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Detection of ocular mucus in normal human conjunctiva and conjunctiva from patients with cicatricial pemphigoid using lectin probes and histochemical techniques
Exp. Eye Re,s. (1988) 46, 485 r
485
D e t e c t i o n of Ocular M u c u s in N o r m a l H u m a n Conjunctiva a n d Conjunctiva from Patients with Cicatricial P e m p h i g o i d U s i n g Lectin Probes a n d H i s t o c h e m i c a l T e c h n i q u e s P E T E R A. WELLS*, CAROLYN D E S I E N A - S H A w , BEVERLY R I C E AND C. STEPHEN F O S T E R
Hilles Immunology Laboratory, Howe Laboratory of Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, MA 02114, U.S.A. (Received 18 May 1987 and accepted in revised form 15 September 1987) Conjunctival biopsies from patients with cicatricial pemphigoid affecting the eonjunctiva and patients undergoing cataract surgery (normal conjunctiva) were snap-frozen, cryostat sectioned and incubated with fluorescein-conjugated lectins; peanut agglutinin (PNA), Helix poraatia agglutinin (HPA), soybean agglutinin (SBA), wheat germ agglutinin (WGA) and succinylated wheat germ agglutinin (S-WGA). Controls consisted of preincubating the lectins with the appropriate blocking sugars before applying the lectins to the sections. PNA and HPA stained the mucus granules contained in the conjunctival goblet cells but did not stain mucus or glycocalyx at the ocular surface distal to the goblet cells. Native WGA and S-WGA had high affinities for conjunctival goblet cells and the apical epithelial cell layers. Native WGA stained mucus and glycocalyx at the ocular surface. This staining of the ocular surface by WGA was confirmed at the transmission electron microscopic level using WGA conjugated to ferritin. Cicatricial pemphigoid patients in this study had reduced numbers of goblet cells ; however, those goblet cells which were observed in cicatricial pemphigoid conjunctiva stained positively with HPA, PNA, WGA, and SWGA as did goblet cells in normal conjunctiva. Key words: mucus; mucin; conjunctiva; human; pemphigoid; lectins; goblet cell; histochemistry.
Introduction Cicatricial p e m p h i g o i d (CP) is a s y s t e m i c disease of u n k n o w n b u t p r e s u m e d a u t o i m m u n e etiology. CP affects the eyes causing a chronic cicatrizing conjunctivitis, progressive c o n j u n c t i v a l subepithelial fibrosis, fornix foreshortening, s y m b l e m p h e r o n f o r m a t i o n , m e i b o m i a n d u c t o b s t r u c t i o n a n d e v e n t u a l l a c r i m a l d u c t c o m p r o m i s e with reduced t e a r flow (Foster, 1987). D i a g n o s t i c criteria for confirmation of cicatricial p e m p h i g o i d affecting the c o n j u n c t i v a includes a c o n j u n c t i v a l b i o p s y a n d demo n s t r a t i o n of i m m u n o r e a c t a n t deposition in t h e c o n j u n c t i v a l epithelial b a s e m e n t m e m b r a n e zone (Foster, 1987). Several studies (Ralph, 1975; Nelson a n d W r i g h t , 1984) have shown t h a t CP p a t i e n t s , in the l a t e r stages of the disease, have r e d u c e d n u m b e r s of c o n j u n c t i v a l g o b l e t cells r e l a t i v e to i n d i v i d u a l s with n o r m a l c o n j u n c t i v a . I t is t h o u g h t t h a t the subepithelial fibrosis which occurs in CP is responsible for a l t e r a t i o n s in t h e c o n j u n c t i v a l e p i t h e l i u m including decreased n u m b e r s of g o b l e t cells a n d s q u a m o u s m e t a p l a s i a of the c o n j u n c t i v a l e p i t h e l i u m (the t r a n s i t i o n of t h e n o r m a l n o n - k e r a t i n i z i n g c o n j u n c t i v a l e p i t h e l i u m with goblet cells to a k e r a t i n i z i n g s q u a m o u s e p i t h e l i u m w i t h o u t goblet cells). P r e v i o u s scanning electron microscopic studies of c o n j u n c t i v a l biopsies from CP p a t i e n t s (Foster, D e S i e n a - S h a w a n d Wells, 1986) have d e m o n s t r a t e d a t h i c k mucus layer o v e r l y i n g the c o n j u n c t i v a l surface. I n order to d e t e r m i n e w h e t h e r a n y q u a l i t a t i v e differences existed in t h e mucin g l y c o p r o t e i n a n d * To whom correspondence should be addressed. 0014-4835/88/040485+ 13 $03.00/0
9 1988 Academic Press Limited
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c a r b o h y d r a t e staining p a t t e r n s of c o n j u n c t i v a from CP p a t i e n t s c o m p a r e d with n o r m a l h u m a n c o n j u n e t i v a , we have i n c u b a t e d b i o p s y specimens with a panel of fluorescein i s o t h i o c y a n a t e (FITC) c o n j u g a t e d lectins.
Materials and Methods Conjunctival biopsies were obtained from patients with cicatricial pemphigoid, patients undergoing cataract surgery (normal conjunctiva) and patients with corneal ulcers, atopy and rosacea blepharoconjunctivitis. Informed consent was obtained from patients in this study. After subconjunctival injection of 2 % xylocaine, a 2 x 2 mm square of limbal conjunctiva was excised and placed onto cotton gauze saturated with sterile saline. Conjunctival specimens for glycol methacrylate embedding were immersed in Karnovsky's fluid (1% paraformaldehyde, 1"25 % glutaraldehyde, 25 mM sodium phosphate and 150 mM sodium cacodylate buffer, pH 7'2) for 24 hr at room temperature. Specimens for transmission electron microscopy were placed in 4 % glutaraldehyde, 0"1 u sodium cacodylate buffer, pH 7"2, at 4~
Staining with FITC-conjugated lectins Biopsy specimens which had been placed onto guaze with sterile saline were embedded in OCT compound (Tissue-Tek, Miles Laboratories) and rapidly frozen at - 2 5 ~ in an IEC Minotome cryostat. Cryostat sections (4 tern thick) were cut from the OCT embedded tissue using a steel knife at - 2 0 ~ and mounted on gelatin-coated slides. The slides were air-dried at room temperature, washed with 0'05 M sodium phosphate buffered saline, pH 7-2 (PBS) and incubated with FITC-conjugated lectins, native WGA, succinylated WGA, HPA, PNA and SBA. The FITC-conjugated lectins (1 mg m1-1 protein concentration) were purchased from EY Laboratories, Inc. (San Mateo, CA), and diluted 1:80, 1:160 or 1:320 with PBS. Control slides were incubated with FITC-conjugated lectins which had been previously incubated with the appropriate blocking sugars. HPA, PNA and SBA were all blocked with 0'2 M lactose (final concentration) dissolved in PBS, pH 7'2. Native and succinylated WGA were blocked with 2 mM diacetylchitobiose (final concentration) dissolved in PBS, pH 7.2. After incubation with the lectin for 15-30 rain, the slides were washed with PBS three times and coverslipped with 90 % glycerol, 10 % PBS containing 0"1% p-phenylene diamine, pH 9"0 (Johnson and Araujo, 1981). The slides were observed and photographed using a Zeiss Photomic I I I photomicroscope fitted with epi-illumination for FITC fluorescence.
Neuraminidase-FITC-PNA staining sequence Unfixed cryostat sections mounted on glass slides were incubated for 30 rain at 37~ with neuraminidase (Sigma Type X from Clostridium perfringens, Sigma Chemical Co.) 10 U m1-1 in PBS, pH 5"0. Controls were incubated for the same amount of time at 37~ with PBS, pH 5"0, without enzyme. The slides were then washed with PBS, pH 7"2 and incubated with F I T C - P N A diluted 1 : 160 for 15-30 rain. Slides were washed with PBS and coverslipped as described above.
Incubation with ferritin-conjugated lectins For electron microscopy, conjunctival biopsies were fixed with 4 % glutaraldehyde at 4~ for 4-7 days. The biopsy specimens were washed with 0'1 M sodium cacodylate buffer, pH 7'2, and unreacted aldehyde groups were removed by incubating the tissue with 200 mM glycine in 50 mM sodium phosphate buffer, pH 7"2, for 60 min at room temperature. Biopsy specimens were then washed with PBS and incubated for 60 rain with ferritin-conjugated lectins ; WGA and PNA (1 mg m1-1 protein concentration, EY Laboratories, San Mateo, CA). Both ferritin-conjugated lectins were diluted to 25/~g m1-1 with PBS, pH 7"2. Control tissue was incubated with the same concentration of ferritin-conjugated lectin which had been blocked with the appropriate sugars as outlined above for the FITC-conjugated lectins. The biopsy specimens were washed with PBS after incubation with ferritin-labelled lectins, fixed for 30 min with 2'5 % glutaraldehyde in 0'l M sodium cacodylate buffer, and postfixed with
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1% osmium tetroxide in 0"l M sodium cacodylate buffer. The tissue was then dehydrated and embedded in Epon-Araldite mixture (Mollenhauer, 1964). Ultrathin sections were cut from the blocks using a diamond knife, mounted on copper mesh grids, stained with 2 % aqueous uranyl acetate and examined using a J E O L JEM-100C transmission electron microscope.
