monoclonal antibodies to human amnion recognize...

Monodonal Antibodies to Human Amnion RecognizeDifferent Components of the Robbit Eye

Doe-Li Hsi,* Danielle Fredj-Reygrobeller.f Chang-Jing G. Yen,* Pierre-Paul Elena,fGerard Moulin, f and Philippe Lapalusf

The epithelium of the eye originates from embryonic ecto-derm, whereas the amnion is an extra-embryonic membranethat bears close relationship with many ectodermal tissues.Shared antigens have been identified between human amnionand cornea using rabbit antisera to human amnion. Threemonoclonal antibodies to human amnion, GB4, GB9, andGB11 were studied by immunofluorescence on the anteriorsegment of the rabbit eye. GB4 recognized the epithelium ofthe conjunctiva and the subcapsular epithelium of the lens.GB9 reacted only with the central four fifths of the cornealepithelium; the peripheral epithelium near the limbus wasnot reactive. GB11 detected the pigmented epithelial cellsin ciliary processes. Invest Ophthalmol Vis Sci 27:620-622,1986

Human amnion is an extra-embryonic ectoderm thatlines the amniotic cavity, wraps around the umbilicalcord, and continues with the fetal skin. Many antigenicsimilarities have been demonstrated between amnionand various ectoderm-derived tissues.1 The eye is es-sentially an ectodermal structure; only the stromas ofthe cornea, iris, and ciliary body are of mesoderm andneural crest ectoderm origin. Several groups of amnionantigens that have been characterized by rabbit antiserato amnion showed cross-reactivity with human cornea.2

Recently, eight monoclonal antibodies to human am-nion have been developed.3 Three of these antibodies,GB4, GB9, and GB11, showed interesting reactivitiesto the rabbit cornea, conjunctiva, ciliary processes, andlens. GB4 recognized cytoplasmic structures in theamniotic epithelium; GB9 reacted with amniotic epi-thelium and sub-epithelial connective tissue; and GB 11detected the supra-basal epithelium of the skin. Eachof these antibodies recognized denned structures in theanterior segment of the rabbit eye.

Materials and Methods. Tissues: Eye specimenswere obtained from five New Zealand Albino and threeFauve de Bourgogne rabbits. After enucleation, a cir-cumlimbal incision was made (2 mm beyond the lim-bus, along the conjunctiva), the whole lens was ex-tracted with the anterior segment of the eye. Theaqueous humor and vitreous body were removed by afilter paper. The eye specimens were then placed be-tween two slices of rabbit liver that were used as sup-porting tissues. Rabbit skin, lung, trachea, kidney,stomach, esophagus, intestine, eye lid, and lip werealso collected. The specimens were frozen in liquid ni-

trogen and stored at -20°C. Studies using these ani-mals were performed in conformance with the ARVOResolution on the Use of Animals in Research.

Antibodies: Three mouse monoclonal antibodies tohuman amnion GB4, GB9, and GB11 were used forthis study. The production and their reactivities to hu-man amnion and other extra-embryonic tissues weredetailed by Hsi and Yeh.4 Undiluted supernatant fluidsof the hybridoma cell lines were used. The culture me-dium of P3-NSl-Ag4-l (NS-1) cell line was used as acontrol. Fluorescein isothiocyanate (FITC) conjugatedrabbit anti-mouse immunoglobulin (Ig) were purchasedfrom Dakopatts (Copenhagen, Denmark).

Immunofluorescence: Frozen sections (5 pm) wereprepared by using a cryostat (Bright Instrument; Hun-tingdon, England) and air-dried at room temperature.Sections were pre-washed for 10 min in 0.15 M phos-phate buffered saline (PBS), pH 7.2, then incubatedwith 50 Ail of the supernatant fluid for 2 hr. After beingwashed in PBS for 5 min, sections were reacted witha 1:40 dilution of 50 /A FITC conjugated rabbit anti-mouse Ig. Some of the sections were counterstainedwith propidium iodide.5 After the last incubation, thesections were washed in PBS for 5 min, then mountedin AF1 mounting media (Citifluor Ltd., London). Sec-tions were studied by epi-illumination with the use ofa Zeiss Universal Microscope (Carl Zeiss, Inc.; Ober-kochen, West Germany) fitted with HBO 50 mercuryarc lamp, epifluorescence condenser III RS and filtersets for FITC and propidium iodide studies.6

