponses at 1 ? g/ml of anti-CD3 mAb in combinationwith the keratocytes (Fig. 3A and B). When the kerato-cyte B7–1 ligand was blocked with anti-B7–1 mAb, pro-liferation of both CD4+ and CD8+ T cells was reduced; themaximal effect was seen with 1 ? g/ml of anti-B7–1 mAb(Fig. 4). The inhibition was not complete, however, indi-cating that although B7–1 on the keratocytes providescostimulation, other molecules contribute to theproliferation.

2.4 Induction of antigen-specific T cellproliferation and cytokine secretion bykeratocytes

To determine the optimal conditions for expression ofHSV-1 antigen on infected keratocytes, the keratocyteswere incubated with IFN- + for 3 days to elicit expressionof MHC class II molecules, then infected with HSV-1strain RE at an MOI of 3 and incubated for various peri-ods of time. Flow cytometry showed expression of HSV-1 antigen 6 h after infection and peak expression at 18 h(data not shown), while the cells maintained their mor-phology.

Herpetic stromal keratitis occurs after the primary epi-thelial keratitis is resolved. To obtain HSV-1-primed Tcells, CD4+ or CD8+ cells were purified from draining cer-vical lymph nodes of BALB/c mice at various times afterinfection. As shown in Fig. 5, HSK was visible at 8–10days postinfection (score, 0.5 to 1+), reached a peak at14–21 days (severity score, 3+), and decreased there-after.

To determine whether the keratocytes provide an HSV-1antigen-specific signal to T lymphocytes, we examinedcellular proliferation (Fig. 6) and cytokine production


Fig. 3. T cell proliferation by keratocyte costimulation. Purified CD4+ (A) and CD8+ (B) T cells (5×104 cells/well) from lymph nodesof BALB/c mice were cultured in complete medium on plates coated with the indicated concentration of anti-CD3 mAb alone orwith irradiated keratocytes (1×104 cells/well). T cell proliferation by anti-CD3 plus anti-CD28 was used as a positive control. Thecultures were incubated for 72 h and pulsed with [3H]thymidine for 18 h. Values are means ± standard deviations of data fromthree independent experiments. CD3, anti-CD3 mAb; CD28, anti-CD28 mAb.

Fig. 4. Inhibition of T cell proliferation by B7–1 mAb. Purified CD4+ (A) and CD8+ (B) T cells (5×104 cells/well) were cultured withirradiated keratocytes and indicated concentrations of anti-B7–1 mAb for 1 h on anti-CD3 mAb (1 ? g/ml)-coated plates. The cellswere incubated for 72 h and pulsed with [3H]thymidine for 18 h. Values are means ± standard deviations of data from three inde-pendent experiments. B7-1, anti B7–1 mAb; CD3, anti-CD3 mAb

(Fig. 7) by CD4+ and CD8+ T cells (obtained at early,peak, and late stages of HSK) in response to HSV-1-infected, irradiated keratocytes. T cells from the early

and late stages of corneal inflammation showed a lowlevel of proliferative response to HSV-1-infected kerato-cytes (Fig. 6A and C), whereas T cells from the peak of


Fig. 5. Severity of herpetic stromal keratitis (HSK). The cor-neas of BALB/c mice were infected with 1×105 PFU of HSV-1 strain RE. The severity of HSK was graded with a slit lampbiomicroscope based on the degree of corneal opacity andneovascularization as described in Sect.4. Values are means± standard deviations of scores at each observation point.