Light microscope histochemical procedures Conjunctival biopsies fixed with Karnovsky's fluid were dehydrated using a graded series of ethanols and embedded in glycol methacrylate (LKB Historesin, L K B Produkter, AB): Sections 4/tm thick were cut with glass knives using a Sorvall JB-4 microtome and mounted on glass slides. The slides were stained with either 1% Alcian Blue 8GX in 3 % acetic acid, pH 2'5, for 2 hr or a high iron diamine solution as described by Spicer (1965) for 24 hr. The high iron diamine solution specifically stains sulfate esters (Spicer, 1965) while the Alcian Blue solution stains both sulfate esters and carboxyl groups. Controls for the Alcian Blue staining consisted of sections which were incubated for 24 hr at 37~ with neuraminidase (Sigma Type X, l0 U m1-1 in PBS, pH 5'0, as described above) prior to the Alcian Blue staining. Controls for the neuraminidase digestion included (l) incubating sections for 24 hr at 37~ with PBS, pH 5"0, or (2) incubating sections for 24 hr with protease (Sigma Type V I I I from Bacillus subtilis) 9"6 U m1-1 in PBS, p H 7'5, and then staining with Alcian Blue 8GX in 3 % acetic acid. Controls for the high iron diamine technique consisted of active methylation to block the sulfate esters as described by Spicer (1965). The slides were incubated for 4 hr at 60~ with absolute methanol containing 0' 1 N HC1 prior to staining for 24 hr with the high iron diamine solution. Positive controls for active methylation consisted of slides incubated for 4 hr at 60~ with absolute methanol (no HC1) and then stained for 24 hr with high iron diamine. After staining with Alcian Blue and high iron diamine, the slides were washed with distilled water and coverslipped with gelvatol. The slides were photographed using standard brightfield illumination. Results
T h e specificities of the lectins utilized for this s t u d y are c o n t a i n e d in Table I. A t t h e light microscopic level, F I T C - c o n j u g a t e d n a t i v e W G A ( F I T C - W G A ) s t a i n e d t h e apical epithelial cells, c o n j u n c t i v a l g o b l e t cells a n d t h e ocular surface of n o r m a l c o n j u n c t i v a l biopsies (Fig. 1). Control sections i n c u b a t e d w i t h F I T C - W G A a n d 2 mM d i a c e t y l c h i t o b i o s e d i s p l a y e d only m i n i m a l b a c k g r o u n d fluorescence (Fig. 2). A t
TABLE I.
Lectin specificities Lectin Wheat-germ agglutinin (WGA) Succinylated WGA (S-WGA) Helix pomatia agglutinin (HPA) Peanut agglutinin (PNA) Soybean agglutinin (SBA)
Dolichos biflorus agglutinin (DBA) Ulex europeus agglutinin (UEA) Rieinus communis agglutinin I (RCA-I) Concanavalin A (Con A)
Carbohydrate specificity /?-D-N-Acetylglucosamine, and Sialic Acid fl-D-N-Acetylglucosamine a-D-N-Acetylgalactosamine> a-D-N-Acetylglucosamine fl-D-N-Galactose-/? (l-3)-D-N-Acetyl galactosamine :> Galactosamine and Galactose a-D-N-Acetylgalactosamine> ~6-D-N-Acetylgalactosamine > a-D-N-Galactose a-D-N-Acetylgalactosamine a-L-Fucose /?-D-N-Galactose Or-D-N-Glucose,~-D-Mannose
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E T AL.
Fro. 1. Normal human c(mjurwti\ a i~('ubaied wilh FIT(' ~V(;A. k'~tlxed erSostat section. Goblet cells ((;(') and the eonjul~t.tival surtace a~'(' positi\ely stained. Stroma (N). • 100. FI(< 2. ('ontrol setti(nl ~f' ~o,'ma] human (.(mjunctiva incubated u ith F I T ( ' WGA and tbe competing sugar. 2m5i diacetxh.hit(~lfio.e N()te the absen(.e (if" fluores(.ent.e exeept for a weak reaction at the conjundivaI surface. • 10(} Fa;. 3. ('onjmu'tival sl~e('imet) fr<>m a patiel~t with cicatrieial pemphigoid. (Iryostat section incubated ~itb F1T(' W(;A. Note the intense staining of the apical epithelial cells and the epithelial cell plasma membrane, x25(t. Fro. 4. Normal human eon,junetiva incubated ~ith FIT(' conjugated S-\VGA. Goblet cells ((IC) and punetate areas in apieal epithelial c'ells are positively labelled. Stroma (S). x I00.
higher magnification, a fibrillar network of positively stained material, interpreted as ocular mucus, was often observed at the ocular surface of normal eonjunetiva after incubation with FIT(; WOA. Biopsy specimens fl'om patients with CP showed a similar staining pattern after' incubation with FIT() WGA. in m a n y of these patients, especially those with advanced disease, decreased numbers of eonjunctival goblet cells were observed; however, those goblet cells which were observed in CP patients stained positively with FITC WGA. Intense staining of the apical epithelial cells and ocular surface also occurred in biopsy specimens from CP patients after incubation with FITC WGA (Fig. 3). Incubation of conjunetival biopsy sections with FITCconjugated succinylated WGA (FITC-S-WGA) yielded a slightly different staining pattern; the apical epithelial cell layers and the goblet cells were positively stained; however, the ocular surface of the eonjunctival epithelium was not stained as intensely by FITC S-WGA (Fig. 4). Except for the decreased numbers of goblet cells
OCULAR MUCUS IN HUMAN CONJUNCTIVA
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mr
489
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(arrow) are positive. Unfixed ~'r\x>~tat <(e~i~H •
I:Ic;. 6. ('onjuHt.tival specimen f'r'om a ci(.atricial t)e~lplliMoidpatient stained with FIT(I HPA. (;oblet <,ells (G(') and ve~icles in the apical epithelial cells are pr • FIe;. 7. ('onjunelival ~pecimen from a eiealri(,ial i)~,mphiKoidpa.tienl stained with FIT(? PNA. Goblet cells ((4(') present in this ('P ]patient'> ('oIIjUl]('IiVaat'e positive. Stroma (S). x 100. Fro. 8 (onjunctiva fi'om a ek.atricia] pemphi~oid patient with squamous metaplasia stained with FITC PXA. Weak staining occurred over the conjunetivai epithelial <.ell plasma membranes and/or extracelhdar substance. ('onjtmetival epithelium (('E): stioma (S). x 100.
in s p e c i m e n s from CP p a t i e n t s , s i m i l a r results were o b t a i n e d in b o t h n o r m a l e o n j u n e t i v a a n d s p e e h n e n s fl'om CP p a t i e n t s u s i n g F I T C S W G A . i n c u b a t i o n of n o r m a l e o n j u n e t i v a l biopsies with F I T C H P A r e s u l t e d in specific l a b e l l i n g of t h e g o b l e t eells (Fig. 5). The e o n j u n e t i v a l e p i t h e l i u m a n d o c u l a r surface were n o t labelled b y FITC-- H P A in e i t h e r n o r m a l e o n j u n e t i v a or e o n j u n e t i v a f r o m CP p a t i e n t s (Fig. 6). I n c u b a t i o n of n o r m a l e o n j u n e t i v a l biopsies w i t h F I T C H P A r e s u l t e d in specific l a b e l l i n g of t h e g o b l e t cells a n d m u c u s s t r a n d s e m a n a t i n g from t h e g o b l e t cells, w i t h o n l y w e a k l a b e l l i n g of t h e e o n j u n e t i v a l epithelial cells (Fig. 7). S t a i n i n g of the e o n j u n e t i v a l g o b l e t cells a n d n m e u s s t r a n d s was b l o c k e d w h e n F I T C P N A was
preineubated with 0'2 M lactose (data not shown). F I T C - P N A stained the eonjunetival epithelial cell membranes and/or extraeellular substance in biopsies from CP patients with squamous metaplasia (Fig. 8). Digestion of see
P. A. W E L L S E T AL.
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TABLE II
Summary of staining patterns observed in normal and pathologic conjunctival specimens* Lectins Area Conjunctival surface Goblet cells Epithelial cells Conjunctival stroma
WGA
S-WGA
+ + + + + + 82 + 82 . . .
HPA
PNA
N-PNA t
SBA
+ :~ + + -
+ :~ + + -II
+ :~ + + +** +
+ + + +
.
