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Investigative Ophthalmology & Visual Science, Vol. 32, No. 8, July 1991Copyright © Association for Research in Vision and Ophthalmology

Class II Antigen Expression by Lacrimal Epithelial Cells

An Updated Working Hypothesis for Antigen Presentation by Epithelial Cells

Austin K. Mircheff,* J. Peter Gierow,* Leslie M. Lee,* Ross W. Lambert,*Ronald H. Akashi,* and Florence M. Hofmant

It has been suggested that aberrant expression of Class II histocompatibility antigens (HLA) is in-volved in T cell activation and leads to autoimmunity. Although Class II antigen expression was foundin various nonlymphoid tissues, including salivary glands, its expression on lacrimal epithelial cells hasnot been reported. In this study, 12 cadaver lacrimal glands were analyzed for HLA-DR and for thenumbers and distributions of T suppressor cells (Ts), T helper cells (Th), B cells, and macrophages.None of these cases exhibited the high numbers of inflammatory cells, tissue damage, and fibrosischaracteristic of Sjogren's syndrome. The HLA-DR-positive epithelial cells were detected in ten cases;they represented from less than 1% to more than 70% of the epithelial cells. In these ten positive cases,there were greater numbers of T cells per millimeter squared (229 ± 94 [mean ± the standard error ofthe mean]) than in the two HLA-DR-negative cases (37 ± 1 [mean ± range]). Three lacrimal glandspecimens tested were negative for immunoglobulin (Ig) G-bearing B cells, and two of the three speci-mens tested had IgA-bearing cells. Acinar cells were isolated from rat and rabbit lacrimal glands andcultured overnight in serum-free media supplemented with several potential mediators of Class IIantigen expression: interferon-7, carbachol, or isoproterenol. Freshly isolated cells did not expressClass II antigens at detectable levels, but in most experiments, they began to express the antigen evenin the absence of putative mediators. In light of results from recent studies of antigen presentation andepithelial cell membrane dynamics, these findings suggest a hypothesis in which Class II antigen-ex-pressing epithelial cells present antigenic peptides that are generated in the intracellular compartmentsin communication with the basal-lateral membrane assembly and recycling pathway. Invest Ophthal-mol Vis Sci 32:2302-2310,1991

The lacrimal glands contribute a substantial por-tion of the volume of the aqueous layer of the preocu-lar tear film, and lacrimal dysfunction is a majorcause of tear film disorders and resulting ocular mor-bidity. Lacrimal insufficiency often is attributed toaging and to an autoimmune disease, Sjogren's syn-drome. It is generally presumed that during progres-sion of the disease in Sjogren's syndrome, inflamma-tory infiltrates inhibit the secretory function of glan-dular epithelial cells; the end stage of the disease is

From the Departments of *Physiology & Biophysics, ""Ophthal-mology, and fPathology, University of Southern California Schoolof Medicine, Los Angeles, California.

Supported by the University of Southern California School ofMedicine Biomedical Research support grant, National Institutesof Health grant EY 05801 (Bethesda, Maryland), and private funds(RHA). JPG was the recipient of a Fight for Sight-Prevent Blindnesspostdoctoral fellowship, Schaumburg, Illinois. RWL was the recipi-ent of a Sigma Xi grant-in-aid of research, Research Triangle Park,North Carolina.

Submitted for publication: January 23, 1990; accepted February15, 1991.

Reprint requests: Austin K. Mircheff, PhD, Department of Physi-ology and Biophysics, University of Southern California, School ofMedicine, 1333 San Pablo Street, Los Angeles, CA 90033.

characterized by complete destruction of the secre-tory epithelium and fibrosis of the gland.1"3 Becauseaging is associated with increasing lymphocytic infil-tration and fibrosis of the lacrimal gland, it has beensuggested that the factors impairing lacrimal secretionmay be similar in aging and autoimmune disease.4

