the distribution and ontogeny of mhc antigens in...

The Distribution and Ontogeny of MHC Antigens inMurine Oculor Tissue

Hsin-min Wang, Henry J. Kaplan, W. C. Chan,* and Martha Johnson

The distribution of H-2 (Class I) and la (Class II) antigens in the mouse eye was determined with theuse of monoclonal antibodies and found to be different. H-2 antigen was observed in the cornealepithelium, conjunctiva! epithelium, choroid, and inner and outer nuclear layer of the retina—in orderof decreased intensity of staining. In contrast, la was detected most strongly, in a patchy distribution,in the choroid, limbus epithelium, and peripheral corneal stroma, followed by the iris and ciliary body.Thus, distinctively different patterns of distribution of H-2 and la were observed in the normal mouseeye. Surprisingly, adult eyes stained more intensely for both H-2 and la antigens than neonatal eyes,implying that the expression of MHC antigens varies with ontogeny. Since unique and importantimmunologic functions have been ascribed to class I and II antigens, their different distribution withinthe eye may indicate that various ocular structures can play distinctive roles in the immune response.Invest Ophthalmol Vis Sci 28:1383-1389,1987

MHC antigens have been implicated as importantcomponents both in the generation and expression ofthe immune response.1 For example, the antigen spe-cific activation of the helper T lymphocyte requirespresentation of both antigen and la (a Class II prod-uct) by the antigen-presenting cell. In a similar man-ner, cytotoxic T cell activity is dependent upon therecognition of antigen and products of the H-2D andH-2K (Class I) regions on the target cell.2 Althoughthe role of MHC antigens in ocular immunologicphenomenon is not known, histocompatibility anti-gens may be important. For example, the immuno-genicity of the cornea may be diminished by the ab-sence of antigen-presenting cells bearing Class IIMHC antigens, ie, Langerhan's cell3; the phenomenaof anterior chamber immunologic privilege for tissueallografts4"6 and anterior chamber associated im-mune deviation (ACAID)7"" may in part result fromthe unique tissue distribution of MHC antigenswithin the eye.

Previous investigators have used immunofluores-cent techniques to study the ocular distribution of

From the Department of Ophthalmology and the *Departmentof Pathology, Emory University School of Medicine, Atlanta,Georgia.

Supported by NIH Grant No. EY-03723 and an UnrestrictedGrant to the Department of Ophthalmology from Research to Pre-vent Blindness, Inc., New York. HJK is a Research to PreventBlindness-Olga Keith Weiss Scholar.

Submitted for publication: January 17, 1986.Reprint requests: Hsin-min Wang, PhD, Department of Oph-

thalmology, Emory Eye Center, 1327 Clifton Road, NE, Atlanta,GA 30322.

HLA-ABC (Class I) and Dr (Class II) antigens in nor-mal neonatal and adult human eyes.1213 However,only a few structures within the eye have been stud-ied, and Dr expression in the posterior segment of theeye has been neglected. We investigated the tissuedistribution and ontogeny of both H-2 (Class I) andla (Class II) antigens in the mouse eye, using rat anti-mouse monoclonal antibodies and the ABC (avidin-biotin) immunoperoxidase method.14

Materials and Methods

Preparation of Purified Monoclonal Antibodies

Hybridoma cell lines which produce monoclonalantibodies against either H-2 (all haplotypes, ATCCNo: TIB 126, Rockville, Maryland)15 or la (I-Abd-8

I-Edk, ATCC No: TIB 120) antigens16 were culturedin Dulbecco's modified Eagle's medium (Sigma, St.Louis, MO) containing 5% fetal bovine serum (FBS,Sterile Systems, Inc., Logan, UT), penicillin-G (100unit/ml, GIBCO, Grand Island, NY) and streptomy-cin (100 Mg/ml, GIBCO) in a humidified incubatorfilled with 5% CO2 and 95% air. The supernatant ofthe confluent culture was pooled. Antibody from 800ml of the pooled supernatant was precipitated by theaddition of an equal volume of 100% saturated(NH4)2SO4, pH 7.0, for 1 hr at 4°C.17 The precipitatewas collected by centrifugation at 48,100 g for 30min, and was redissolved in a minimum volume ofdistilled water. The solution was dialyzed overnightagainst two changes of 2 1 0.04 M phosphate buffer,containing 0.03 M NaCl, pH 8.0.18 It was cleared ofany precipitate by centrifugation and concentrated by