Results. GB4: GB4 reacted with the conjunctival ep-ithelium. The reactivity began at the limbus where thestratified squamous epithelium of the cornea changedto the simple columnar epithelium of the conjunctiva(Fig. 1), continued to the inner surface of the eye lid,and disappeared where the epithelium changed to thestratified squamous epithelium of the skin. GB4 alsoreacted with the subcapsular epithelium of the lens (Fig.2); the epithelial cells at the equatorial zone of the lenswere nonreactive. Among the other rabbit tissues ex-amined, the reactivities could be identified on the basalepithelium of the hair follicles and some of the sweatglands in skin, the epithelium of trachea, bronchi, andepithelium lining the calyces of the kidney. In someinstances, the reactivity could also be identified on afew stromal cells in the sub-epithelial connective tissuesof these specimens.

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Fig. 1. The reactivity of GB4 on the rabbit conjunctiva (XI50). The reactivity of GB4 can be identified in the epithelium of the conjunctiva.Note that the comeal epithelium (C) and the stromal tissues (S) were nonreactive.

Fig. 2. The reactivity of GB4 on the rabbit lens (X150). The reactivity of GB4 can be detected in the subcapsular epithelium of the lens (L).Some very weak reactivities can also be observed in the iris (I). The lens epithelium at the equatorial zone did not react with GB4 (not shown).

Fig. 3. The reactivity of GB9 on the rabbit cornea (X150). GB9 recognized the upper layers of corneal epithelium (C). The corneal epitheliumat the limbus (Lb) did not react with this antibody.

Fig. 4. The reactivity of GBl 1 on the rabbit ciliary processes (X150). The epithelial cells situated on the basement membrane of'the ciliaryprocesses reacted with GBl 1. The upper nonpigmented epithelium was nonreactive. Some reactivities can also be found in a few cells in thestromal tissues.

GB9: GB9 reacted with the central four fifths of thecorneal epithelium; its reactivity stopped abruptly nearthe limbus region (Fig. 3). No morphological differencecould be discerned between the GB9-positive and GB9-negative corneal epithelium. In the middle of the cor-nea, the basal layer of the corneal epithelium reactedmore weakly than the upper layers. None of the otherrabbit tissues examined reacted with GB9.

GB11: GBl 1 reacted with the pigmented epithelialcells of ciliary processes (Fig. 4). The epithelial cells ofthe iris were nonreactive. The reactivity was more ob-viously observed in the albino rabbit eyes. Some cellsin the stromal tissues of ciliary processes and iris also

weakly reacted. No other rabbit tissues examined re-acted with this antibody, except the skin; the reactivityof GBl I could be seen in the cytoplasm of the supra-basal epithelium of the skin which was similar to thepattern found in human epidermis.4

Discussion. The three monoclonal antibodies de-scribed in this communication provided extremelyspecific epithelial markers for the anterior segment ofthe rabbit eye. GB4 reacted with the conjunctival ep-ithelium and the subcapsular epithelium of the lens;GB9 recognized the corneal epithelium; and GB11 de-tected the pigmented epithelium of the ciliary processes.In clinical ophthalmology, inflammation and lesions


622 INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / April 1986 Vol. 27

of the anterior segment of the eye, such as acid andalkaline burns to the epithelium and stroma of the cor-nea, are frequent pathological cases. These conditionscan be easily produced in animals, particularly in rab-bits for experimental studies.7 These three monoclonalantibodies should provide valid means to follow themigration, proliferation, and differentiation of the ep-ithelial cells during wound healing.8'9 In extra-capsularsurgery of cataract, lens is removed with its epithelium.However, secondary cataract often occurs when theremaining epithelial cells at equatorial zone of the lensproliferate and eventually cover the posterior capsule.10

The results of this study demonstrate that GB4 reactedwith the subcapsular epithelium of the lens, but notwith the cells located at the equatorial zone. Thus, GB4will be particularly useful in studying the posteriorcapsule epithelialization during the formation of sec-ondary cataract.

Although the biochemical nature and the functionof the antigens recognized by GB4, GB9, and GB11await further studies, these antibodies offer valuabletools for ophthalmologists to investigate the epi-thelial differentiation in the anterior segment of therabbit eye.