The lacrimal glands are important components ofthe mucosal-associated lymphoid system,5"7 and theirparticipation in this system depends, in part, on theirability to recruit T and B lymphocytes.7 There iscurrently little information concerning the normalmechanisms of lymphocytic recruitment or the waysthese mechanisms might be perturbed to cause exces-sive lymphoproliferation. One theory for the etiologyof autoimmune disease places special emphasis on theinappropriate or aberrant expression of Class II histo-compatibility antigens by nonlymphoid cells. TheClass II antigens, HLA-DR in humans and major his-tocompatibility complex (MHC) la in rodents, aretransmembrane cell surface structures composed oftwo polypeptides of molecular weights 33 and 28 kilo-daltons.8 These antigenic determinants are found onresting mature B cells, macrophages, and dendriticcells throughout the body.9"12 Class II antigens func-

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No. 8 LACRIMAL EPITHELIAL HLA-DR EXPRESSION / Mircheff er ol 2303

tion in antigen presentation to T cells in conjunctionwith the T cell receptor and foreign antigens.13 Ac-cording to one theory,14"16 the inappropriate expres-sion of Class II antigens by a cell population allowsthat population to present its own surface constitu-ents, rendering it a target for an immune response.

There are now numerous instances in which au-toimmune disease has been correlated with inappro-priate Class II antigen expression. These include thy-roid epithelial cells in autoimmune thyroiditis17 andsalivary duct cells in Sjogren's syndrome.18"20 How-ever, Class II antigen expression by epithelial cells ofthe lacrimal glands has not been discussed to ourknowledge, either for humans or for relevant animalmodels.

Before attempting to consider the role inappropri-ate Class II antigen expression might play in eitherage- or autoimmune-related lymphoproliferation inthe lacrimal glands, it seemed essential to confirmthat lacrimal gland epithelial cells can, in fact, expressClass II antigens. We report here the presence of ClassII antigens on epithelial cells and the correlation be-tween this antigen expression and the numbers of Tlymphocytes and macrophages in human lacrimalglands. Furthermore, in an attempt to evaluate possi-ble mechanisms for Class II antigen expression, weused isolated rat and rabbit lacrimal acinar cells main-tained in short-term culture. In most experiments, theacinar cells began expression of Class II antigens evenin the absence of interferon-7 or other potential medi-ators of Class II antigen expression. On the basis ofthese observations, we present a hypothesis for anti-gen presentation by Class II antigen-expressing epithe-lial cells that incorporates concepts derived from re-cent studies of antigen presentation by macrophagesand of plasma membrane dynamics in secretory epi-thelial cells.

Materials and Methods

Materials

Cadaver lacrimal glands were obtained as excess tis-sue from the Department of Anatomy and Cell Biol-ogy within 24 hr post mortem (IRB number 06077).Male Sprague-Dawley rats, 5-7 weeks old, were ob-tained from Harlan Sprague-Dawley (Indianapolis,IN). Male New Zealand white rabbits (2 kg) were ob-tained from Irish Farms, Norco, CA. All animals wereused in accordance with the ARVO Resolution on theUse of Animals in Research.

The following substances were used: OCT (Miles,Elkhart, IN); Ham's F-12 medium, Dulbecco'sModified Eagle's Medium (DME), and fetal calfserum (Irvine Scientific, Irvine, CA); purified collage-nase (Gibco, Gaithersburg, MD); hyaluronidase

(Worthington, (Freehold, NJ); Hank's medium andDNase type 1 (Sigma, St. Louis, MO); and humanrecombinant interferon (Collaborative Research,Bedford, MA).

The following antibodies were obtained: Ox-6 (im-munoglobulin [Ig] G, mouse monoclonal against ratla; Sera-lab, Sussex, UK) and Leu 2 (anti-CD8, T sup-pressor cells), Leu 3 (anti-CD4, T helper cells), Leu 14(anti-CD22, B cells), Leu M5 (anti-CDlie, macro-phages), and anti-HLA-DR (all from Becton-Dickin-son, San Jose, CA). Samples of the supernatant ofmonoclonal culture 2C4, which contains an antibodydirected against the rabbit MHC Class II antigen la,21

were provided by Drs. Katherine L. Knight (StritchSchool of Medicine, Loyola University) and RobertA. Prendergast (The Johns Hopkins UniversitySchool of Medicine). Biotinylated horse anti-mouseantibody, biotinylated goat anti-rabbit antibody, andavidin-peroxidase complex were obtained from Vec-tor (Burlingame, CA). Polyclonal rabbit antibodiesto human IgA and IgG were from Dako (Carpen-teria, CA).