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1384 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / August 1987 Vol. 28

st

1a St

Fig. 1. Staining of mouse corneal frozen sections with the mono-clonal antibody against H-2 (Class I) antigen. The black granules inthe epithelium are positive staining, (a) adult cornea, (b) youngcornea, (c) neonatal cornea. Arrows indicate the positive reaction.St = stroma, Ep = epithelium (original magnification X600).

Amicon ultrafiltration with a pM-10 membrane(Amicon Co., Danvers, MA) to 20 ml. The concen-trated solution was passed through a DEAE-Cellulose(Sigma, St. Louis, MO) column (2.6 X 20 cm). Theeffluent fraction with highest protein concentrationwas collected, pooled and concentrated again withAmicon ultrafiltration. A control was obtained bypurifying the pooled supernatant from the p3 X 63cell line as detailed above.

Preparation of Frozen Sections For Immunohistology

Mice were used in accordance with the ARVO Res-olution on the Use of Animals in Research. Balb/Cmice of three different age groups, ie, adult (>6 weeksold), young (14-18 days) and neonatal (<7 days) weresacrificed with metofane (Pitman-Moore, Inc., NJ).Eyes were enucleated and oriented on a thin squarecork board so that the cornea and retina would beobtained on a single section. The eye was coveredcompletely with OCT compound (Miles Scientific,Naperville, IL) and snap-frozen in an isopentane-dryice bath. Four n frozen sections were cut at —21°Cusing a cryostat (Tissue-Tek II, Miles Scientific) andplaced on a chrome alum precoated slide. They werethen quickly fixed by dipping into cold acetone.

Staining of Eye Sections With MonoclonalAntibodies Against H-2 or la Antigens

The vectastain ABC (Avidin-Biotin Complex) Im-munoperoxidase staining procedure (Vector Lab,

Burlingame, CA) was followed with some modifica-tion. Four tx frozen sections from mouse eyes werefixed in acetone for 10 min. After washing for 15 minin 0.05 M Tris-saline, pH 7.6, the sections were incu-bated for 20 min with 1:5 diluted normal rabbitserum (NRS) in Tris-saline containing BGEN (bo-vine serum albumin [3%], gelatin (0.25%), EDTA [5mM] and NP-40 [0.025%]). Excess serum was blottedfrom the sections, and 50 fd of a 1:5 dilution of puri-fied rat monoclonal antibody against either themouse H-2 or la antigen (in Tris-saline-BGEN) wasadded and incubated overnight at 4°C in a moisturechamber. The sections were washed 3 times for 10min in Tris-saline, and incubated for 30 min withbiotinylated rabbit anti-rat IgG, diluted with a mouseskin extract, which was prepared as follows: 1 ml of0.5 M Tris, pH 7.6 and 5.7 ml of distilled water weremixed with 3.3 ml of the supernatant of a mouse skinextract obtained from shaking minced mouse skinovernight at 4°C with 10 ml of 0.45 M NaCl solution.The slides were again washed 2 times for 8 min withTris-saline. They were incubated for 10 min in 3%H2O2 in methanol and washed 2 times for 10 minwith Tris-saline. The sections were then incubated for30 min with vectastains ABC reagent (Avidin biotin-ylated peroxidase complex, Vector Lab., Burlingame,CA). They were washed for 40 min in Tris-saline,dipped in 0.1 M acetate buffer, pH 5.2 for 1 min, andincubated for 5 to 10 min in AEC (3-amino-9-ethyl-carbazole, Sigma) solution, which was made from 3ml of AEC (4 mg/ml) in N,N-dimethyformamide, 42ml of acetate buffer and 60 MI of 30% H2O2. Theywere then washed in tap water for 5 min, counter-stained with Mayer's hematoxylin for 4 min, andquickly dipped in 1% ammonia water. After 10 minof washing under tap water they were covered with aglass coverslip, using a drop of warm glycerine jelly,containing 8% gelatin and 50% glycerine.