Key words: monoclonal antibodies, conjunctiva, cornea, cil-iary process, lens

From INSERM U210* and Laboratoire de Pharmacologief Facultede Medecine, Avenue de Vallombrose, 06034 Nice-Cedex, France.

Submitted for publication: June 5, 1985. Reprint requests: Pr. Ag.P. Lapalus, Laboratoire de Pharmacologie, Faculte de Medecine, Av-enue de Vallombrose, 06034 Nice-Cedex, France.

References1. Hsi BL, Yeh CJG, and Faulk WP: Human amniochorion: tissue-

specific markers, transferrin receptors and histocompatibilityantigens. Placenta 3:1, 1982.

2. Hsi BL, Yeh CJG, and Faulk WP: Characterization of antibodiesto antigens of the human amnion. Placenta 5:513, 1984.

3. Hsi BL and Yeh CJG: Monoclonal antibodies to human amnion.J Reprod Immunol, in press.

4. Ockleford CD, Hsi BL, Wakely J, Badly RA, Whyte A, andFaulk WP: Propidium iodide as a nuclear marker in immuno-fluorescence. I. With tissue and cytoskeleton studies. J ImmunolMethods 43:261, 1981.

5. Yeh CJG, Hsi BL, and Faulk WP: Propidium iodide as a nuclearmarker in immunofluorescence. II. Use with cellular identifi-cation and viability studies. J Immunol Methods 43:269, 1981.

6. Moses RA, Parkinson G, and Schuchardt R: A standard largewound of the corneal epithelium in the rabbit. Invest OphthalmolVis Sci 18:103, 1979.

7. Fredj-Reygrobellet D, Moulin G, Elena PP, and Lapalus P:Wound healing of the epithelia in albino rabbit eye anterior seg-ment. Poster ISER Congress Alicante (Spain), 1984.

8. Shapiro MS, Friend J, and Thoft RA: Corneal re-epithelializationfrom the conjunctiva. Invest Ophthalmol Vis Sci 21:135, 1981.

9. Pettit TH: Study of lens regeneration in the rabbit. InvestOphthalmol 2:243, 1963.

10. McDonald JE, Roy FH, and Hanna C: After cataract of therabbit: Autoradiography and electron microscopy. AnnOphthalmol 6:37, 1974.

Tear Immunoglobulins Measured by ELISA

P. K. Coyle* and Patrick A. 5ibony*t

The authors applied an ELISA to measure IgG, IgA, andIgM concentration in tears from 20 normal subjects. Thisassay was more sensitive than any other previously reportedtechnique to quantitate tear immunoglobulin. Only 2 nl oftears were required and concentrations as small as 1 ng/mlcould be detected. IgM was present in all samples at a geo-metric mean level of 5.6 Mg/ml. Mean IgA level was 186 ng/ml and mean IgG was 6.7 Mg/ml- No correlation was foundbetween tear and serum levels, suggesting that local synthesiswas responsible for most of the tear immunoglobulin. ThisELISA offers a sensitive and reliable method to analyze verysmall volumes of tears. It can be modified to test for manydifferent antigens and antibodies. Invest Ophthalmol Vis Sci27:622-625, 1986

Previous investigators have applied a variety of tearcollection methods and immune assays to measure tearimmunoglobulins.1"9 In addition to inherent variationbetween individual subjects, these studies have some-times produced a wide range of immunoglobulin con-

centrations in part because of the lack of standardiza-tion, relative insensitivity of the assays, and differencesin assay and collection techniques. The relatively largevolumes of tears which most assays require has maderesearch in this area difficult. We applied a modifiedELISA immunoassay to study the concentration ofimmunoglobulins in normal tears. We found this tech-nique could reliably measure immunoglobulins insmaller volumes of tears with greater sensitivity thanhas been previously reported using other techniques.The purpose of this study is to present our findings onimmunoglobulin concentration in normal tears usinga modified ELISA assay.

Materials and Methods. Subjects: Simultaneous tearand serum samples were collected from 20 subjectswithout eye disease (11 men and 9 women) ranging inage from 21 to 62 yr (mean 35 yr). None was takingany medications that could interfere with tear produc-tion. Serum was collected from an additional ten nor-


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