Other reagents were from standard suppliers.

Tissue Preparation

Human lacrimal glands were placed in physiologicsaline at 4°C and transported to the laboratory. Fatand readily accessible connective tissue were re-moved. Glandular tissue was blotted dry, snap frozen,and stored at -70°C.

The rats were killed by CO2 narcosis. The rabbitswere sedated with an intramuscular injection of keta-mine and xylazine and then given lethal injections ofsodium pentobarbital. Lacrimal glands to be used forcryosections were rinsed in physiologic saline, thenplaced in OCT, snap frozen, and stored at -70°C.The glands to be used for isolation of acinar cells wereplaced in sterilized Ham's F-12 medium supple-mented with penicillin (100 U/ml), streptomycin (0.1mg/ml), L-glutamine (2 mM), linoleic acid (0.084 ng/ml), HEPES (10 mM), soy bean trypsin inhibitor(0.05 mg/ml), and bovine serum albumin (5 mg/ml).All solutions were sterilized by filtration through 0.2-/xm pore-size filters (Costar, Cambridge, MA). Acinarcells were isolated with the modification of Oliver'sprocedure22 described by Hann et al.23 They weremaintained in 50% Ham's F-12 medium with 50%low-glucose DME. The medium did not containserum, but it was supplemented with penicillin (100U/ml), streptomycin (0.1 mg/ml), glutamine (4 mM),hydrocortisone (5 nM), transferrin (5 Mg/ml), insulin(5 jig/ml), epiderma growth factor (10 ng/ml), buty-rate (2 mM), and linoleic acid (0.084 Mg/ml). Agentstested for their ability to induce Class II antigen ex-


2304 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / July 1991 Vol. 32

pression were: interferon-7 (1000 U/ml); carbachol(10 JUM and 1 mM); and isoproterenol (10 tiM and 1mM). Overnight culture was done in a 37°C water-jacketed incubator under an atmosphere of 95% airand 5% CO2. After culturing, acinar cells were trans-ferred to Falcon centrifuge tubes (Becton Dickinson,Lincoln Park, NJ), diluted to 15 ml with medium,and sedimented at 100 X g in a preparative centrifuge.They were washed once by resuspension and centrifu-gation, then resuspended in Hank's medium, pH 7.4,containing 5% bovine serum albumin. Aliquots of200 ix\ were sedimented onto polylysine-coated slidesat 800 rpm for 5 min in a cytocentrifuge (Shandon,Pittsburgh, PA). Cell preparations were allowed to dryovernight and then used for immunocytochemistry.

Immunocytochemistry

Immunoperoxidase staining of cryostat sectionsand cytocentrifuge preparations was done as de-scribed previously.24 Briefly, the sections were fixed inacetone for 5 min, then incubated in phosphate-buf-fered saline (PBS), pH 7.4, for 5 min. Human, rat, andrabbit specimens were pretreated sequentially withavidin and biotin according to the vendor's instruc-tions (Vector) to reduce nonspecific backgroundstaining. Tissue samples were incubated with primaryantibodies in a humidified chamber for 30 min at25 °C, then washed for 10 min with 1% bovine serumalbumin in PBS. Depending on the source of the pri-mary antibody, the tissues were then incubated eitherwith biotinylated horse anti-mouse antibody or bio-tinylated goat anti-rabbit antibody for 30 min, rinsed,and washed. They then were incubated with avidin-biotin-peroxidase complex for 20 min and againrinsed and washed. Amino-ethyl carbazole, whichproduces a red precipitate in the presence of peroxi-dase, was added to all the slides and incubated for 10min, followed by Mayer's hematoxylin for 3 min. Theslides were rinsed in tap water for 10 min andmounted in glycerol and PBS. Controls for the stain-ing procedure included both the use of an irrelevantantibody and the use of PBS in place of primary anti-body.