Results

Immunohistochemical staining of adult mouseeyes demonstrated that H-2 (Figs, la and 3a) and la(Figs. 2a and 4a) antigens were distributed differently.Strong staining for H-2 antigen was observed in theconjunctival epithelium, corneal epithelium and cho-roid; moderate staining in the ciliary body and iris;weak staining in the outer nuclear layer, inner nu-clear layer and ganglion cell layer of the retina; andno staining in the corneal endothelium and stroma,the retinal pigment epithelium and photoreceptorlayer (Figs. 1, 3, 5). In contrast, la antigen was de-tected most strongly, but in a patchy distribution, inthe choroid, conjunctival epithelium and peripheral


No. 8 MHC ANTIGENS IN THE MOUSE EYE / Wong er ol. 1085

corneal stroma, and less intensively in the iris andciliary body. A weak, even distribution, probably rep-resenting background staining, was noted in the cor-neal epithelium (Figs. 2, 4, 6). The detailed stainingpattern of each ocular tissue follows.

Cornea, Limbus and Conjunctiva

The corneal epithelium stained intensely with themonoclonal antibody to H-2 (Figs. 1, 5a). Within thecorneal epithelium the most superficial layer stainedlightest and the epithelial cell plasma membrane wasmost intensively stained. Since the inside of the cellremained clear, the contour of the cells was easilyobserved. The junctions between cells, as well as thatbetween the cell plasma membranes and Bowman'smembrane stained prominently. No difference wasnoted between the central and peripheral portions ofthe corneal epithelium. No staining was noted in thecorneal stroma or endothelium.

In contrast to anti-H-2, the monoclonal antibodyto la produced a very weak and even pattern of stain-ing in the corneal epithelium (Fig. 2a); no staining ofthe central corneal stroma or endothelium was ob-served. In the peripheral corneal stroma, as well as thelimbus, a spot-like staining pattern was frequentlynoted (Fig. 6c). This was most prominent in the ante-rior rather than posterior stroma. It was frequentlyassociated with a few spots of heavy staining for la atthe basal layer of the epithelium, near the limbus.

Iris and Ciliary Body

As demonstrated in Figures 5d and e, patchy stain-ing for H-2 was observed in the iris and ciliary body.It appeared most intense in the inner rather than theouter cell layers of the ciliary body. The pattern of lastaining was somewhat different. Patchy, but ratherstrong staining was observed in both the ciliary bodyand iris (Fig. 6e, d). Unfortunately, the poor resolu-tion of histologic details in our frozen sections did notallow us to more precisely identify the cells beingstained.

Choroid

As shown in Figure 5c, H-2 antigen was abun-dantly present in the choroid, especially in the vicin-ity of Bruch's membrane. Frequently, intense stain-ing was noted in the endothelium of the blood vessels.A strong patchy pattern of staining was observed withthe monoclonal antibody to la (Fig. 6a). Most of thestaining appeared in the plasma membrane of cellsscattered throughout the choroid, and not near largeblood vessels.

St

St

Fig. 2. Staining of mouse corneal frozen sections with the mono-clonal antibody against la (Class II) antigen. Weak staining of theepithelium is observed in the adult (a) but not in the young (notshown) or neonatal (b) cornea. St = stroma, Ep = epithelium (origi-nal magnification X600).

Retina

A weak uniform pattern of staining for H-2 wasobserved in the outer nuclear layer, outer plexiformlayer, inner nuclear layer, inner plexiform layer andretina ganglion cell layer (not shown). In general, thenuclear layers stained less intensely than the plexi-form layers. The photoreceptor layer did not stain at

Sc

w'Ch' Sc

Ch

RC

RC

3 a K

Fig. 3. Staining of mouse retinal frozen sections with the mono-clonal antibody against H-2 antigen. Strong staining is observed inthe adult choroid (Ch, between the solid arrows) (a), patchy stain-ing in the young choroid (open arrow) (b), and no staining in theneonate (c). SC = sclera, RC = rods and cones, ONL = outernuclear layer (original magnification X600).