* Key to staining intensity; for each lectin, a single plus denotes positive staining of the histological structure, two pluses denotes more intense positive staining, 2-3 times the intensity of staining graded with a single plus, a minus sign denotes no staining of the histological structure other than background fluorescence. N-PNA; Neuraminidase FITC-PNA staining sequence. :~ HPA, PNA and N-PNA stained the ocular surface only in areas adjacent to conjunctival goblet cells. 82 WGA and S-WGA stained the apical epithelial cells more intensely compared to the basal epithelial cell layers. II The plasma membranes and/or extracellular substance in the conjunctival epithelium stained weakly in CP patients with squamous metaplasia. ** The epithelial cell plasma membranes in specimens from cataract and CP patients stained positive when cryostat sections were treated with neuraminidase prior to FITC-PNA.
FIe;. 9. Conjunctiw~ from a cicatricial pemphigoid patient stained with FITC SBA. Positive staining occurred over the apical epithelial cells and elements within the subepithelial stroma. Conjunctival epithelium (CE); stroma (S). x ltt0. Fro. 10, Control section from the same pemphigoid patient incubated with FITC-SBA and the competing sugar, 0'2 5i lactose. (~onjunetival epithelium (CE); stroma (S). x 100. n e u r a m i n i d a s e p r i o r t o F I T C P N A i n c u b a t i o n r e s u l t e d in i n c r e a s e d l a b e l l i n g o f t h e c o n j u n e t i v a l e p i t h e l i a l cell m e m b r a n e s in b o t h n o r m a l b i o p s y s p e c i m e n s a n d s p e c i m e n s f r o m C P p a t i e n t s ( d a t a s u m m a r i z e d in T a b l e I I ) . I n c r e a s e d F I T C - P N A s t a i n i n g also o c c u r r e d o v e r t h e s u b e p i t h e l i a l s t r o m a w h e n s e c t i o n s w e r e p r e t r e a t e d with neuraminidase. The neuraminidase FITC-PNA staining could be inhibited by t h e a d d i t i o n o f 0'2 M l a c t o s e t o t h e l e c t i n . Incubation of human conjunetival biopsies with FITC-SBA resulted in intense s t a i n i n g o f t h e e o n j u n e t i v a l e p i t h e l i u m , e s p e c i a l l y t h e a p i c a l cell l a y e r s a n d b l o o d
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':~~, ~ii~~!iiiili~~i~!i!ilii~!ii!!!i84ii~ill~
FIG. 11. Normal human conjunctiva stained with ferritin-conjugated WGA. Arrows point to ferritinWGA particles labelling mucins and the glycocalyx of the epithelial surface cell. Conjunctival epithelial surface cell (CE). • 47000. Fro. 12. Control normal human conjunctiva incubated with ferritin-WGA and the competing sugar, 2 mM diacetylchitobiose. Ferritin-WGA binding to the glyeocalyx (arrow) and to mucins is diminished. Conjunctival epithelium (CE). • 47000.
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FIG. 13. Conjunctiva from a cicatricial pemphigoid patient incubated with ferritin-WGA. Electron opaque ferritin-WGA particles (arrows) are observed labelling the conjunctival surface. Conjunctival epithelium (CE). • vessels in the conjunctival stroma (Fig. 9). This labelling was completely blocked by preincubation of the FITC SBA with 0"2 M lactose (Fig. 10). The staining pattern observed in CP conjunctiva after incubation with F I T C - S B A was identical to the staining pattern observed in normal conjunctiva with this lectin. The light microscopic results obtained using this panel of FITC conjugated lectins are summarized in Table II. The ocular surface of normal conjunctiva was positively stained by WGA conjugated to ferritin (Fig. 11). At the ultrastructural level, WGA-ferritin staining appeared to be localized over the glycocalyx of the conjunctival surface ceils and also over mucins at the conjunctival surface. This staining of the conjunctival surface was abolished when WGA-ferritin was preincubated with diacetylchitobiose (Fig. 12). A similar labelling of the conjunctival surface was obtained when specimens from CP patients were incubated with WGA-ferritin (Fig. 13). Incubation of conjunctival biopsy specimens with PNA-ferritin did not result in labelling of the conjunctival surface regardless of whether the biopsy specimens were from individuals with normal conjunctiva or CP patients. Sections of human conjunctiva cut from blocks of tissue embedded in glycol methacrylate and stained with Alcian Blue in acetic acid showed goblet cells with positively stained mucus granules. This staining was similar in both normal conjunctiva and conjunctiva from diseased eyes provided t h a t goblet cells were present in the tissue sample (Fig. 14). Alcian Blue staining of the conjunctival goblet cells was greatly decreased but not abolished when the sections were digested with neuraminidase for 24 hr (Fig. 15). Digestion with protease or incubation with PBS for 24 hr did not diminish the positive Alcian Blue staining of the goblet cell granules.
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FIG. 14. Conjunctiva from a patient with corneal ulcer and increased numbers of conjunctival goblet cells. Sections were incubated for 24 hr with PBS, pH 5"0, and then with Alcian Blue 8GX in 3 % acetic acid. Goblet cells (GC) are positively stained. Stroma (S). Glycol methacrylate section. • 125. FIG. 15. Conjunctiva from the same patient as Fig. 14 incubated for 24 hr with neuraminidase in PBS, pH 5"0, and then stained with Alcian Blue 8GX in acetic acid. Staining of the goblet cells is decreased. x 125. Fro. 16. Normal human conjunctiva incubated at 60~ with absolute methanol for 4 hr and stained with high iron diamine. Positive staining occurred over the goblet cells (GC). Stroma (S). Glycol methacrylate section, x 125. FIG. 17. Conjunctiva from the same patient as Fig. 16 incubated with methanol+HC1 (active methylation) and stained with high iron diamine. Staining of the goblet cell granules is abolished, indicating that the staining observed in Fig. 16 is due to sulfated mucins, x 125. Sections of h u m a n c o n j u n c t i v a which were stained with the high iron d i a m i n e (HID) reagent also showed positively stained goblet cell granules (Fig. 16). Positive s t a i n i n g with H I D was completely abolished when the sections were actively m e t h y l a t e d (methanol + HC1) prior to t r e a t m e n t with the H I D r e a g e n t (Fig. 17). Sections which were p r e t r e a t e d with m e t h a n o l alone ( m e t h y l a t i o n control) did n o t show a n y decrease in H I D s t a i n i n g of the goblet cell granules. The results of these histochemical studies are s u m m a r i z e d in Table I I I .