The slides were examined using a 1 -mm2 grid. Mac-rophages, T lymphocytes, and B cells could be re-solved in almost all cases. They were counted in threeseparate areas per tissue; the results are expressed asmean numbers of cells per millimeter squared ± thestandard deviation. It was not possible to resolve indi-vidual Class II-positive epithelial cells, and they werescored as: - (negative); + / - (< 1% of epithelial cellspositive); ++ (1-30% of epithelial cells positive); and+++ (> 70% of the epithelial cells positive).

Results

Lacrimal glands from five women (age range, 71-91 yr; mean, 83 yr) and two men (age range, 87-88 yr)were examined. The HLA-DR was detected on lym-phoid cells, as expected, and on 1-100% of the epithe-lial cells in 10 of the 12 samples tested (Fig. 1). Boththe cytoplasm and the surface of the acinar cells ap-peared to stain for HLA-DR. The staining in anacinar cluster was not uniform but appeared in a mo-saic pattern, suggesting differences in the levels ofHLA-DR expression by individual epithelial cells.Two of the 12 lacrimal tissues showed no HLA-DR-positive epithelial cells; the lymphocytes in these tis-sues stained positively for HLA-DR (Fig. 2).

As illustrated in Figures 3-5, T helper and suppres-sor lymphocytes and macrophages were distributedamong the acinar clusters, with 3- to 11-fold morelymphoid cells than macrophages. The data in Table1 indicate that there were wide ranges in the numbersof helper T lymphocytes, suppressor T lymphocytes,and macrophages. The ratio of T helper to T suppres-sor cells for the positive specimens did not suggest anyparticular pattern. Although the number of sampleswith no epithelial HLA-DR expression was too smallto permit generalization, it may be noteworthy thatthe two negative cases examined had fewer lymphoidcells than nine of the ten samples with positive epithe-lial cells.

Three of the human lacrimal gland samples alsowere surveyed for IgA- and IgG-producing lympho-cytes. All three samples were negative for IgG. Onesample (from a 71-year-old woman) was also negativefor IgA; this was one of the two samples that did nothave HLA-DR-positive epithelial cells. The two othersamples examined (from an 87-year-old man and a91-year-old woman), both of which had HLA-DR-positive epithelial cells, were also positive for IgAcells.

In a series of experiments designed to test the abili-ties of various agents to induce Class II antigen ex-pression, lacrimal acinar cells were isolated from maleSprague-Dawley rats and cultured overnight in con-trol medium and in media supplemented with inter-feron-7, isoproterenol, or carbachol. Preliminary ex-periments also were done with a combination of 1ng/ml of TPA and 1 ixg/m\ of A23187 as potentialmediators of Class II antigen expression; however thiscombination was cytotoxic. In one experiment, con-trol cells and cells maintained in the presence of 10nM carbachol were negative for Class II antigen (Fig.6), but Class II antigen-positive cells could be detectedafter overnight culture in the presence of either iso-proterenol or interferon-7 (Fig. 7). In two additional



Fig. I. Human iacnmai gland tissue from an 88-yr-old man isstained with anti-HLA-DR and shows intensely positive acinarcells. Magnification X100.

Fig. 2. Human lacrimal gland tissue from an 88-yr-old woman isstained with anti-HLA-DR and shows no DR-positive epithelialcells but occasional DR-positive leukocytes (arrows, magnificationX250).

•x.

Fig. 3. Human lacrimal gland tissue from an 88-yr-old man isstained with anti-CD4 and shows low numbers of positive T helpercells ... .onp the acini. Magnification X100.

Fig. 4. Human lacrimal gland tissue from an 88-yr-old man isstained with anti-CD8 and shows T suppressor cells distributedamong the acini. Magnification X100.

Fig. 5. Human lacrimal gland tissue from an 88-yr-old man isstained with anti-CDll and shows a modest number of macro-phages. Magnification X100.

Fig. 6. Isolated rat lacrimal acinar cells from overnight cultureshow no Class II antigen expression. Magnification X400.