1386 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / Augusr 1987 Vol. 28

Ch

Ch

RC

t

RC

4 a

RC

c ;

Fig. 4. Staining of mouse retinal frozen sections with the mono-clonal antibody against la antigen. Patchy staining is observed inthe adult choroid (ch) (open arrows) (a) but not in the young (b) orneonatal choroid (c). The retina is not stained in any age group.The choroid is between the two small solid arrows. RPE = retinalpigment epithelium, RC = rods and cones (original magnificationX600).

Fig. 5. Staining of adult mouse eye frozen sections with themonoclonal antibody against H-2 antigen. There is strong stainingof the corneal epithelium (Ep) (a); moderate staining around thelarge conjunctival blood vessels (BV) (b)-the black granule is apositive reaction (arrows); staining in the choroid (Ch) but none inthe retinal pigment epithelium (RPE), rods or cones (RC) (c);patchy staining in the iris (I) (d) and the ciliary body (CB) (e)(original magnifications a-d X600; e X240).

all. No staining of any cells within the retina or retinalpigment epithelium was noted with the monoclonalantibody to la (Fig. 4a).

Comparison of MHC Expression by the Cornea!Epithelium and Choroid of Different Age Groups

Corneal epithelium: The staining characteristics ofthe corneal epithelium was studied in three differentage groups—the adult (>6 weeks), the young mouse(14-18 days) and the neonate (<7 days). As seen inFigure la, b and c, the corneal epithelium stained ineach of these age groups. However, it was noted thatthe intensity of staining increased with the age of themouse and paralleled the increase in thickness of thecorneal epithelium. There were only two cell layers inthe neonatal corneal epithelium, whereas the youngand adult mouse had many more. The intensity ofH-2 staining was weak, but even, in the neonate;moderate in the outer layer, and weak in the basallayer, of the young; strong throughout all layers of theadult.

Only in the adult corneal epithelium was weaklypositive staining for la observed (Fig. 2a). In neitherthe neonate nor young mouse was la detected(Fig. 2b).

Choroid: A strong belt-like staining pattern wasobserved in the choroid of the adult mouse with themonoclonal antibody to the H-2 antigen (Fig. 3a).This was not observed in either the neonate or youngmouse. Instead, a patchy pattern of distribution wasobserved in the young mouse (Fig. 3b) and no stain-ing in the neonate (Fig. 3c). In contrast, although astrong, patchy pattern of staining for la was notedthroughout the choroid in the adult mouse (Fig. 4a),no detectable la was present in either the youngmouse or the neonate (Fig. 4b and c).

Discussion

We studied the tissue distribution and ontogeny ofboth H-2 (Class I) and la (Class II) antigens in normalmouse eyes immunohistologically, using the ABCimmunoperoxidase method. The staining patterns ofour antigen-specific monoclonal antibodies were ini-tially confounded by both antigen non-specific ab-sorption of our second antibody and the presence ofendogenous peroxidase. We found that biotinylatedrabbit anti-rat IgG (the second antibody), and notperoxidase,19 nonspecifically bound to the mousecorneal stroma and sclera, specifically to the collag-enous extracellular matrix. By treating the biotinyl-ated rabbit anti-rat IgG with neutral salt-soluble col-lagen extract from mouse skin,20-21 we were able toeliminate this non-specific staining. Endogenous per-


No. 8 MHC ANTIGENS IN THE MOUSE EYE / Wang er ol. 1387

oxidase was removed from mouse eye sections bytreatment with 3% hydrogen peroxide in methanol.This also decolorized the red blood cells in vessels,which simplified interpretation of our sections sincethe AEC substrate can produce a color similar to redblood cells.

As a control for each monoclonal antibody, we ei-ther deleted the primary antibody or replaced it witha purified fraction of the p3 X 63 cell culture super-natant. No staining was observed with either control,indicating that the staining patterns demonstrated inour tissue sections were specific for the monoclonalantibodies employed. In the adult mouse eye the cor-neal epithelium, conjunctiva and limbus stainedmost intensely for H-2. The junction between epithe-lial cells was strongly stained, suggesting that the H-2antigen is membrane-bound.22 These results are con-sistent with previously published studies using indi-rect immunofluorescence in the human cornea.13

However, in contrast to the human eye, where it ap-pears that the expression of HLA-ABC is greater inthe peripheral than central corneal epithelium,23 weobserved no difference. Staining was also not ob-served in the corneal stroma or endothelium, imply-ing that under normal conditions these tissues ex-pressed minimal or no H-2 antigen, although in vitroculture of human corneal stromal24 and endothelialcells25 can result in the expression of Class I MHCantigens.