Discussion Mucins (mucin glycoproteins) secreted b y c o n j u n c t i v a l goblet cells (Kessing, 1968), c o n j u n c t i v a l epithelial cells (Greiner et al., 1980, Greiner, W e i d m a n , K o r b a n d
494
P.A. WELLS ET AL. TABLE I I I
Summary of the staining patterns obtained with histochemical techniques* Treatment Alcian Blue (AB) Protease-ABt Neuraminidase-AB High iron diamine (HID) Methanol-HID:~ Methanol+HC1-HID (active methylation):~
Goblet cell staining + + + + +
++ ++ + ++ ++
+ + + +
* Grading system ; four pluses denotes intense staining of the goblet cells as observed when sections were stained with AB or HID without any pretreatment. Three pluses, two pluses, and one plus indicate 75, 50, and 25 % of the four plus staining intensity. A minus sign denotes an absence of observable staining. t Sections were digested with protease or neuraminidase for 24 hr. Methanol or active methylation 4 hr at 60~ Allansmith, 1985), and possibly by the lacrimal gland (Allen, Wright and Reidl 1972 ; Jensen, Falbe-Hansen, Jacobsen and Michelsen, 1969) contribute to the innermost mucus layer of the trilaminar preocular tear film (Holly and Lemp, 1977). These mucins overlie the surface cells of the cornea and conjunctiva. Tear film mucins play important roles in maintaining the wettability of the corneal surface (Lemp, Holly, I w a t a and Dohlman, 1970; Holly and Lemp, 1971), and in the defense of the ocular surface against bacteria and other infectious agents (Gibbons, 1982). Lectins consisting of proteins and glycoproteins from various plants and animals can be used to label mucins in human conjunctival biopsies. These lectins are capable of binding to specific carbohydrate residues found on glycoproteins and other glycoconjugates (Goldstein and Hayes, 1978). Previous light microscopic studies (Kawano, Uehara, Sameshina and Ohba, 1984) utilized fluorescein-conjugated WGA, SBA, PNA, Ricinus communis agglutinin-I (RCA-I), and Limulus polyphemus agglutinin (LPA) to label normal human conjunctival goblet cell granules. The lectins, Ulex europaeus agglutinin (UEA), Dolichos biflorus agglutinin (DBA), and concanavalin A (Con-A) did not label human conjunctival goblet cell granules (Kawano et al., 1984). In our study, conjunctival goblet cells in specimens from individuals with normal conjunctiva, and CP patients, were labelled with native WGA and succinylated WGA. Native WGA binds to terminal or internal trimeric, dimeric and monomeric Nacetylglucosamine residues and to terminal sialic acid residues (Goldstein an d Hayes, 1978). In contrast, S-WGA binds only to terminal N-acetylglucosamine residues and does not bind to sialic acid (data supplied by the manufacturer, E Y Laboratories). At the light microscopic level, native WGA also labelled the apical epithelial cells and the conjunctival surface in both cataract patients and CP patients. The binding of WGA to the conjunctival surface was confirmed at the TEM level by incubating tissue with WGA-ferritin. The binding of native WGA to the conjunctival surface, goblet cells, and the apical epithelial cells indicates the presence of N-acetylglucosamine and/or sialic acid residues at these tissue sites. Conjunctival goblet cell granules in specimens from cataract patients and in specimens from CP patients without squamous metaplasia were positively labelled with F I T C - H P A and FITC-PNA. Labelling of human conjunctival goblet cell
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granules with HPA indicates the presence of terminal N-acetylgalactosamine residues on the mucin glycoproteins. Labelling of the goblet cell granules with PNA indicates the presence of terminal D-galactose-/?(1-3)-N-acetylgalactosamine disaccharides or terminal D-galactose residues on the mucin macromolecules (Goldstein and Hayes, !978). Pretreatment of cryostat sections of human conjunctiva with neuraminidase caused increased staining of the non-goblet epithelial cell plasma membranes with FITC-PNA. The unmasking of PNA binding sites by neuraminidase digestion can be attributed to the removal of terminal sialic acid residues which inhibit PNA binding to penultimate D-galactose-fl(1-3)-N-acetylgalactosamine disaccharides in sialylated glycoproteins and glycolipids (Corfield and Schauer, 1982; Goldstein and Hayes, 1978). Previous histochemical studies (Srinavasan, Worgul, Iwamoto and Merriam, 1977 ; Matsumoto and Mimura, 1974; Kessing, 1968) have indicated that the mucus granules found in eonjunetival goblet cells contain neutral periodic acid-Schiff (PAS) positive mucins and acidic sialylated mueins (sialomucins). Wright and Mackie (1977) detected sulfomucins in human conjunctival goblet cells using Alcian Blue at pH 1"0. Our results from incubating glycol methacrylate sections of human conjunctiva with high iron diamine showed positive staining of the goblet cells. The abolition of high iron diamine staining when the sections were pretreated by active methylation established that the goblet cell granules contain sulfated mucins (sulfomucins) (Spicer, 1965). In addition, our results using glycol methacrylate embedded specimens exposed to neuroaminidase and Alcian Blue agreed with the previous studies and confirmed that neuraminidase-sensitive sialic acid residues were present in human conjunctival goblet cell granules The results of the lectin binding and histochemical studies indicate that human goblet cell mucins consist of glycoproteins with neutral, sulfated and sialylated oligosaccharide chains. It is possible that these three types of oligosaccharide chains exist on a single mucin glycoprotein, or that the human conjunctival goblet cell synthesizes several distinct mucin glyeoproteins. The lectin staining patterns which were observed in the conjunetiva of patients with CP and in normal human conjunctiva were similar. Although many CP patients had reduced numbers of goblet cells, those goblet cells which were observed in CP conjunctiva stained positively with WGA, S-WGA, HPA, and PNA as did goblet cells in normal conjunctiva. Previous studies employing SDS PAGE (Wells, Ashur and Foster, 1986) have shown that ocular mucus glycoproteins collected from the inferior fornix of patients with CP, atopy, rosacea blepharitis, Stevens-Johnson syndrome and other types of conjunctival inflammation have electrophoretic mobilities identical to those of normal ocular mucus glycoproteins. Together, the results of the lectin binding studies and the SDS-PAGE studies indicate that the conjunctival goblet cells from patients with CP do not synthesize or secrete abnormal ocular mucins. These studies effectively rule out a model for dry eye in CP which involves the secretion of abnormal mucins from the conjunctiva. The dry eye symptoms and increased viscosity of the ocular mucus which is observed in many CP patients may be related to either (1) a decrease in the surfactant properties of the mucus due to the addition of lipids, proteins, or nucleic acids to the ocular mucus, (2) a general decrease in mucus production due to decreased numbers of goblet cells, or (3) a decrease in lacrimal gland secretions due to scarring and obstruction of the lacrimal ducts. Further studies are planned at the transmission electron microscopic level to determine the amount of mucus at the ocular surface in CP patients compared to individuals with normal conjunctiva. 18
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This study was supported by United States Public Health Service grant EY 05813 and a grant-in-aid from the National Society to Prevent Blindness.
REFERENCES Allen, M., Wright, P. and Reid, L. (1972). The human lacrimal gland. Arch. Ophthalmol. 88, 493-7. Corfield, A. P. and Schauer, R. (1982). Occurrence of sialic acids. In Sialic Acids: Chemistry, Metabolism and Function. (Ed. Schauer, R.). Pp. 5-37. Springer-Verlag. New York. Foster, C. S. (1987). Cicatricial pemphigoid. Trans. Am. Ophthalmol. Soc. 84, 527-663. Foster, C. S., DeSiena-Shaw, C. and Wells, P. A. (1986). Scanning electron microscopy of conjunctival surfaces in patients with ocular cicatricial pemphigoid. Am. J. Ophthalmol. 102, 584-91. Gibbons, R. J, (1982). Review and discussion of role of mucus in mucosal defense. In Recent Advances in Mucosal Immunity. (Eds Strober, W., Hanson, L. A. and Sell, K. W.). Pp. 343-51. Raven Press: New York. Goldstein, I. J. and Hayes, C. E. (1978). The lectins : carbohydrate-binding proteins of plants and animals. Adv. Carbohydrate Chem. Biochem. 35, 127-340. Greiner, J.V., Kenyon, K.R., Henriquez, A.S., Korb, D.R.,Weidman, T.A. and Allansmith, M. R. (1980). Mucus secretory vesicles in the conjunctival epithelial cells of wearers of contact lenses. Arch. Opthalmol. 98, 1843-6. Greiner, J. V., Weidman, T. A., Korb, D. R. and Allansmith, M. R. (1985). Histochemical analysis of secretory vesicles in nongoblet conjunctival epithelial cells. Acta Ophthalmol. 63, 89-92. Holly, F. J. and Lemp, M. A. (1971). Wettability and wetting of the corneal epithelium. Exp. Eye Res. 11,239-50. Holly, F. J. and Lemp, M. A. (1977). Tear physiology and dry eyes, Surv. Ophthalmol. 22, 69-87. Jensen, O. A., Falbe-Hansen, I., Jacobsen, T. and Michelsen, A. (1969). Mucosubstances of the acini of the human lacrimal gland (orbital part), I. Histochemical identification. Acta Ophthalmol. 47, 605-19. Johnson, G. D. and de C, Nogueira Araujo, G. M. {1981). A simple method of reducing the fading of immunofluorescence during microscopy. J. Immunol. Meth. 4:}, 349-50. Kawano, K., Uehara, F. Sameshima, M. and Ohba, N. (1984). Application of lectins for detection of goblet cell carbohydrates of the human conjunctiva. Exp. Eye Res. 38, 439-47. Kessing, S.V. (1968). Mucus gland system of the conjunctiva. Acta Ophthal. Suppl. 95, 1-133. Letup, M. A., Holly, F. J., Iwata, S. and Dohlman, C. H. (1970). The precorneal tear film. I. Factors in spreading and maintaining a continuous tear film over the corneal surface. Arch. Ophthalmol. 83, 89-94. Matsumoto, K. and Mimura Y. (1974). Histochemical studies on the conjunctival goblet cells. Connect. Tissue 6, 85-90. Mollenhauer, H. H. (1964}. Plastic embedding mixtures for use in electron microscopy. Stain Technol. 39, 111-4. Nelson, J. D. and Wright, J. C. (1984). Conjunctival goblet cell densities in ocular surface disease. Arch. Ophthalmol. 102, 1049-51. Ralph, R.A. (1975). Conjunctival goblet cell density in normal subjects and in dry eye syndromes. Invest. Ophthalmol. 14, 299-302. Spicer, S. S. (1965). Diamine methods for differentiating mucosubstances histochemically. J. Histochem. Cytochem. 13, 211-34. Srinavasan, B. D., Worgul, B. V., Iwamoto, T. and Merriam, G. R. (1977). The conjunctival epithelium. II. Histochemical and ultrastructural studies on human and rat conjunctiva. Ophthalmic Res. 9, 987-1018.
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Wells, P. A., Ashur, M. L . and Foster, C. S. (1986). SDS-polyacrylamide gel electrophoresis of individual ocular mucus samples from patients with normal and diseased conjunctiva. Curt. Eye Res. 5,823-31. Wright, P. and Mackie, I . A . (1977): Mucus in the healthy and diseased eye. Trans. Ophthalmol. Soc. U.K. 97, 1-7.
