190
Monoclonal Antibodies to Periodontal Ligament Cells William T. DuBois, * Jeffery Edmondson,f Stephen B. Milam,T William B. Winborn,f Robert Hay, * David L. Carnes,s Kenneth S. Kornman, * and Robert J. Klebef
Ten mouse monoclonal antibodies were prepared against cultured bovine periodontal ligament cells to be used as reagents for the study of periodontal disease and wound healing. Using standard immunohistochemical methods, these antibodies were found to recognize cell surface antigens in formalin-fixed bovine periodontium. Three of the 10 monoclonal antibodies (i.e., PDL-1, PDL-2, and PDL-10) cross-reacted with cells found in primate periodontium. While the isolated monoclonal antibodies appeared to distinguish subpopulations of cells located in the supporting tissues of teeth, immunohistological examination of other organs (dermis, kidney, skeletal muscle, thyroid, and parotid gland) indicated that a number of cell types of mesenchymal origin share an antigen(s) found on periodontal cells. The monoclonal antibodies described in this report should prove to be useful in studies of periodontal disease and guided tissue regeneration by providing both analytical reagents and immunochemical methods for isolating selected cell populations of the periodontium. J Periodontol, 1991; 62:190-196.
Key Words: Monoclonal antibodies; wound healing; guided tissue regeneration; periodontal diseases; periodontium/anatomy; periodontium/histology.
Periodontal disease results in the destruction of the supporting tissue surrounding the tooth and can lead to loss of function or extraction of the tooth affected. Current therapy is designed to prevent further destruction of normal tissue and enhance the reparative process. One approach, termed guided tissue regeneration, is designed to specifically recruit cells from the periodontal ligament to repair the damaged area with new cementum, ligament, and bone.1"4 The evaluation of this therapy is empirical because of the difficulty in determining the identity of the cell populations which participate in the wound healing process.5'6 Monoclonal antibodies, which are specific for distinct cell types, would be of great benefit in the analysis of periodontal disease. The ability of monoclonal antibodies to react with specific cell types within a mixed population provides a powerful tool to isolate and identify selected cell types.7 Monoclonal antibodies are important reagents in the study of cell population dynamics during developmental and pathological processes.8-" Monoclonal antibodies can now be used to demonstrate cell populations that were not detected by routine histological procedures that depended on chemical dyes.12 In the case of the lymphocytic series, monoclonal antibody methods can detect at least 11 types of T*Department of Periodontics, University of Texas
Health Science TX. ^Department of Cellular and Structural Biology. §Department of Endodontics. 'American Type Culture Collection, Rockville, MD.
San
Antonio,
Center,
cells13
as well as several other white blood cell subgroups13,14 that were only theoretical entities prior to the advent of monoclonal antibody technology. While monoclonal antibodies have been prepared to many cell types found in blood,13-14 a comprehensive series of monocloncal antibodies have not been developed for the analysis of cell population dynamics in most other tissues. The goal of this study has been to produce monoclonal antibodies that recognize cell types of interest in oral biology.
MATERIALS AND METHODS
Cell Cultivation Cell lines were maintained with Dulbecco's medium containing 10% newborn bovine calf serum plus 100 units/ml penicillin and 100 micrograms/ml streptomycin (hereafter referred to as culture medium). During the cell fusion procedure and selection for hybrid clones, medium containing 10% fetal bovine calf serum and/or Hybri-Max CPSR-311 were
employed. mouse myeloma cell line, FOX-NY,
and other percell lines were obtained from the American Type Culture Collection. Bovine periodontal ligament (PDL) cells were adapted to culture by placing minced pieces of bovine periodontal ligament in culture medium. The periodontal ligament specimens were obtained from freshly extracted The
manent
'Sigma
Chemical
Co., St. Louis, MO.
Volume 62 Number 3
DUBOIS, EDMONDSON, MILAM, WINBORN, HAY, CARNES, KORNMAN,
posterior teeth approximately 15 mm apical to the gingival crest. After several days, cells were observed migrating away from attached PDL fragments. Early cultures contained several morphologically distinct cell types while established cultures that were used in monoclonal antibody preparation contained mainly fibroblasts with many branched processes. Immediately prior to use in immunization, the cells were trypsinized and washed with sterile saline. The following human cell lines were obtained from the American Type Culture Collection, HeLa, HepG2, HuTu, RD, and A549. SV40-transformed IMR-90 cells were obtained from the Coriell Institute for Medical Research, Camden, NJ. A chondrosarcoma cell line of human origin15
was
obtained from Dr. Wendell Winters of this institution.