Fig. 6. Proliferation of HSV-1-specific T cells induced byherpes-infected keratocytes. Corneas of BALB/c mice wereinfected with 1×105 PFU of HSV-1 strain RE. CD4+ and CD8+

T cells were purified from the cervical lymph nodes of indi-vidual mice on postinfection day 7 (A), 21 (B), and 30 (C), andthen incubated with HSV-1 strain RE-infected keratocytes(MOI of 3) for 18 h. In each experiment, three mice wereused as donors of lymphocytes. The cells were further incu-bated for 72 h and pulsed with [3H]thymidine for 18 h. T cell+ keratocyte + CD3 1 ? g/ml, positive control; normal T cell +infected keratocyte, negative control. Values are means ±standard deviations of data from three independent experi-ments. CD3, anti-CD3 mAb; B7–1, anti-B7–1 mAb.

corneal inflammation showed a markedly enhanced pro-liferative response (Fig. 6B). Blocking keratocyte B7–1with anti-B7–1 mAb lowered T cell proliferation. Theseresults indicate that HSV-1-infected keratocytes canpresent HSV-1 antigen to HSV-specific T cells and alsocan provide a costimulatory signal in terms of B7–1 aswell as other surface molecules. HSV-1 infection for upto 18 h did not down-regulate the MHC class II expres-sion on the keratocyte surface induced by IFN- + treat-ment (data not shown).

To examine cytokines produced by HSV-1-primed T cellsstimulated with HSV-1-infected keratocytes, we assayedthe cell culture supernatants for the Th1 cytokines IL-2and IFN- + and the Th2 cytokine IL-4 (Fig. 7). High levelsof IL-2 and IFN- + were detected in cultures of CD4+ Tcells obtained during the peak of corneal inflammation.Blocking the B7–1 costimulatory molecule on the kerato-cytes with anti-B7–1 Ab reduced the levels of the Th1cytokines in these cultures. The level of IL-4 in the as-sayed cultures was low. The results indicate that HSV-1-infected keratocytes induce secretion of the Th1 cyto-kines IL-2 and IFN- + , but not the Th2 cytokine IL-4, fromHSV-specific CD4+ T cells.

2.5 Infiltrating immune cells in cornealinflammation

The normal, noninflamed cornea does not contain leuko-cytes. In the inflamed corneas of mice infected withHSV-1 strain RE, infiltrating cells have been shown tobe predominantly PMN and CD4+ T lymphocytes [4]. Todetect infiltrating immune cells in our stromal inflamma-tion model, we performed immunohistological experi-

ments at the early, peak, and late stages of stromalinflammation. At the early inflammation stage, infiltratingimmune cells were not seen (Fig. 8A). As corneal inflam-mation progressed, however, blood vessels graduallyinvaded from the periphery to the central cornea, and atthe peak stage of inflammation, both PMN and CD4+ Tcells were observed in the corneal stroma; CD8+ T cellswere rare (Fig. 8B). As corneal inflammation waned, theinfiltrating cells gradually disappeared. These results


Fig. 7. Secretion of Th1 cytokines by T cells. HSV-1 primed T cells were cultured with HSV-1 strain RE-infected keratocytes. Theculture supernatants were collected at 72 h and analyzed for IL-2, IFN- + , and IL-4 by ELISA. Cytokine levels (pg/ml) were deter-mined from standard curves and represented as mean values. Values are means ± standard deviations of data from six wellseach, representing a single experiment.

Fig. 8. Hematoxylin and eosin-stained sections of corneas with (A) mild and (B) severe herpetic stromal keratitis (HSK). (A) Cor-nea with mild HSK shows typical three-layered structure: epithelium (top), stroma (middle), and endothelium (bottom). Arrow-heads indicate stromal keratocytes. Original magnification, x400. (B) Cornea with severe HSK shows a thickened stroma heavilyinfiltrated with blood vessels and inflammatory leukocytes, as well as a disrupted endothelium. Arrowheads indicate stromalkeratocytes. Arrows with tails indicate infiltrating lymphoid cells. Original magnification, x400.

indicate that CD4+ T cells are available for antigen pre-sentation by keratocytes in corneas with HSV-1-inducedstromal inflammation and may, therefore, play a role thedevelopment of stromal inflammation under these condi-tions.