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D e t e c t i o n of Ocular M u c u s in N o r m a l H u m a n Conjunctiva a n d Conjunctiva from Patients with Cicatricial P e m p h i g o i d U s i n g Lectin Probes a n d H i s t o c h e m i c a l T e c h n i q u e s P E T E R A. WELLS*, CAROLYN D E S I E N A - S H A w , BEVERLY R I C E AND C. STEPHEN F O S T E R
Hilles Immunology Laboratory, Howe Laboratory of Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, MA 02114, U.S.A. (Received 18 May 1987 and accepted in revised form 15 September 1987) Conjunctival biopsies from patients with cicatricial pemphigoid affecting the eonjunctiva and patients undergoing cataract surgery (normal conjunctiva) were snap-frozen, cryostat sectioned and incubated with fluorescein-conjugated lectins; peanut agglutinin (PNA), Helix poraatia agglutinin (HPA), soybean agglutinin (SBA), wheat germ agglutinin (WGA) and succinylated wheat germ agglutinin (S-WGA). Controls consisted of preincubating the lectins with the appropriate blocking sugars before applying the lectins to the sections. PNA and HPA stained the mucus granules contained in the conjunctival goblet cells but did not stain mucus or glycocalyx at the ocular surface distal to the goblet cells. Native WGA and S-WGA had high affinities for conjunctival goblet cells and the apical epithelial cell layers. Native WGA stained mucus and glycocalyx at the ocular surface. This staining of the ocular surface by WGA was confirmed at the transmission electron microscopic level using WGA conjugated to ferritin. Cicatricial pemphigoid patients in this study had reduced numbers of goblet cells ; however, those goblet cells which were observed in cicatricial pemphigoid conjunctiva stained positively with HPA, PNA, WGA, and SWGA as did goblet cells in normal conjunctiva. Key words: mucus; mucin; conjunctiva; human; pemphigoid; lectins; goblet cell; histochemistry.
Introduction Cicatricial p e m p h i g o i d (CP) is a s y s t e m i c disease of u n k n o w n b u t p r e s u m e d a u t o i m m u n e etiology. CP affects the eyes causing a chronic cicatrizing conjunctivitis, progressive c o n j u n c t i v a l subepithelial fibrosis, fornix foreshortening, s y m b l e m p h e r o n f o r m a t i o n , m e i b o m i a n d u c t o b s t r u c t i o n a n d e v e n t u a l l a c r i m a l d u c t c o m p r o m i s e with reduced t e a r flow (Foster, 1987). D i a g n o s t i c criteria for confirmation of cicatricial p e m p h i g o i d affecting the c o n j u n c t i v a includes a c o n j u n c t i v a l b i o p s y a n d demo n s t r a t i o n of i m m u n o r e a c t a n t deposition in t h e c o n j u n c t i v a l epithelial b a s e m e n t m e m b r a n e zone (Foster, 1987). Several studies (Ralph, 1975; Nelson a n d W r i g h t , 1984) have shown t h a t CP p a t i e n t s , in the l a t e r stages of the disease, have r e d u c e d n u m b e r s of c o n j u n c t i v a l g o b l e t cells r e l a t i v e to i n d i v i d u a l s with n o r m a l c o n j u n c t i v a . I t is t h o u g h t t h a t the subepithelial fibrosis which occurs in CP is responsible for a l t e r a t i o n s in t h e c o n j u n c t i v a l e p i t h e l i u m including decreased n u m b e r s of g o b l e t cells a n d s q u a m o u s m e t a p l a s i a of the c o n j u n c t i v a l e p i t h e l i u m (the t r a n s i t i o n of t h e n o r m a l n o n - k e r a t i n i z i n g c o n j u n c t i v a l e p i t h e l i u m with goblet cells to a k e r a t i n i z i n g s q u a m o u s e p i t h e l i u m w i t h o u t goblet cells). P r e v i o u s scanning electron microscopic studies of c o n j u n c t i v a l biopsies from CP p a t i e n t s (Foster, D e S i e n a - S h a w a n d Wells, 1986) have d e m o n s t r a t e d a t h i c k mucus layer o v e r l y i n g the c o n j u n c t i v a l surface. I n order to d e t e r m i n e w h e t h e r a n y q u a l i t a t i v e differences existed in t h e mucin g l y c o p r o t e i n a n d * To whom correspondence should be addressed. 0014-4835/88/040485+ 13 $03.00/0
9 1988 Academic Press Limited
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c a r b o h y d r a t e staining p a t t e r n s of c o n j u n c t i v a from CP p a t i e n t s c o m p a r e d with n o r m a l h u m a n c o n j u n e t i v a , we have i n c u b a t e d b i o p s y specimens with a panel of fluorescein i s o t h i o c y a n a t e (FITC) c o n j u g a t e d lectins.
Materials and Methods Conjunctival biopsies were obtained from patients with cicatricial pemphigoid, patients undergoing cataract surgery (normal conjunctiva) and patients with corneal ulcers, atopy and rosacea blepharoconjunctivitis. Informed consent was obtained from patients in this study. After subconjunctival injection of 2 % xylocaine, a 2 x 2 mm square of limbal conjunctiva was excised and placed onto cotton gauze saturated with sterile saline. Conjunctival specimens for glycol methacrylate embedding were immersed in Karnovsky's fluid (1% paraformaldehyde, 1"25 % glutaraldehyde, 25 mM sodium phosphate and 150 mM sodium cacodylate buffer, pH 7'2) for 24 hr at room temperature. Specimens for transmission electron microscopy were placed in 4 % glutaraldehyde, 0"1 u sodium cacodylate buffer, pH 7"2, at 4~
Staining with FITC-conjugated lectins Biopsy specimens which had been placed onto guaze with sterile saline were embedded in OCT compound (Tissue-Tek, Miles Laboratories) and rapidly frozen at - 2 5 ~ in an IEC Minotome cryostat. Cryostat sections (4 tern thick) were cut from the OCT embedded tissue using a steel knife at - 2 0 ~ and mounted on gelatin-coated slides. The slides were air-dried at room temperature, washed with 0'05 M sodium phosphate buffered saline, pH 7-2 (PBS) and incubated with FITC-conjugated lectins, native WGA, succinylated WGA, HPA, PNA and SBA. The FITC-conjugated lectins (1 mg m1-1 protein concentration) were purchased from EY Laboratories, Inc. (San Mateo, CA), and diluted 1:80, 1:160 or 1:320 with PBS. Control slides were incubated with FITC-conjugated lectins which had been previously incubated with the appropriate blocking sugars. HPA, PNA and SBA were all blocked with 0'2 M lactose (final concentration) dissolved in PBS, pH 7'2. Native and succinylated WGA were blocked with 2 mM diacetylchitobiose (final concentration) dissolved in PBS, pH 7.2. After incubation with the lectin for 15-30 rain, the slides were washed with PBS three times and coverslipped with 90 % glycerol, 10 % PBS containing 0"1% p-phenylene diamine, pH 9"0 (Johnson and Araujo, 1981). The slides were observed and photographed using a Zeiss Photomic I I I photomicroscope fitted with epi-illumination for FITC fluorescence.
Neuraminidase-FITC-PNA staining sequence Unfixed cryostat sections mounted on glass slides were incubated for 30 rain at 37~ with neuraminidase (Sigma Type X from Clostridium perfringens, Sigma Chemical Co.) 10 U m1-1 in PBS, pH 5"0. Controls were incubated for the same amount of time at 37~ with PBS, pH 5"0, without enzyme. The slides were then washed with PBS, pH 7"2 and incubated with F I T C - P N A diluted 1 : 160 for 15-30 rain. Slides were washed with PBS and coverslipped as described above.
Incubation with ferritin-conjugated lectins For electron microscopy, conjunctival biopsies were fixed with 4 % glutaraldehyde at 4~ for 4-7 days. The biopsy specimens were washed with 0'1 M sodium cacodylate buffer, pH 7'2, and unreacted aldehyde groups were removed by incubating the tissue with 200 mM glycine in 50 mM sodium phosphate buffer, pH 7"2, for 60 min at room temperature. Biopsy specimens were then washed with PBS and incubated for 60 rain with ferritin-conjugated lectins ; WGA and PNA (1 mg m1-1 protein concentration, EY Laboratories, San Mateo, CA). Both ferritin-conjugated lectins were diluted to 25/~g m1-1 with PBS, pH 7"2. Control tissue was incubated with the same concentration of ferritin-conjugated lectin which had been blocked with the appropriate sugars as outlined above for the FITC-conjugated lectins. The biopsy specimens were washed with PBS after incubation with ferritin-labelled lectins, fixed for 30 min with 2'5 % glutaraldehyde in 0'l M sodium cacodylate buffer, and postfixed with
OCULAR MUCUS IN HUMAN CONJUNCTIVA
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1% osmium tetroxide in 0"l M sodium cacodylate buffer. The tissue was then dehydrated and embedded in Epon-Araldite mixture (Mollenhauer, 1964). Ultrathin sections were cut from the blocks using a diamond knife, mounted on copper mesh grids, stained with 2 % aqueous uranyl acetate and examined using a J E O L JEM-100C transmission electron microscope.