Table 1. Summary of expression of HLA-DR on epithelial cells and quantitation of leukocyte subtypes presentin human lacrimal gland samples

Case

88-yr-old F71-yr-oldF79-yr-old F79-yr-old M86-yr-old F88-yr-old M87-yr-old M91-yr-oldF94-yr-old M66-yr-old F38-yr-old M75-yr-old M

HLA-DR+epithelial cells

_

-±

+++

++++++

++++++

Leu2( suppressor/

18 ± 55± 1

25 ± 1375 ± 1897 ±2076 ± 132± 1

144 ± 3370 ± 19

108 ± 17119± 13152 ±31

Leu3(^helper)

20 ± 534 ± 557 ± 1513± 690 ± 1189 ± 1130 ± 727 ± 1329 ± 109± 5

930 ± 113195 ± 6

LeuM5(macrophage)

10 ± 311 ± 218 ± 23± 1

18± 327 ± 146± 17± 2

85 ±2134 ± 15

225 ± 2769 ± 9

Leu 14(B-cell)

ndndnd<1nd

10%*nd

0nd<1

225 ± 4317 ± 2 4

Values presented are mean numbers of positive cells per mm3; nd, notdone; F, female; M, male.

Scoring for DR-positive epithelial cells: - negative; ± <1%; + <30%; + +<70%; + + + >70%.

* Positively staining cells in this sample appeared in clusters and were diffi-cult to identify and count; values given for this sample are estimated percent-ages of the total tissue area occupied by the clusters.

experiments, however, 20-50% of the control cellsshowed Class II antigen expression, making it difficultto quantify the effects of the potential mediators ofClass II antigen expression.

Because of the preparation-to-preparation variabil-ity of Class II antigen expression by rat lacrimal acinarcells in control media and because of difficulties inobtaining consistent yields of viable cultured cellsfrom the rat preparation, we undertook a parallel se-ries of experiments with rabbit lacrimal cells in short-term culture. Cryostat sections of rabbit lacrimalglands had Class II antigen-positive macrophages andlymphocytes (Fig. 8), but no Class II antigen-positiveepithelial cells were detected. Freshly isolated acinarcells (Fig. 9) also were negative for Class II antigenexpression. In four separate experiments, 70-80% ofthe acinar cells in control medium had become posi-tive for Class II antigen expression by 72 hr in culture,and the frequency of positive cells was not altered no-tably by carbachol (10 fiM and 1 mM), isoproterenol(10 nM and 1 mM), or interferon-7 (1000 U/ml). Thestaining appeared to occur at both the plasma mem-branes and at intracellular sites. An additional experi-ment was done to survey the time course of inductionof Class II antigen expression. After 3 hr in culturemedium, the earliest time examined, 70-80% of theepithelial cells stained positively (Fig. 10), and the fre-quency of positive cells was not altered by carbachol,isoproterenol, or interferon-7. The only obviouschange to occur over the ensuing 15 hr was an in-creased tendency of the cells to form clusters (Fig. 11).This phenomenon made it difficult to interpret appar-ent differences in the intensity of staining of cells thathad been maintained in culture for different intervals.

Discussion

These observations show that epithelial cells of hu-man, rat, and rabbit lacrimal glands, like those of thethyroid gland,17 salivary glands,18"20 hepatic duct,25

mammary gland,26 small intestine,27 and retinal pig-ment epithelium,2829 are capable of expressing ClassII HLAs.

In general, the degree of epithelial HLA-DR expres-sion tends to correlate with the extent of lymphocyticinfiltration in various autoimmune diseases.14"20-25"28

This relationship is consistent with the hypothesisthat aberrant Class II antigen expression may be capa-ble of triggering an autoimmune response. However,as will be discussed, the cause-and-effect relationshiphas not been proved.

The observation (Table 1) that the total numbers oflymphocytes were smaller in the two samples withHLA-DR-negative epithelial cells than in nine of theten samples with positive epithelial cells suggests ageneral, but by no means absolute, correlation be-tween epithelial Class II antigen expression and num-bers of lymphocytes in the lacrimal gland. A similarrelationship was suggested by preliminary experi-ments with lacrimal glands from NZB/W mice; theseanimals develop lacrimal lymphocytic infiltrates simi-lar to those observed in Sjogren's syndrome.30 Al-though these results were difficult to quantify becauseof an excessive level of background staining, la wasnot apparent on epithelial cells at 7 wk and 12 wk, butit could be detected on small numbers of the epithelialcells at 20 wk,31 a time interval over which the num-ber of la-positive leukocytes roughly doubles (18 permm2 at 7 wk, 15 per mm2 at 12 wk, and 30 per mm2 at



Fig. 7. Isolated rat lacrimal acinar cells from the same prepara-tion show occasional Class II antigen-positive cells (arrows) afterovernight culture in the presence of 1000 U/ml interferon y. Mag-

Fig. 8. Normal rabbit lacrimal gland tissue shows no Class 11antigen-positive acinar cells, whereas results for leucocytes are posi-tive. Magnification X100.