3 Discussion

Corneal stromal keratocytes are fibroblast-like cells thatproduce collagen and other proteoglycans necessary tomaintain the clarity and integrity of the cornea. When lib-erated into tissue culture, keratocytes grow rapidly, con-tinue to produce collagen and other ground substances,

and adopt a typical fibroblast morphology. The fact thatthese cells can go from a quiescent state to one ofhyperactivity, in terms of protein synthesis and multipli-cation, may imply that they undergo dedifferentiation intissue culture. In such a dedifferentiated state, thesecells could be capable of expressing membrane markersnot ordinarily expressed in vivo and could also becomecapable of functions such as antigen processing andpresentation in vitro as compared to the in vivo situation.To begin to define the functional and immunologicalproperties of corneal stromal keratocytes, we studiedpure cultures of these cells and defined their membranesurface markers and capacity to serve as APC in vitro.Such investigations will help clarify the immunopatho-


logic processes that occur in the corneal stroma, forexample, herpetic stromal keratitis. In this disease pro-cess in vivo, initiated by herpes virus infection, cornealstromal keratocytes express viral antigens and are thetargets for immune destruction by cytotoxic T cells.Whether or not this process represents a bona fide invivo expression of the antigen-presenting cell propertiesof keratocytes remains to be determined.

It should be noted that the membrane marker studiesand antigen-presenting function of the corneal keratocy-tes reported here are demonstrated using cultured cells.The relationship of these findings to keratocytes in vivowill be the focus of future studies. It is well documentedthat keratocytes in the normal cornea are quiescent cellswith the minimal metabolic activity necessary to maintaincorneal stromal integrity. In the pathologic cornea, kera-tocytes undergo morphologic and metabolic activationand they may then express costimulatory molecules andpresent antigens as we found to be the case for culturedcells.

The results of this study bear on two aspects of theimmune response: antigen presentation and autoimmu-nity. Professional APC such as dendritic cells, Langer-hans cells, and macrophages internalize complex proteinantigens, process these antigens into small peptides,and present them on the cell surface in association withMHC class I and II molecules [19, 20]. In addition, theprofessional APC also express on their plasma mem-branes a variety of costimulatory molecules such asB7–1, B7–2, CD40, and 4–1BBL [19–23]. The combinedpresentation of antigen on an MHC molecule and inter-action of costimulatory molecules on the APC with a Tlymphocyte is necessary for the initiation of T cell-mediated immune responses [21, 22]. Numerous investi-gations of the antigen-presenting capacity of cells thatare not identified as professional APC have beenreported [24–26]. Epithelial cells, thyroid gland cells,pancreatic cells, and others have been shown to haveinducible APC function [25, 27–30]. In this study weinvestigated the capacity of corneal stromal keratocytesto present herpes viral antigens. We found that thesecells constitutively express B7–1 and also express lowlevels of CD40 and 4–1BBL. Furthermore, we showedthat these cells are responsive to IFN- + and expressMHC class II molecules on their membranes. Based onthese findings, we tested the capacity of these cells topresent antigens to T lymphocytes and found that, invitro, corneal keratocytes could provide both antigenicand costimulatory signals to T lymphocytes resulting incellular proliferation and cytokine production.

Other investigators have shown that cells in the corneacan constitutively express MHC class I molecules and

can be induced to express MHC class II molecules in thepresence of IFN- + [14, 31, 32]. These earlier studies pro-vided support for the possibility that keratocytes mayserve as regional APC in the cornea. The corneal epithe-lium contains a small population of Langerhans cellswhich may present antigens in this cell layer [33]. How-ever, these cells generally do not enter the cornealstroma.

The cells in our cultures did not stain with mAb to CD45,CD11b, or CD11c, or F4/80 mAb, unlike splenocyteswhich did stain with all of these antibodies. We believethat our cultured keratocytes were not bone marrow-derived. We failed to identify any cells in the normal cor-neal stroma expressing CD45, CD11b, CD11c, or F4/80by immunohistochemical staining (data not shown), inmarked contrast to the findings of Hamrah et al. [34]. Itshould be noted, however, that this situation changesdramatically under pathological circumstances, such asfollowing HSV-1 infection.