Light microscope histochemical procedures Conjunctival biopsies fixed with Karnovsky's fluid were dehydrated using a graded series of ethanols and embedded in glycol methacrylate (LKB Historesin, L K B Produkter, AB): Sections 4/tm thick were cut with glass knives using a Sorvall JB-4 microtome and mounted on glass slides. The slides were stained with either 1% Alcian Blue 8GX in 3 % acetic acid, pH 2'5, for 2 hr or a high iron diamine solution as described by Spicer (1965) for 24 hr. The high iron diamine solution specifically stains sulfate esters (Spicer, 1965) while the Alcian Blue solution stains both sulfate esters and carboxyl groups. Controls for the Alcian Blue staining consisted of sections which were incubated for 24 hr at 37~ with neuraminidase (Sigma Type X, l0 U m1-1 in PBS, pH 5'0, as described above) prior to the Alcian Blue staining. Controls for the neuraminidase digestion included (l) incubating sections for 24 hr at 37~ with PBS, pH 5"0, or (2) incubating sections for 24 hr with protease (Sigma Type V I I I from Bacillus subtilis) 9"6 U m1-1 in PBS, p H 7'5, and then staining with Alcian Blue 8GX in 3 % acetic acid. Controls for the high iron diamine technique consisted of active methylation to block the sulfate esters as described by Spicer (1965). The slides were incubated for 4 hr at 60~ with absolute methanol containing 0' 1 N HC1 prior to staining for 24 hr with the high iron diamine solution. Positive controls for active methylation consisted of slides incubated for 4 hr at 60~ with absolute methanol (no HC1) and then stained for 24 hr with high iron diamine. After staining with Alcian Blue and high iron diamine, the slides were washed with distilled water and coverslipped with gelvatol. The slides were photographed using standard brightfield illumination. Results
T h e specificities of the lectins utilized for this s t u d y are c o n t a i n e d in Table I. A t t h e light microscopic level, F I T C - c o n j u g a t e d n a t i v e W G A ( F I T C - W G A ) s t a i n e d t h e apical epithelial cells, c o n j u n c t i v a l g o b l e t cells a n d t h e ocular surface of n o r m a l c o n j u n c t i v a l biopsies (Fig. 1). Control sections i n c u b a t e d w i t h F I T C - W G A a n d 2 mM d i a c e t y l c h i t o b i o s e d i s p l a y e d only m i n i m a l b a c k g r o u n d fluorescence (Fig. 2). A t
TABLE I.
Lectin specificities Lectin Wheat-germ agglutinin (WGA) Succinylated WGA (S-WGA) Helix pomatia agglutinin (HPA) Peanut agglutinin (PNA) Soybean agglutinin (SBA)
Dolichos biflorus agglutinin (DBA) Ulex europeus agglutinin (UEA) Rieinus communis agglutinin I (RCA-I) Concanavalin A (Con A)
Carbohydrate specificity /?-D-N-Acetylglucosamine, and Sialic Acid fl-D-N-Acetylglucosamine a-D-N-Acetylgalactosamine> a-D-N-Acetylglucosamine fl-D-N-Galactose-/? (l-3)-D-N-Acetyl galactosamine :> Galactosamine and Galactose a-D-N-Acetylgalactosamine> ~6-D-N-Acetylgalactosamine > a-D-N-Galactose a-D-N-Acetylgalactosamine a-L-Fucose /?-D-N-Galactose Or-D-N-Glucose,~-D-Mannose
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E T AL.
Fro. 1. Normal human c(mjurwti\ a i~('ubaied wilh FIT(' ~V(;A. k'~tlxed erSostat section. Goblet cells ((;(') and the eonjul~t.tival surtace a~'(' positi\ely stained. Stroma (N). • 100. FI(< 2. ('ontrol setti(nl ~f' ~o,'ma] human (.(mjunctiva incubated u ith F I T ( ' WGA and tbe competing sugar. 2m5i diacetxh.hit(~lfio.e N()te the absen(.e (if" fluores(.ent.e exeept for a weak reaction at the conjundivaI surface. • 10(} Fa;. 3. ('onjmu'tival sl~e('imet) fr<>m a patiel~t with cicatrieial pemphigoid. (Iryostat section incubated ~itb F1T(' W(;A. Note the intense staining of the apical epithelial cells and the epithelial cell plasma membrane, x25(t. Fro. 4. Normal human eon,junetiva incubated ~ith FIT(' conjugated S-\VGA. Goblet cells ((IC) and punetate areas in apieal epithelial c'ells are positively labelled. Stroma (S). x I00.
higher magnification, a fibrillar network of positively stained material, interpreted as ocular mucus, was often observed at the ocular surface of normal eonjunetiva after incubation with FIT(; WOA. Biopsy specimens fl'om patients with CP showed a similar staining pattern after' incubation with FIT() WGA. in m a n y of these patients, especially those with advanced disease, decreased numbers of eonjunctival goblet cells were observed; however, those goblet cells which were observed in CP patients stained positively with FITC WGA. Intense staining of the apical epithelial cells and ocular surface also occurred in biopsy specimens from CP patients after incubation with FITC WGA (Fig. 3). Incubation of conjunetival biopsy sections with FITCconjugated succinylated WGA (FITC-S-WGA) yielded a slightly different staining pattern; the apical epithelial cell layers and the goblet cells were positively stained; however, the ocular surface of the eonjunctival epithelium was not stained as intensely by FITC S-WGA (Fig. 4). Except for the decreased numbers of goblet cells
OCULAR MUCUS IN HUMAN CONJUNCTIVA
Ff~;.5. Not'~ual}mma~:':>~ju~:r
mr
489
VIT( HPA.(;oh!:.t,.~.lls((;(').andn::a.,is.trands
(arrow) are positive. Unfixed ~'r\x>~tat <(e~i~H •
I:Ic;. 6. ('onjuHt.tival specimen f'r'om a ci(.atricial t)e~lplliMoidpatient stained with FIT(I HPA. (;oblet <,ells (G(') and ve~icles in the apical epithelial cells are pr • FIe;. 7. ('onjunelival ~pecimen from a eiealri(,ial i)~,mphiKoidpa.tienl stained with FIT(? PNA. Goblet cells ((4(') present in this ('P ]patient'> ('oIIjUl]('IiVaat'e positive. Stroma (S). x 100. Fro. 8 (onjunctiva fi'om a ek.atricia] pemphi~oid patient with squamous metaplasia stained with FITC PXA. Weak staining occurred over the conjunetivai epithelial <.ell plasma membranes and/or extracelhdar substance. ('onjtmetival epithelium (('E): stioma (S). x 100.
in s p e c i m e n s from CP p a t i e n t s , s i m i l a r results were o b t a i n e d in b o t h n o r m a l e o n j u n e t i v a a n d s p e e h n e n s fl'om CP p a t i e n t s u s i n g F I T C S W G A . i n c u b a t i o n of n o r m a l e o n j u n e t i v a l biopsies with F I T C H P A r e s u l t e d in specific l a b e l l i n g of t h e g o b l e t eells (Fig. 5). The e o n j u n e t i v a l e p i t h e l i u m a n d o c u l a r surface were n o t labelled b y FITC-- H P A in e i t h e r n o r m a l e o n j u n e t i v a or e o n j u n e t i v a f r o m CP p a t i e n t s (Fig. 6). I n c u b a t i o n of n o r m a l e o n j u n e t i v a l biopsies w i t h F I T C H P A r e s u l t e d in specific l a b e l l i n g of t h e g o b l e t cells a n d m u c u s s t r a n d s e m a n a t i n g from t h e g o b l e t cells, w i t h o n l y w e a k l a b e l l i n g of t h e e o n j u n e t i v a l epithelial cells (Fig. 7). S t a i n i n g of the e o n j u n e t i v a l g o b l e t cells a n d n m e u s s t r a n d s was b l o c k e d w h e n F I T C P N A was
preineubated with 0'2 M lactose (data not shown). F I T C - P N A stained the eonjunetival epithelial cell membranes and/or extraeellular substance in biopsies from CP patients with squamous metaplasia (Fig. 8). Digestion of see
P. A. W E L L S E T AL.
490
TABLE II
Summary of staining patterns observed in normal and pathologic conjunctival specimens* Lectins Area Conjunctival surface Goblet cells Epithelial cells Conjunctival stroma
WGA
S-WGA
+ + + + + + 82 + 82 . . .
HPA
PNA
N-PNA t
SBA
+ :~ + + -
+ :~ + + -II
+ :~ + + +** +
+ + + +
.