Adult mouse corneal epithelium stained lightly,but evenly, with our antibody to la. A previous studyof human corneal epithelium did not demonstratethe presence of HLA-Dr.26 Since Class II MHC anti-gens have been detected in epithelial cells from otherorgans—salivary gland, gut, lactating mammarygland27 and renal tubules28'29—it is not unreasonableto expect a small amount of Class II antigen in thecorneal epithelium. The positive spot-like staining wenoted in the epithelium of the limbus may be asso-ciated with Langerhan's cells, which have been docu-mented in this location.3'26'3031 However, we ob-served spot-like deposits of la in the peripheral cor-neal stroma. These have not been previously reportedand we do not know with what cell type it is asso-ciated. Although there is no in situ expression of la oncorneal endothelial cells it does appear that they havethe potential to express la during an inflammatoryreaction.32

Class I MHC antigens were only weakly expressedin the iris and ciliary body of the mouse eye. How-ever, scattered throughout these structures were fullsize cells that expressed la intensely. It is not knownwhat types of cells express this Class II MHC antigenor if they play an important part in immune re-

6 a

Fig. 6. Strong patchy staining is observed with the monoclonalantibody against la antigen in the adult mouse choroid (Ch) (a); inthe epithelium (solid arrows) and anterior stroma (open arrow) ofthe limbus and peripheral cornea (c); in the iris (I) (d) and ciliarybody (CB) (e), as indicated by the solid arrows. However, a weak,even pattern of staining is observed in the cornea epithelium (Ep)(b). RPE = retinal pigment epithelium, RC = rods and cones, Sc= sclera, St = stroma (original magnification X600).

sponse. Additional immunohistologic and functionalstudies are in progress to further clarify these cell pop-ulations.

Both H-2 and la are expressed strongly in the cho-roid of the normal adult mouse eye. The patchy dis-tribution of la positive cells, which appearedthroughout the choroid, may identify a cell similar tothe Langerhan's cell which is involved in antigenprocessing. Preliminary observations in a model ofHSV-induced keratouveitis in the mouse suggeststhat the patchy expression of la within the choroidincreases during an active immune response (Wangand Kaplan, submitted for publication).

In contrast to the choroid, the retina did not dem-onstrate la and only weakly expressed H-2. This isconsistent with previous observations that the centralnervous system only expresses MHC antigens weakly,if at all.33 Nevertheless, central nervous system astro-cytes, oligodendrocytes, microglia and other neuronscan be induced to express H-2 and la after exposureto gamma interferon.34-35 Likewise, the RPE in theeye can be induced to express la in experimentalEAU and retinitis pigmentosa.36"38 Since it has re-cently been shown that la positive astrocytes and vas-


1388 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / August 1987 Vol. 28

cular endothelial cells within the central nervous sys-tem can serve as antigen presenting cells, it is notunreasonable to postulate a similar role for these cellswithin the retina.35

Since the MHC antigens were expressed moststrongly in the corneal epithelium and choroid, thesetissues were chosen to study the effects of age onMHC antigen expression. In general, as the mousebecame more mature the quantity of expressed ClassI and II antigens increased. The neonatal mouse eyeexpressed H-2 very weakly and la not at all. Theseobservations are consistent with other reports thatindicate: (1) a defect in antigen presenting cell func-tion in neonatal mice39; (2) an increase in the numberof Langerhan's cells in the limbal and conjunctivalepithelium of the eye with maturity40; and (3) thedetection of HLA-ABC and Dr antigens in thehuman fetus only after 8 weeks of gestation.41

With the development of better immunohistologictechniques the cell populations stained for Class I andII MHC antigens will be more specifically defined.Furthermore, the variable expression of these anti-gens during the immune response may signal a possi-ble role for these cells in either the induction or ex-pression of immunity.Key words: MHC antigens, ontogeny, monoclonal antibod-ies, immunoperoxidase method, murine ocular tissue

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