Expression of costimulatory molecules in the cornea hasbeen investigated using an indirect approach. Chen andHendricks [12] reported that anti-B7–1 antibody treat-ment ameliorated HSK, a chronic corneal stromal im-mune response. These results suggested that B7-1-expressing cells are present in the corneal stroma. In thatHSK is an immune inflammatory process characterizedby infiltrating leukocytes which could provide a costimu-latory molecule stimulus, however, it is impossible toconclude which B7-1-expressing cells might be respon-sible for HSK. We have found that B7–1 is constitutivelyexpressed on the plasma membranes of cultured kerato-cytes and that B7–1 transcripts are uniformly present inthese cells during culture in vitro. CD40 and 4–1BBLexpression at low levels was also noted. This is the firstinvestigation of the presence of these costimulatory mol-ecules on corneal stromal cells.

Support for a pivotal role of corneal keratocytes in anti-gen processing and presentation in HSK comes from thework of Cubitt et al. [17]. These investigators reportedthat IL-8, a proinflammatory chemokine that recruits Tcells and neutrophils into inflammatory sites, is producedby both corneal epithelial cells and corneal stromal kera-tocytes. Thus, a role for corneal stromal keratocytes inantigen processing and presentation, T cell costimula-tion, and T cell recruitment is supported, but not proven,by these observations in an in vitro system. Investiga-tions of the corneal stromal keratocyte in situ are neces-sary to confirm that these cells function in vivo as they doin vitro.

Two groups of investigators have reported that HSK inmice, which develops as an immune response to herpes-


virus, may evolve into an autoimmune disease directedagainst corneal stromal antigens [35, 36]. They reportedthat T cell clones could be isolated from mice in whichHSK was induced and that these T cell clones recog-nized peptides with similar amino acid sequencesderived from corneal stromal antigens and herpes viralantigens. In one of these studies [35], the induction oftolerance to the putative self antigen prevented thedevelopment of HSK in the tolerant mice. Thus, thischronic, debilitating viral infection of corneal stromalcells is speculated to represent an autoimmune re-sponse that develops subsequent to the development ofHSK. More recently, investigators studying human cor-neas with HSK failed to isolate T cell clones reactive withcorneal self antigens [37]. Further experimentation willbe needed to determine if there are significant differ-ences between HSK in humans and mice and if autoim-munity develops in the mouse but fails to do so inhumans.

The results of our experiments open up new lines ofinvestigation of HSK including the possibility of identify-ing corneal stromal autoantigens and determining if thekeratocytes serve as the antigen-presenting cell in HSK,leading to the development of autoimmune disease inthis tissue. Based on our findings that keratocytes arecapable of antigen presentation and costimulation andthe findings of others that these cells produce proinflam-matory chemokines [38, 39], it is not impossible toexpect that these cells may, as well, be capable of pre-senting self antigens in the context of self MHC and thatthis would lead to the development of a chronic autoim-mune process in the corneal stroma. Presently, cDNAlibraries from keratocytes are being subjected to cloningand subtraction analysis so that we may identify somaticgene sequences that are similar to those of sequencesknown to exist in HSV-1. Within these studies, it may bepossible to synthesize HSV-1 peptides and corneal kera-tocyte peptides and determine which of these may beresponsible for HSK on the one hand and the autoim-mune sequelae of HSK on the other. Studies such as thiswill greatly aid in the development of therapeutic strate-gies to prevent and/or treat this sight-threatening condi-tion.