* Key to staining intensity; for each lectin, a single plus denotes positive staining of the histological structure, two pluses denotes more intense positive staining, 2-3 times the intensity of staining graded with a single plus, a minus sign denotes no staining of the histological structure other than background fluorescence. N-PNA; Neuraminidase FITC-PNA staining sequence. :~ HPA, PNA and N-PNA stained the ocular surface only in areas adjacent to conjunctival goblet cells. 82 WGA and S-WGA stained the apical epithelial cells more intensely compared to the basal epithelial cell layers. II The plasma membranes and/or extracellular substance in the conjunctival epithelium stained weakly in CP patients with squamous metaplasia. ** The epithelial cell plasma membranes in specimens from cataract and CP patients stained positive when cryostat sections were treated with neuraminidase prior to FITC-PNA.
FIe;. 9. Conjunctiw~ from a cicatricial pemphigoid patient stained with FITC SBA. Positive staining occurred over the apical epithelial cells and elements within the subepithelial stroma. Conjunctival epithelium (CE); stroma (S). x ltt0. Fro. 10, Control section from the same pemphigoid patient incubated with FITC-SBA and the competing sugar, 0'2 5i lactose. (~onjunetival epithelium (CE); stroma (S). x 100. n e u r a m i n i d a s e p r i o r t o F I T C P N A i n c u b a t i o n r e s u l t e d in i n c r e a s e d l a b e l l i n g o f t h e c o n j u n e t i v a l e p i t h e l i a l cell m e m b r a n e s in b o t h n o r m a l b i o p s y s p e c i m e n s a n d s p e c i m e n s f r o m C P p a t i e n t s ( d a t a s u m m a r i z e d in T a b l e I I ) . I n c r e a s e d F I T C - P N A s t a i n i n g also o c c u r r e d o v e r t h e s u b e p i t h e l i a l s t r o m a w h e n s e c t i o n s w e r e p r e t r e a t e d with neuraminidase. The neuraminidase FITC-PNA staining could be inhibited by t h e a d d i t i o n o f 0'2 M l a c t o s e t o t h e l e c t i n . Incubation of human conjunetival biopsies with FITC-SBA resulted in intense s t a i n i n g o f t h e e o n j u n e t i v a l e p i t h e l i u m , e s p e c i a l l y t h e a p i c a l cell l a y e r s a n d b l o o d
OCULAR MUCUS IN HUMAN CONJUNCTIVA
491
':~~, ~ii~~!iiiili~~i~!i!ilii~!ii!!!i84ii~ill~
FIG. 11. Normal human conjunctiva stained with ferritin-conjugated WGA. Arrows point to ferritinWGA particles labelling mucins and the glycocalyx of the epithelial surface cell. Conjunctival epithelial surface cell (CE). • 47000. Fro. 12. Control normal human conjunctiva incubated with ferritin-WGA and the competing sugar, 2 mM diacetylchitobiose. Ferritin-WGA binding to the glyeocalyx (arrow) and to mucins is diminished. Conjunctival epithelium (CE). • 47000.
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FIG. 13. Conjunctiva from a cicatricial pemphigoid patient incubated with ferritin-WGA. Electron opaque ferritin-WGA particles (arrows) are observed labelling the conjunctival surface. Conjunctival epithelium (CE). • vessels in the conjunctival stroma (Fig. 9). This labelling was completely blocked by preincubation of the FITC SBA with 0"2 M lactose (Fig. 10). The staining pattern observed in CP conjunctiva after incubation with F I T C - S B A was identical to the staining pattern observed in normal conjunctiva with this lectin. The light microscopic results obtained using this panel of FITC conjugated lectins are summarized in Table II. The ocular surface of normal conjunctiva was positively stained by WGA conjugated to ferritin (Fig. 11). At the ultrastructural level, WGA-ferritin staining appeared to be localized over the glycocalyx of the conjunctival surface ceils and also over mucins at the conjunctival surface. This staining of the conjunctival surface was abolished when WGA-ferritin was preincubated with diacetylchitobiose (Fig. 12). A similar labelling of the conjunctival surface was obtained when specimens from CP patients were incubated with WGA-ferritin (Fig. 13). Incubation of conjunctival biopsy specimens with PNA-ferritin did not result in labelling of the conjunctival surface regardless of whether the biopsy specimens were from individuals with normal conjunctiva or CP patients. Sections of human conjunctiva cut from blocks of tissue embedded in glycol methacrylate and stained with Alcian Blue in acetic acid showed goblet cells with positively stained mucus granules. This staining was similar in both normal conjunctiva and conjunctiva from diseased eyes provided t h a t goblet cells were present in the tissue sample (Fig. 14). Alcian Blue staining of the conjunctival goblet cells was greatly decreased but not abolished when the sections were digested with neuraminidase for 24 hr (Fig. 15). Digestion with protease or incubation with PBS for 24 hr did not diminish the positive Alcian Blue staining of the goblet cell granules.
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FIG. 14. Conjunctiva from a patient with corneal ulcer and increased numbers of conjunctival goblet cells. Sections were incubated for 24 hr with PBS, pH 5"0, and then with Alcian Blue 8GX in 3 % acetic acid. Goblet cells (GC) are positively stained. Stroma (S). Glycol methacrylate section. • 125. FIG. 15. Conjunctiva from the same patient as Fig. 14 incubated for 24 hr with neuraminidase in PBS, pH 5"0, and then stained with Alcian Blue 8GX in acetic acid. Staining of the goblet cells is decreased. x 125. Fro. 16. Normal human conjunctiva incubated at 60~ with absolute methanol for 4 hr and stained with high iron diamine. Positive staining occurred over the goblet cells (GC). Stroma (S). Glycol methacrylate section, x 125. FIG. 17. Conjunctiva from the same patient as Fig. 16 incubated with methanol+HC1 (active methylation) and stained with high iron diamine. Staining of the goblet cell granules is abolished, indicating that the staining observed in Fig. 16 is due to sulfated mucins, x 125. Sections of h u m a n c o n j u n c t i v a which were stained with the high iron d i a m i n e (HID) reagent also showed positively stained goblet cell granules (Fig. 16). Positive s t a i n i n g with H I D was completely abolished when the sections were actively m e t h y l a t e d (methanol + HC1) prior to t r e a t m e n t with the H I D r e a g e n t (Fig. 17). Sections which were p r e t r e a t e d with m e t h a n o l alone ( m e t h y l a t i o n control) did n o t show a n y decrease in H I D s t a i n i n g of the goblet cell granules. The results of these histochemical studies are s u m m a r i z e d in Table I I I .
