Stephan Lolov⁽¹⁾, Dinka Petrova⁽²⁾, Stanimir Kyurkchiev⁽³⁾, George Edrev^(4)
(1)Institute of Biology and Immunology of Reproduction. Bulgarian Academy of Sciences - Sofia. Bulgaria
⁽²⁾⁽³⁾Institute of Biology and Immunology of Reproduction; Bulg. Acad. Sci. - Sofia. Bulgaria
⁽⁴⁾Dept. Otorhinolaryngology. Transport Medical Institute - Sofia. Bulgaria

	[Immunology]	[Otorhinolaryngology]

PRESENTATION

Abstract

Cholesteatoma is a destructive lesion of the human middle ear with unknown etiopathogenesis. Immunohistochemical assays with specific monoclonal antibodies have been used to register expression and distribution of several well-known antigens. In this study, the authors have looked for abnormal or unknown cholesteatoma’s constituents generating monoclonal antibodies.

BALB/c mice were immunized with subcutaneous implantation of the cholesteatoma’s sac and matrix. Hybridomas have been generated following procedures known in the art. The screening for clones with specific antibodies production was done using enzyme linked immunosorbend assay (ELISA) against saline tissue extract and immunofluorescence (IF) with paraffin embedded tissue sections.

The monoclonal antibody designated 1F8 showed positive reaction in ELISA and in IF when tested against both cholesteatoma and ear canal skin. At the same time when supernatants from different cell and tissue cultures have been used as an antigen in ELISA, 1F8 reacted only with cholesteatomas’ supernatant. In Western blot it recognizes three small bands and a prominent one, with MW of approximately 70 kD. Using immunohistochemistry the antigen was localized in the subepithelial connective tissue fibers of the skin and the cholesteatoma. The antigen of IF8 was detected in all tested cholesteatomas. Studies are now in progress to characterize the antigen in more details.

Key words:

cholesteatoma; monoclonal antibodies; immunohistochemistry

Introduction

Cholesteatoma is a destructive lesion of the human middle ear with unknown etiopathogenesis. The process of its formation is an object of intensive research and several controversial theories have been formulated:

• transition of epidermoid cell rests in the temporal bone (epidermoid formation) into congenital cholesteatoma ^{9, 12, 13, 16, 17};
• epidermoid metaplasia of middle ear epithelium ³;
• pathological aberration of the normal physiological process of auditory epithelial migration ^{2, 14, 18, 19}.

Immunohistochemical assays with specific monoclonal antibodies have been used to register expression and distribution of several well-known antigens (fibronectin ^{4, 20, 24}, interleukin (IL)-1 alpha and IL-2 ^{1, 8, 10, 21, 22}, epidermal growth factor, transforming growth factor-beta 1 and 2 ²³, tumor necrosis factor-alpha ^{15, 25}, keratinocyte growth factor ^{7, 11}, c-myc protein ⁶, different cell surface peptidases, parathyroid hormone-related protein ⁵ and many others), which may modulate cell growth and differentiation, as well as invasion of healthy tissues and bone resorption. The existence of a defect in the normal biology and/or biochemistry of cholesteatoma’s constituents have been suggested, but nothing specific for this entity have been reported.

In this study, we have looked for abnormal or unknown cholesteatoma’s constituents generating monoclonal antibodies.

Materials and methods

Some of the methods used were based on the classical protocols with just minor modifications:

SDS Gel Electrophoresis	Laemmli, U. K. (1970)26
Protein Concentration Determination	Bradford
Silver Staining of Gels	WH Heidcamp (1996)
Electroblotting of SDS-PAA gels	WH Heidcamp (1996)
Thawing /Freezing Hybridomas and Myelomas	Peter MacCallum (1998)
Cloning by Limiting Dilution	Nanci Donacki
Ascites Production	Peter MacCallum (1998)

Immunization

The mice were given an anaesthetic dose of 0.01 % Halothanum thymolo (Spofa) and immobilized on surgical board. After removal of the fur by shaving and dampening with ethanol, a V-shaped cutaneous incision was made in the dorsal region. About 1 mm³ piece of xenogenous tissue, handled with forceps was inserted through a small slit in the animal’s subcutaneous tissue until becomes completely embedded. After that, the skin was carefully sutured.

Two and four weeks later, the procedure of tissue implantation was repeated, each time with a histologicaly proved cholesteatoma, from a different patient. Three days before the fusion, a final boost was done intraperitonealy with saline extract of cholesteatoma (10 m g total protein).

Saline tissue extract

As soon as possible after excision, cholesteatoma’s tissue and skin from external meatus of seven patients were minced and cells were disrupted with ten cycles of freezing to -20^oC and thawing to room temperature in the presence of PBS with PMSF, aprotinin and trypsin inhibitor. After brief spin at 1850 G the supernatants were collected and stored at -20^oC until used.

Fusion

P3X63-Ag8.653 cells (non secreting, 8-azaguanine resistant, HPRT – myeloma; 2.5 x 10⁷ cells) were added to spleen cells (9.2 x10⁷) from the immunized BALB/c mice. PEG/DMSO solution was added under agitation folowing well established protocol (Peter MacCallum). The cell pellet was resuspended in RPMI 1640 medium supplemented with 10% fetal calf serum and plated into five tissue culture plates and incubated in the presence of hypoxanthine, aminopterin, and thymidine medium in a humidified 37^oC, 5% CO₂ incubator.