4 Materials and methods

4.1 Antibodies

The following reagents were employed for the identificationof cell membrane markers: PE-conjugated I-Ad (AMS-32.1);PE-conjugated streptavidin; PE-conjugated mAb to CD45(LCA, Ly5), CD11b, and CD11c, and F4/80 mAb; biotinlyatedmAb specific for mouse B7–1 (16–10A1), B7–2 (GL1), and

CD40 (3/23); purified anti-CD3 mAb (145.2C11), anti-CD28mAb (37.51), anti-B7–1 mAb (16–10A1), biotin-conjugatedanti-mouse ICAM-1 (3E2), and anti-Fc mAb (2.4G2); and PE-conjugated mouse IgG2b, hamster IgG, rat IgG2a, and ratIgG2b isotype control Ab (BD Pharmingen, San Diego, CA).Rabbit anti-HSV Ab (DAKO, Carpinteria, CA) was used forHSV-1 Ag detection.

4.2 Isolation and culture of keratocytes

Pure cultures of keratocytes were obtained from the corneasof BALB/c mice (Harlan Laboratories, Indianapolis, IN) usingpreviously described procedures [40–42]. Briefly, corneaswere aseptically removed and the epithelial and endothelialsurfaces scraped with a sterile scalpel blade to completelyremove the unwanted cells. Histological analysis confirmedthat this procedure removes the epithelium and any Langer-hans cells contained within it and also removes the endothe-lium. The remaining corneal stroma was cut into small frag-ments and placed into tissue culture flasks containing Dul-becco’s modified Eagles medium supplemented with 10%FBS. Within a week, keratocytes grew out of the cornealstroma and these were subcultured three times to expandthe number of cells and to ensure their purity. Attempts tostain cells in the keratocyte cultures for membrane markerssuch as CD11c, F4/80, OX-2, CD205, and CD45, which areexpressed on bone marrow-derived cells, uniformly failed.Using both flow cytometry and immunofluorescencemicroscopy, we have not been able to identify a hematopoi-etic lineage cell subpopulation in the keratocyte culturesused in these experiments.

The third subculture generation, a pure population of kerato-cytes having a uniform morphology similar to that of fibro-blasts, readily formed monolayers. These cells were usedthroughout the study. Further confirmation that the culturedcells were keratocytes came from ancillary studies in ourlaboratories. The cytoplasm of the cultured keratocytes canbe stained immunohistochemically for collagen subunits,and supernatants from keratocyte cultures contain maturecollagen fibrils. In other studies using bone marrow-derivedAPC, we have observed that these cells do not form conflu-ent monolayers and always grow as individual cells or smallclusters of cells. Cells having this growth pattern were notseen in the keratocyte cultures. Furthermore, cultured bonemarrow-derived APC constitutively express CD11c, F4/80,OX-2, CD205, CD45, and MHC class II markers.

For MHC class II expression, keratocytes were stimulatedwith 500 U/ml of recombinant murine interferon gamma(R&D Systems, Inc., Minneapolis, MN) for 0–5 days at 37°Cin a humidified CO2 incubator. To induce costimulatory mole-cule expression, keratocytes were stimulated with 15 ? g/mlof LPS (Sigma Chemical Co., St. Louis, MO) or 2 ng/ml ofPMA (Sigma) plus 0.15 ? M ionomycin (Sigma) for 1, 2, or3 days.


4.3 Flow cytometry

Cultured keratocytes (1×105) were harvested and washedtwice in washing buffer (HBSS containing 1% FBS and0.1 % sodium azide), then incubated with PE-conjugated I-Ad or biotinylated anti-mouse B7–1, B7–2, or CD40. Biotin-conjugated antibodies were detected by PE-conjugatedstreptavidin. For HSV-1 Ag staining, IFN- + -treated keratocy-tes were washed with DMEM-5% FBS, infected with HSV-1strain RE at an MOI of 3, and incubated for 6, 12, or 18 h.The cells were then stained with FITC-conjugated rabbitanti-HSV-1 Ab, washed three times with washing buffer, andanalyzed by flow cytometry (FACS Vantage SE). Theseexperiments were repeated at least three times and isotypecontrols were performed with each experiment.