Fig. 9. Freshly isolated rabbit lacrimal acinar cells show no ClassII antieen expression. Magnification X400.

Fig. 11. Rabbit lacrimal acinar cells after 18 hr in control culturemedium showed positive results for Class II antigen expression andmarked clustering. Magnification X400.

20 wk; FM Hofman and AK Mircheff, unpublished).Thus, it appears that in lacrimal glands, as in otherepithelial tissues, the degree of epithelial Class II anti-

Fig. 10. Rabbit lacrimal acinar cells after 3 hr in control culturemedium showed positive results for Class II antigen expression.Magnification X400.

gen expression tends to correlate with the degree oflymphocytic infiltration.

Despite the apparent relationship between epithe-lial Class II antigen expression and lymphocytic infil-tration, the observations summarized in Table 1 sug-gest that epithelial expression of Class II antigens doesnot lead inexorably to the vigorous autoimmune re-sponse characteristic of Sjogren's syndrome. With theexception of the 38-year-old man, all of the cases de-scribed in Table 1 showed total numbers of lympho-cytes in the range of non-Sjogren's lacrimal glands asdescribed in a younger population of donors by Wiec-zorek et al.5 Furthermore, none of the cases in ourstudy exhibited the tissue destruction and fibrosis typi-cal of Sjogren's syndrome.2-3 The IgG-bearing B cells,a hallmark of Sjogren's infiltrates,32 were not detect-able in the three samples we analyzed.

Attempts to understand the cause-and-effect rela-tionship between epithelial Class II antigen expres-


2308 INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / July 1991 Vol. 32

sion and lymphocytic activation are complicated bythe fact that there may be several different mecha-nisms for induction of epithelial cell Class II antigenexpression. One mechanism, now believed to operatein several different epithelial tissues, including the thy-roid gland,33 the salivary gland,20 and the retinal pig-ment epithelium,29 involves interferon-7 released byactivated T cells and macrophages in inflammatoryinfiltrates.

The observation of a lacrimal gland sample withunusually low numbers of lymphocytes and an unusu-ally large number of HLA-DR-positive epithelial cells(from an 87-year-old man, Table 1) raises the possibil-ity that factors other than locally released cytokinesmight also be able to induce epithelial HLA-DR ex-pression. Because thyroid-stimulating hormone, thehormone which controls thyroid hormone release, in-duces Class II antigen expression by thyroid epithelialcells in culture15 and prolactin induces Class II anti-gen expression by mammary epithelial cells,26 we at-tempted to determine whether lacrimal epithelial cellClass II antigen expression might be influenced byinterferon-7, carbachol, or isoproterenol, the lattertwo neurotransmitter receptor agonists which triggerlacrimal secretion by different intracellular signaltransduction pathways.34 Our results were variablebecause some rat acinar cell preparations and all rab-bit acinar cell preparations contained cells that ex-pressed Class II antigen in culture even in the absenceof added potential mediators. Although these experi-ments yielded little information about the control ofClass II antigen expression by lacrimal acinar cells,they confirmed that these cells are capable of express-ing the Class II antigen. In most experiments, the cellsspontaneously began expression Class II antigenwhen placed into culture media, and therefore we canconclude that either: (1) Class II antigen expression isinhibited by the normal milieu of the lacrimal glandor (2) intracellular or autocrine mediators released asthe cells adapt to the conditions of cell culture havethe effect of inducing Class II antigen expression. Ineither case, it appears that the normal lacrimal acinarcell may be ready to begin expressing Class II antigensin response to various potential stimuli, including, forexample, interferon-7, other cytokines, or excessiveor sustained (compared with pulsatile) secretomotorstimulation.