Screening

First clones were seen after 7-10 days. The first screening procedure was started after 2 weeks, with a second and third, a few days later. Only wells with the confirmed presence of one or two big clones, were tested for the secretion of desired antibody. Both enzyme linked immunosorbend assay (ELISA) and indirect immunofluorescence (IF) were used as screening procedures.

Hybridomas secreting specific antibodies were passed through limiting dilution and retested until all wells were considered positive.

ELISA

1. Saline tissue extract was diluted to 10 µg /ml in PBS and 50 µl was added to each well of 96 well PVC plate (Titertek). Plate was left overnight (ON) covered with saran wrap, at 4œC.
2. The unbound Ag was washed out by inverting the plates and flicking the wells dry. The plate was rinsed by adding PBS to each well and inverting it again. The rinse was repeated twice and 150 µl of blocking solution (10% heat inactivated calf serum in PBS and 0.02% azide for longer storage) was added to every well, leaving the plate for 1 hr at room temperature (RT) or ON at 4œC.
3. The cell culturing supernatant from the hybridoma tested was added (50 µl/well) and the plate was left for 2 hr at RT.
4. Unbound antibody was washed 4 times with PBS + 0.1% Tween 20 and 50 µl of appropriately diluted second antibody (peroxidase labeled goat anti-mouse imunoglobulin, DAKO) was added to all wells. The plate was left for 1 hr at RT.
5. The plate was washed 4 times with PBS + 0.1% Tween 20 and 50 µl of substrate (5 mg ortophenilen diamin; 10 ml 100mM Na Citrate pH 4.2; 10 µl 30% hydrogen peroxide) was added to every well. After 20 min the color development was stopped by adding 50 µl 10% H₂SO₄. The absorbtion was recorded on an ELISA reader (Dynatech AG, USA) at 492 nm.

Immunohistochemistry

1. All tissues were fixed in 10% neutral buffered formalin and embedded in paraffin. Sections were 5-10 µm thick. They were incubated 3 times in xylene and hydrated by placing in 95%, 70%, 50%, ethanol for 5 minutes each.
2. Endogenous peroxidase was quenched by placing slides in 3% hydrogen peroxide in methanol for 20 minutes.
3. Slides were blocked for 20 min with 10% goat serum and washed 4 times with PBS + 0.1% Tween.
4. The cell culturing supernatant from the hybridoma tested was added (20 µl/section) and the slides were incubated in a humidified chamber ON or for 2 hr at RT. The antibody was restricted to the wet region of the slide by surface tension alone.
5. Slides were blocked again for 20 min with 10% goat serum.

Fluorescence visualization:

1. FITC labeled rabbit anti-mouse immunoglobulin was used in a dilution of 1:200 and the slides were incubated in a humidified chamber for 1 hr. Sections were then washed 4 times with PBS + 0.1% Tween.
2. Temporary glycerol mounting was done and results were observed and documented under UV-irradiation on Leitz fluorescence microscope.

Peroxidase visualization (Avidin-biotin affinity method, ABA):

1. Biotinylated goat anti-mouse imunoglobulin was used in a dilution of 1:1000 and the slides were incubated in a humidified chamber for 1 hr. Sections were then washed 4 times with PBS + 0.1% Tween.
2. Peroxidase-conjugated avidin was used in a dilution of 1:200 and the slides were incubated in a humidified chamber for 1 hr. Sections were then rinsed 4 times with PBS + 0.1% Tween.
3. Slides were incubated in fresh DAB solution. (10 mg DAB + 20 µl 30% H₂O₂ in 20 ml 0.1 M Tris pH 7.2; 200 µl 1 M imidazole). The reaction was stoped by washing in running water when a uniform brown color first becomes visible on the sections.
4. Sections were counterstained by haematoxilin, dehydrated by placing in 50%, 70%, 95% ethanol, and 3 times in xylene for 5 minutes each. Samples were permanently mounted.

Western blot

1. After electroblotting of SDS-PAA gels, transferred proteins were stained with Ponceau S and respective lines were marked.
2. Unoccupied protein binding sites on the nitrocellulose (0.2 µm, Sigma) were blocked with 30 min incubation in PBS with 0.1% Tween 20.
3. The immobilized proteins were probed with cell supernatant from the hybridoma tested or from unrelevant hybridoma (negative control) - 2 hr incubation at RT.
4. The membrane was washed three times, 5 minutes each in PBS with 0.05% Tween 20 and transferred to a solution of peroxidase-conjugated goat anti-mouse serum - 1:1000, 1 hr at RT.
5. For peroxidase visualization, membtrane was incubated in fresh DAB solution. (10 mg DAB + 20 µl 30% H₂O₂ in 20 ml 0.1 M Tris pH 7.2; 200 µl 1 M imidazole). The reaction was stoped by washing in running water when slight background becomes visible on the negative control.