Discussion Mucins (mucin glycoproteins) secreted b y c o n j u n c t i v a l goblet cells (Kessing, 1968), c o n j u n c t i v a l epithelial cells (Greiner et al., 1980, Greiner, W e i d m a n , K o r b a n d
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P.A. WELLS ET AL. TABLE I I I
Summary of the staining patterns obtained with histochemical techniques* Treatment Alcian Blue (AB) Protease-ABt Neuraminidase-AB High iron diamine (HID) Methanol-HID:~ Methanol+HC1-HID (active methylation):~
Goblet cell staining + + + + +
++ ++ + ++ ++
+ + + +
* Grading system ; four pluses denotes intense staining of the goblet cells as observed when sections were stained with AB or HID without any pretreatment. Three pluses, two pluses, and one plus indicate 75, 50, and 25 % of the four plus staining intensity. A minus sign denotes an absence of observable staining. t Sections were digested with protease or neuraminidase for 24 hr. Methanol or active methylation 4 hr at 60~ Allansmith, 1985), and possibly by the lacrimal gland (Allen, Wright and Reidl 1972 ; Jensen, Falbe-Hansen, Jacobsen and Michelsen, 1969) contribute to the innermost mucus layer of the trilaminar preocular tear film (Holly and Lemp, 1977). These mucins overlie the surface cells of the cornea and conjunctiva. Tear film mucins play important roles in maintaining the wettability of the corneal surface (Lemp, Holly, I w a t a and Dohlman, 1970; Holly and Lemp, 1971), and in the defense of the ocular surface against bacteria and other infectious agents (Gibbons, 1982). Lectins consisting of proteins and glycoproteins from various plants and animals can be used to label mucins in human conjunctival biopsies. These lectins are capable of binding to specific carbohydrate residues found on glycoproteins and other glycoconjugates (Goldstein and Hayes, 1978). Previous light microscopic studies (Kawano, Uehara, Sameshina and Ohba, 1984) utilized fluorescein-conjugated WGA, SBA, PNA, Ricinus communis agglutinin-I (RCA-I), and Limulus polyphemus agglutinin (LPA) to label normal human conjunctival goblet cell granules. The lectins, Ulex europaeus agglutinin (UEA), Dolichos biflorus agglutinin (DBA), and concanavalin A (Con-A) did not label human conjunctival goblet cell granules (Kawano et al., 1984). In our study, conjunctival goblet cells in specimens from individuals with normal conjunctiva, and CP patients, were labelled with native WGA and succinylated WGA. Native WGA binds to terminal or internal trimeric, dimeric and monomeric Nacetylglucosamine residues and to terminal sialic acid residues (Goldstein an d Hayes, 1978). In contrast, S-WGA binds only to terminal N-acetylglucosamine residues and does not bind to sialic acid (data supplied by the manufacturer, E Y Laboratories). At the light microscopic level, native WGA also labelled the apical epithelial cells and the conjunctival surface in both cataract patients and CP patients. The binding of WGA to the conjunctival surface was confirmed at the TEM level by incubating tissue with WGA-ferritin. The binding of native WGA to the conjunctival surface, goblet cells, and the apical epithelial cells indicates the presence of N-acetylglucosamine and/or sialic acid residues at these tissue sites. Conjunctival goblet cell granules in specimens from cataract patients and in specimens from CP patients without squamous metaplasia were positively labelled with F I T C - H P A and FITC-PNA. Labelling of human conjunctival goblet cell
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granules with HPA indicates the presence of terminal N-acetylgalactosamine residues on the mucin glycoproteins. Labelling of the goblet cell granules with PNA indicates the presence of terminal D-galactose-/?(1-3)-N-acetylgalactosamine disaccharides or terminal D-galactose residues on the mucin macromolecules (Goldstein and Hayes, !978). Pretreatment of cryostat sections of human conjunctiva with neuraminidase caused increased staining of the non-goblet epithelial cell plasma membranes with FITC-PNA. The unmasking of PNA binding sites by neuraminidase digestion can be attributed to the removal of terminal sialic acid residues which inhibit PNA binding to penultimate D-galactose-fl(1-3)-N-acetylgalactosamine disaccharides in sialylated glycoproteins and glycolipids (Corfield and Schauer, 1982; Goldstein and Hayes, 1978). Previous histochemical studies (Srinavasan, Worgul, Iwamoto and Merriam, 1977 ; Matsumoto and Mimura, 1974; Kessing, 1968) have indicated that the mucus granules found in eonjunetival goblet cells contain neutral periodic acid-Schiff (PAS) positive mucins and acidic sialylated mueins (sialomucins). Wright and Mackie (1977) detected sulfomucins in human conjunctival goblet cells using Alcian Blue at pH 1"0. Our results from incubating glycol methacrylate sections of human conjunctiva with high iron diamine showed positive staining of the goblet cells. The abolition of high iron diamine staining when the sections were pretreated by active methylation established that the goblet cell granules contain sulfated mucins (sulfomucins) (Spicer, 1965). In addition, our results using glycol methacrylate embedded specimens exposed to neuroaminidase and Alcian Blue agreed with the previous studies and confirmed that neuraminidase-sensitive sialic acid residues were present in human conjunctival goblet cell granules The results of the lectin binding and histochemical studies indicate that human goblet cell mucins consist of glycoproteins with neutral, sulfated and sialylated oligosaccharide chains. It is possible that these three types of oligosaccharide chains exist on a single mucin glycoprotein, or that the human conjunctival goblet cell synthesizes several distinct mucin glyeoproteins. The lectin staining patterns which were observed in the conjunetiva of patients with CP and in normal human conjunctiva were similar. Although many CP patients had reduced numbers of goblet cells, those goblet cells which were observed in CP conjunctiva stained positively with WGA, S-WGA, HPA, and PNA as did goblet cells in normal conjunctiva. Previous studies employing SDS PAGE (Wells, Ashur and Foster, 1986) have shown that ocular mucus glycoproteins collected from the inferior fornix of patients with CP, atopy, rosacea blepharitis, Stevens-Johnson syndrome and other types of conjunctival inflammation have electrophoretic mobilities identical to those of normal ocular mucus glycoproteins. Together, the results of the lectin binding studies and the SDS-PAGE studies indicate that the conjunctival goblet cells from patients with CP do not synthesize or secrete abnormal ocular mucins. These studies effectively rule out a model for dry eye in CP which involves the secretion of abnormal mucins from the conjunctiva. The dry eye symptoms and increased viscosity of the ocular mucus which is observed in many CP patients may be related to either (1) a decrease in the surfactant properties of the mucus due to the addition of lipids, proteins, or nucleic acids to the ocular mucus, (2) a general decrease in mucus production due to decreased numbers of goblet cells, or (3) a decrease in lacrimal gland secretions due to scarring and obstruction of the lacrimal ducts. Further studies are planned at the transmission electron microscopic level to determine the amount of mucus at the ocular surface in CP patients compared to individuals with normal conjunctiva. 18
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This study was supported by United States Public Health Service grant EY 05813 and a grant-in-aid from the National Society to Prevent Blindness.
REFERENCES Allen, M., Wright, P. and Reid, L. (1972). The human lacrimal gland. Arch. Ophthalmol. 88, 493-7. Corfield, A. P. and Schauer, R. (1982). Occurrence of sialic acids. In Sialic Acids: Chemistry, Metabolism and Function. (Ed. Schauer, R.). Pp. 5-37. Springer-Verlag. New York. Foster, C. S. (1987). Cicatricial pemphigoid. Trans. Am. Ophthalmol. Soc. 84, 527-663. Foster, C. S., DeSiena-Shaw, C. and Wells, P. A. (1986). Scanning electron microscopy of conjunctival surfaces in patients with ocular cicatricial pemphigoid. Am. J. Ophthalmol. 102, 584-91. Gibbons, R. J, (1982). Review and discussion of role of mucus in mucosal defense. In Recent Advances in Mucosal Immunity. (Eds Strober, W., Hanson, L. A. and Sell, K. W.). Pp. 343-51. Raven Press: New York. Goldstein, I. J. and Hayes, C. E. (1978). The lectins : carbohydrate-binding proteins of plants and animals. Adv. Carbohydrate Chem. Biochem. 35, 127-340. Greiner, J.V., Kenyon, K.R., Henriquez, A.S., Korb, D.R.,Weidman, T.A. and Allansmith, M. R. (1980). Mucus secretory vesicles in the conjunctival epithelial cells of wearers of contact lenses. Arch. Opthalmol. 98, 1843-6. Greiner, J. V., Weidman, T. A., Korb, D. R. and Allansmith, M. R. (1985). Histochemical analysis of secretory vesicles in nongoblet conjunctival epithelial cells. Acta Ophthalmol. 63, 89-92. Holly, F. J. and Lemp, M. A. (1971). Wettability and wetting of the corneal epithelium. Exp. Eye Res. 11,239-50. Holly, F. J. and Lemp, M. A. (1977). Tear physiology and dry eyes, Surv. Ophthalmol. 22, 69-87. Jensen, O. A., Falbe-Hansen, I., Jacobsen, T. and Michelsen, A. (1969). Mucosubstances of the acini of the human lacrimal gland (orbital part), I. Histochemical identification. Acta Ophthalmol. 47, 605-19. Johnson, G. D. and de C, Nogueira Araujo, G. M. {1981). A simple method of reducing the fading of immunofluorescence during microscopy. J. Immunol. Meth. 4:}, 349-50. Kawano, K., Uehara, F. Sameshima, M. and Ohba, N. (1984). Application of lectins for detection of goblet cell carbohydrates of the human conjunctiva. Exp. Eye Res. 38, 439-47. Kessing, S.V. (1968). Mucus gland system of the conjunctiva. Acta Ophthal. Suppl. 95, 1-133. Letup, M. A., Holly, F. J., Iwata, S. and Dohlman, C. H. (1970). The precorneal tear film. I. Factors in spreading and maintaining a continuous tear film over the corneal surface. Arch. Ophthalmol. 83, 89-94. Matsumoto, K. and Mimura Y. (1974). Histochemical studies on the conjunctival goblet cells. Connect. Tissue 6, 85-90. Mollenhauer, H. H. (1964}. Plastic embedding mixtures for use in electron microscopy. Stain Technol. 39, 111-4. Nelson, J. D. and Wright, J. C. (1984). Conjunctival goblet cell densities in ocular surface disease. Arch. Ophthalmol. 102, 1049-51. Ralph, R.A. (1975). Conjunctival goblet cell density in normal subjects and in dry eye syndromes. Invest. Ophthalmol. 14, 299-302. Spicer, S. S. (1965). Diamine methods for differentiating mucosubstances histochemically. J. Histochem. Cytochem. 13, 211-34. Srinavasan, B. D., Worgul, B. V., Iwamoto, T. and Merriam, G. R. (1977). The conjunctival epithelium. II. Histochemical and ultrastructural studies on human and rat conjunctiva. Ophthalmic Res. 9, 987-1018.
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Wells, P. A., Ashur, M. L . and Foster, C. S. (1986). SDS-polyacrylamide gel electrophoresis of individual ocular mucus samples from patients with normal and diseased conjunctiva. Curt. Eye Res. 5,823-31. Wright, P. and Mackie, I . A . (1977): Mucus in the healthy and diseased eye. Trans. Ophthalmol. Soc. U.K. 97, 1-7.
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