4.4 RT-PCR

RNA was extracted from normal, IFN- + (500 U/ml)-stimulated, and LPS (15 ? g/ml)-stimulated keratocytesusing Trizol reagent (GibcoBRL). RNA (1 ? g) was reversetranscribed into cDNA using a PCR cDNA synthesis kit(Clontech Laboratories, Inc., Palo Alto, CA). PCR was per-formed using the sense/antisense primers. PCR conditionswere as follows: for I-Ad, B7–1, B7–2, and CD40, 94°C for5 min, followed by 35 cycles of 1 min at 94°C, 1 min at55°C, and 1 min at 72°C, with a final extension at 72°C for5 min. For detection of 4–1BBL and GAPDH mRNA, anneal-ing was performed at 60°C. The PCR primers used were asfollows: I-Ad, forward 5’-CTGTTTATCAGTCTCCTGGAG-3’;,reverse 5’-GCACACACCACAGTTTCTGTC-3’; B7–1, for-ward 5’-GCTGCCTTGCCGTTACAACTC-3’, reverse 5’-GGAAATTGTCGTATTGATGCC-3’; B7–2, forward 5’-TGG-GACTGCATATCTGCCGTG-3’, reverse 5’-GACCTTGCTT-AGACGTGCAGG-3’; CD40, forward 5’-CAAGCCACTGC-ACAGCTCTTG-3’, reverse 5’-AGATGACATTAGTCTGAC-TCG-3’; 4–1BBL, forward 5’-GAGAGAATAATGCAGACCAG-3’, reverse 5’-GTCATCTACCTGAGGCTTTG-3’; GAPDH, for-ward 5’-GTCATGAGCCCTTCCACGATG-3’, reverse 5’-GAATCTACTGGCGTCTTCACC-3’. PCR products werevisualized by ethidium bromide following electrophoresis on1% agarose gels.

4.5 Virus

The HSV-1 RE strain was grown on CV-1 cells in MEM sup-plemented with 2% heat-inactivated FBS (Hyclone), penicil-lin (100 U/ml), and streptomycin (100 ? g/ml). Virus was tit-tered on CV-1 cells by plaque assay. Virus was aliquoted in1-ml vials and stored at -70 °C.

4.6 Mice and virus infection

Five- to seven-week-old female BALB/c (H-2d) mice (HarlanLaboratories, Indianapolis, IN) were housed in the Louisiana

State University Health Sciences Center animal care facility,and maintained on laboratory chow and water ad libitum.Mice were anesthetized with ketamine hydrochloride (1 mg/kg, Vetamine; Phoenix Scientific Inc., St. Joseph, MO), andxylazine (0.5 mg/kg, Ben Venue Laboratories, Bedford, OH).Corneas were scarified with a 26-gauge needle in a criss-cross pattern and infected with 1×105 PFU of HSV-1 strainRE. On various days after infection, the severity of clinicalstromal keratitis was scored with a slit lamp biomicroscope(Topcon Corporation, Japan) as follows: 0, normal cornea;0.5+, slight neovascularization in the peripheral cornea, orslight edema and opacity; 1+, edema and opacity of thestroma confined to less than one half the diameter of thecornea or mild neovascularization; 2+, edema and opacity ofthe stroma extending more than one half the diameter of thecornea, or moderate neovascularization; 3+, severe edemaand opacity, or neovascularization in the whole cornea, irisnot visible; 4+, corneal perforation.