All steps were done with constant shaking.

Photomicrography

Kodak Technical Pan was the film of choice for Western blot documentation. It was processed to give high contrast. Thus the small differences were accentuated.

Color negative film (AGFA HDC 100) was developed and printed commercially. To avoid the loss of control over the printing process and to receive comparable results:

• all photos were taken on the same film under fixed conditions (illumination and duration);
• the exposure and color balance during automatic printing were constant for the whole film.

Results and discussion

It should be kept in mind that some routine immunization techniques involve the risk of causing conformational changes to the antigen during its purification, cross-linking with carrier or when emulsified in an adjuvant. This can result in the production of antibodies against inactive, denatured antigens. In the present study a new immunization approach was used - the whole cholesteoma’s tissue (sac and matrix) was deposited several times into the subcutaneous tissue of experimental animal. This technique provides an easy way to immunize animals with an uncharacterized and unlocalized antigen in a native conformation. At the same time, xenogenous tissue implantation would result in a prolonged presence of antigen, which would lead to an augmentation of the immune response. So, the use of adjuvant with all its side effects like sterile abscesses, granuloma formation and unwanted antibodies generation is avoided by the present approach.

 

A	B
Silver-stained gel of a 10% SDS-PAGE of saline tissue extract from cholesteatoma (A) and ear canal skin (B). M-lanes contained protein standards with their sizes listed in kilodaltons.	There were no significant differences between cholesteatoma and skin, when several concentrations were compared under reducing conditions (lanes 1 - 1 µg total protein; lanes 2 - 0.5 µg protein; lanes 3 - 0.25 µg protein; lanes 4 - 0.125 µg protein).

Just before the booster immunization a test bleeding was performed and antibody titers were assayed by ELISA. The sigmoid curves obtained by testing serial dilutions of mouse serum demonstrated stronger polyclonal response against cholesteatoma’s extract.

 

A	B
C	D
Indirect immunofluorescence of ear canal skin (A) and cholesteatoma (B) after incubation with cell culturing supernatant from the hybridoma 1F8.	The negative controls of skin (C) and cholesteatoma (D) were treated with supernatant from myeloma cell line.

 

Hematoxilin-eosin staining of cholesteatoma with cholesterol clefts.
There were no specific reaction when serial sections were incubated with supernatant from myeloma cell line (negative control). Brown nodules (arrow) represented not color reaction product, but haemosiderin.
The serial sections, after incubation with culture supernatant from hybridoma 1F8, demonstrated specific brown staining of the connective tissue.

 

When the skin from external meatus was used as an antigen in immunohistochemistry, there was uniform specific staining in the subepithelial space (thin arrow) and more fibrous in deeper layers (thick arrow).
The serial section was incubated with supernatant from myeloma cell line and used as negative control. Some darkly stained areas of epithelium (asterisk), observed also in previous picture, represented staining artifacts.

 

Hematoxilin-eosin stained squames of dead keratin in an acquired cholesteatoma.
The serial paraffin section, incubated with antibody 1F8, followed by visualization with ABA method. Note the brown stained granular structures (specific reaction) and blue dyed squames (hematoxilin counterstaining).
The negative control was incubated with supernatant from myeloma cell line and subjected to ABA procedure.

 

Evaluation of the specificity of monoclonal antibody 1F8 (lane 3) and some others (BX - lane 1 and BY - lane 2) by Western blot analyses using 12% PAA gel and cholesteatoma’s saline extract (100 µg /lane) under denaturing conditions. The lane K was incubated with supernatant from myeloma cell line and used as negative control. To verify transfer efficiency, Ponceau S staining was performed (lane P). The dye front (f) was also marked. 1F8 recognizes three small bands and a prominent one, with MW of approximately 70 kD.

 

When supernatants from different cell cultures (U937, Jurkat, P3U1, HUVEC and SK MEL1 - data not shown) and tissue cultures have been used as an antigen in ELISA, 1F8 reacted only with cholesteatomas’ supernatant. At the same time when saline tissue extracts were absorbed on the PVC plate, the reaction against skin was stronger.

We succeeded to detect the antigen of 1F8 in all cholesteatomas’ cases (seven) studied.

Conclusions

The data of the present work suggest that generation of monoclonal antibodies using tissue implantation as an immunization procedure is a promising approach to obtain an immunological tool for unknown antigen isolation and characterization. Unfortunately, we failed to detect cholesteatoma’s specific antigen. Other immunization and fusion experiments are now in progress.

The monoclonal antibody designated 1F8 showed positive reaction in ELISA and in IF when tested against both cholesteatoma and ear canal skin. It recognizes several bands in Western blot after SDS-PAGE. The antigen of 1F8 was detected in all tested cholesteatomas. It is expressed only in skin and cholesteatoma (data not shown) and possesses some interesting characteristics.

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Discussion Board

Any Comment to this presentation?

[ABSTRACT] [PRESENTATION] [Discussion Board]

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	[Immunology]	[Otorhinolaryngology]

Stephan Lolov, Dinka Petrova, Stanimir Kyurkchiev, George Edrev
Copyright © 1999-2000. All rights reserved.

Last update: 17/01/00