If lacrimal epithelial cells begin expressing Class IIantigens, then it is likely that there are several otherevents that would have to occur if they are to activatelymphocytes and instigate an autoimmune response.Recent work with macrophages and other antigen-presenting cells indicates that Class II antigens nor-mally do not present native plasma membrane constit-uents of the antigen-presenting cell. When macro-

phages and dendritic cells activate T lymphocytes,they do so by presenting antigenic fragments thathave been generated during the proteolytic processingof larger molecules. The processing events occur in anendocytic compartment, and the antigenic fragmentsreturn to the cell surface bound to a specific domainof the Class II molecule.35"37

Recent work on the membrane dynamics of secre-tory epithelial cells suggests the possibility that theremight be circumstances in which Class II antigen-ex-pressing lacrimal epithelial cells are able to presentintracellularly generated antigenic fragments in amanner analogous to the presentation of foreign anti-gens by macrophages. There are now several indica-tions that the lacrimal acinar cell basal-lateral plasmamembrane, ie, the membrane domain in immediatecontact with the glandular interstitium, is highly dy-namic. Cholinergic stimulation triggers a rapid redis-tribution process in which Na+/K+ adenosine triphos-phatase38 and muscarinic receptors39 are mobilizedfrom cytoplasmic pools, presumably associated withthe Golgi complex, and inserted into the basal-lateralmembrane. In addition /?-adrenergic stimulation hasbeen found to accelerate the internalization of basal-lateral membrane constituents.40 It now appears thatthe stimulation-associated insertion and internaliza-tion phenomena both are superimposed on a highsteady-state rate of basal-lateral membrane recycling;isolated lacrimal acinar cells take up and release thefluid phase marker, Lucifer yellow, with half-times ofless than 5 min.41

This background information suggests that theremay be several different mechanisms by which poten-tially antigenic fragments are generated inside theacinar cell and enter membrane compartments thatcommunicate with the membrane assembly and recy-cling pathway traversed by Class II antigens and otherbasal-lateral membrane constituents. These include aback flux of partially degraded proteins from lyso-somes to recycling compartments, a mistargeting oflysosomal proteases to the recycling compartments,and a premature activation of newly synthesized lyso-somal proteases during their transit through the Golgicomplex.

This working hypothesis is still speculative. It incor-porates the observations that lacrimal epithelial cellsare capable of expressing Class II antigens and thatthey maintain an extensive traffic of membrane con-stituents between the Golgi complex, rapidly recy-cling intracellular compartments, and the basal-lat-eral plasma membrane. Although there is no directevidence for the subcellular targeting errors we postu-late to account for the generation of potentially anti-genic peptides by lysosomal proteases, we are aware ofone precedent for the inappropriate accumulation of



lysosomal proteases in a compartment containingpeptides destined for other cellular loci. This occurs inrats treated with supramaximal doses of cerulein, anagonist for the cholecystokinin receptor. In this ani-mal model, lysosomal proteases and secretory zymo-gens, including trypsinogen, accumulate in aqueousvacuoles in the apical cytoplasm of pancreatic acinarcells. It is believed that limited proteolysis by the lyso-somal proteases results in activation of secretory zy-mogens, releasing activated proteases into the acinarcytoplasm and triggering an acute pancreatitis.42 Weanticipate that the mistargeting events that would gen-erate potentially antigenic peptides in lacrimal epithe-lial cells normally are rare and that the low rate atwhich they occur could be one of several factors ac-counting for the observation that epithelial Class IIantigen expression alone is insufficient to trigger au-toimmune reactions.

Key words: Sjogren's syndrome, dacryoadenitis, membranerecycling, antigen presentation

Acknowledgment

The authors thank Drs. Katherine L. Knight (StritchSchool of Medicine, Loyola University) and Robert A.Prendergast (The Johns Hopkins University School of Medi-cine) for generously providing samples of the 2C4 monoclo-nal supernatant, Drs. Samuel C. Yiu and Louanne E. Hannfor advice on the isolation of lacrimal acinar cells, and Ms.Kris Johnson and Ms. Tina Sugimoto for help with the im-munocytochemical staining procedures.

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