4.7 Proliferation assays

CD4+ and CD8+ T cells were purified by mini-MACS micro-beads (Miltenyi Biotec., Auburn, CA) from cervical lymphnodes of BALB/c mice. Purified T cells (5×104 cells/well)were cultured in complete medium (RPMI-1640, 10% FBS)on plates coated with various concentrations of anti-CD3mAb or on irradiated (3000R) keratocytes (1×105 cells/well).To block B7–1 on keratocytes, irradiated keratocytes wereincubated with purified anti-mouse B7–1 mAb for 1 h at37°C before purified T cells were added. For HSV-specific Tcell proliferation, CD4+ and CD8+ T cells were purified fromcervical lymph nodes of HSV-1 strain RE-infected BALB/cmice. The T cells were harvested at the early, peak, andwaning stages of stromal inflammation. The T cells (1×105

cells/well) were cultured for 3 days with irradiated keratocy-tes that had been stimulated with IFN- + (500 U/ml), thenincubated for 18 h with HSV-1 RE strain at an MOI of 3. TheT cell/keratocyte cultures were further incubated for 3 daysat 37 °C in a 5% CO2 environment and pulsed with 1 ? Ci[3H]thymidine (Amersham, Piscataway, NJ) for the last 18 h.The cells were harvested on glass fiber filter paper (What-man Inc., New Jersey) using a cell harvester (Otto Hiller Co.,Madison, WI). Incorporated radioactivity was counted usinga liquid scintillation counter (Beckman).

4.8 Cytokine assay

Cytokine production by T cells was determined in cell cul-ture supernatants harvested after 72 h of stimulation withHSV-1 strain RE-infected keratocytes. Cell culture superna-tants were evaluated for IL-2, IFN- + , and IL-4 by enzyme-linked immunosorbent assay (ELISA) as described by themanufacturer (Endogen, Woburn, MA). Optical densitieswere read on a kinetic microplate reader (Bio Tek Instru-ments, Winooski, VT), and quantified using standards.


4.9 Histopathology and immunohistochemical staining

Eyes were fixed in 10% buffered formalin, embedded in par-affin, sectioned at 7 ? m, and stained with hematoxylin andeosin. Uninfected corneas and corneas with HSK wereexamined microscopically. Evidence for inflammatory andimmune cells was recorded with a photomicroscope. Eyesto be used for immunohistochemical staining were frozen inoptimum cutting temperature medium (OCT), sectioned at7 ? m in a cryostat, fixed in cold acetone for 5 min, and thenstained using a double immunofluorescent staining method.Rat mAb directed against a mouse CD4 T cell marker (L3T4,BD Pharmingen) and a mouse CD8 T cell marker (Ly-2, BDPharmingen) were diluted 1:200, pipetted onto the tissuesections, and incubated for 1 h. After three rinses in Tris-buffered saline (PBS), pH 7.4, the sections were incubatedfor 1 h in goat anti-rat immunoglobulin antibody labeled witha fluorochrome (Alexa Fluor7; Molecular Probes, Eugene,OR). The slides were then washed three times and cover-slipped (GEL/MOUNT7, Biomedia Corp., Foster City, CA).Control sections were incubated in nonimmune rat immuno-globulin diluted to the same concentration as the primaryantibody, and then the second antibody. The slides wereexamined with a fluorescence microscope (Nikon E600) andthe images were photographed (Fujichrome film, MS 100/1000).

Acknowledgements: The authors thank Ms. Carole Hothfor typing the manuscript. This work was supported by U.S.Public Health Service grants R01DE12156 (BSK) from theNational Institute for Dental Research, and R01EY06311(JMH), R01EY02672 (HEK), and P30EY02377 (departmentalcore grant) from the National Eye Institute, National Insti-tutes of Health, Bethesda, Maryland, and support from theKorean Research Foundation (KRF 2001–015-0P0553) andthe SRC Fund to the IRC from the KOSEF and the KoreanMinistry of Science and Technology. Dr. Hill is supported inpart by a Senior Scientific Investigator award (2001) fromResearch to Prevent Blindness, Inc., New York, NY.

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Correspondence: Byoung S. Kwon, LSU Eye Center, 2020Gravier Street, Suite B, New Orleans, LA 70112, USAFax: +1-504-412–1315e-mail: bkwon — lsuhsc.edu


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