Monoclonal Antibody Production Balb-C mice were immunized by intraperitoneal injection with 107 trypsinized cultured periodontal ligament cells in Freund's complete adjuvant each week for 4 to 6 weeks. The animals were sacrificed and their spleens harvested for the fusion procedure. Spleen cells were fused with FOXNY myeloma cells in the presence of 50% polyethylene glycol" as described previously.16 Hybridoma clones were selected with hypoxanthine:aminopterin:thymidine (HAT) media.17'18 Ten to 14 days post-fusion, culture supernatants were initially screened by ELISA for antibody activity to formalin fixed bovine PDL cells. Cells in positive wells were cloned by limiting dilution and the clones isolated were re-screened by immunofluorescence. Hybridoma cell lines in culture media containing 10% DMSO were frozen in liquid nitrogen. PDL-10 has been submitted to the American Type Culture Collection.
ELISA Procedure The ELISA procedure employed at this stage of hybridoma selection has been described in detail previously.16 In brief, bovine PDL cells were grown to confluence in 96-well plates and fixed with neutral-buffered-formalin. Wells were blocked with 1% bovine serum albumin in Tween-PBS (0.05% Tween-20 in 0.15 M NaCl + 0.05 M sodium phosphate, pH 7.2) for 15 minutes and washed. One hundred microliters of culture supernatant were added to each test well and incubated at 37° for 30 minutes. Positive wells were identified following reaction with rabbit anti-mouse-IgG peroxidase conjugate and o-dianisidine peroxidase substrate. As a positive control, polyclonal serum collected from the immunized mouse at the time of sacrifice was employed; the negative control was media conditioned by the FOX-NY parental cell line. Immunofluorescence
Assay
Following cloning by limiting dilution, hybridomas were retested by immunofluorescence as follows. Bovine PDL cells were grown on acid washed glass slides and fixed ^Gibco, Grand Island,
NY.
Table 1:
Antigenic Recognition by
Monoclonal PDL1 PDL2 PDL3 PDL4 PDL5 PDL8 PDL9 PDL10
191
PDL Monoclonal Antibodies
Immunofluorescence Pattern Fibrillar Fibrillar diffuse
Antibody
KLEBE
Punctate, diffuse
Molecular Weight Estimation (kD) 63 63 163; 108; 73 63
Diffuse fibrillar
63; 57 73
109; 63; 57 100; 73
Perinuclear; punctate Strong fibrillar; punctate -
Immunofluorescence patterns were obtained by reacting cultured bovine PDL cells with ascites fluid at a dilution of 1:500 in PBS followed by FITC anti-mouse IgG conjugate at a 1:20 dilution. The appearance of antigen on the cell surface is given above. Note that different patterns are generated by each of the antibodies indicating that a different antigenic determinant is recognized (note that PDL1 and PDL2 are subclones). Molecular weight estimation was carried out by Western blotting as described in text.
Table 2: Localization of Antigens in the PDL Monoclonal Cementum/Cemento- CementoExtracellular Dentin blasts Fibroblasts Matrix cytes Antibody PDL1 PDL2 PDL4 PDL5 PDL9 PDL10
-
-
+ + +
+
+ +
(±) (±) (±)
+ +
-++(+)+ + (±) -
+
+
+
Gingival
Connective Junctional Osteoblasts Osteocytes Tissue Epithelium PDL1 PDL2 PDL4 PDL5 PDL9 PDL10
+ +
+ +
+
+ + + +
+ + +
-
Gingival Epithelium
+ +
+ +
+
+
-
Bovine periodontal tissue was decalcified, embedded, sectioned, and reacted with an avidin-biotin immunoperoxidase reagent as described in the text. Note the similarity between the distribution of the antigens recognized by PDL1, PDL2, and PDL10 with vitronectin. PDL5 and PDL9 reacted with discrete cells within and immediately surrounding the periodontal
ligament. ( + positive reaction; negative reaction; ( ± ) cells positive while other cells are negative). =
=
=
some
-
with neutral buffered formalin. The fixed cells were incubated with hybridoma culture supernatant, washed, and stained with a fluorescein-conjugated goat anti-mouse IgG. Epifluorescence microscopy revealed several hybridomas that produced antibodies which reacted with cell surface antigens on the formalin fixed bovine PDL cells that did not react with a panel of human cell lines (HeLà, HepG2, HuTu, RD, A549, SV-IMR-90, and a human
chondrosarcoma). Ascites Fluid Production Those hybridomas that were
positive by immunofluoresselected for further study. One to ten million hybridoma cells were injected intraperitoneally into pristane-primed Balb-C mice. One to 2 weeks later tumors had cence were
192
J Periodontol March 1991
PERIODONTAL LIGAMENT CELLS
1: Reaction ofperiodontal tissues with anti-periodontal ligament monoclonal antibodies. Bovine periodontal tissues were reacted with monoclonal antibodies conjugated with avidinlbiotin as described in the text. PDL-1 stain of periodontal Sites of positive antibody reaction are indicated by arrows (unlabeled). A PDLPDL-10 stain of periodontal ligament; D PDL-1 stain of gingival epithelium; C ligament; 10 stain of gingival epithelium; E and F PDL-5 and PDL-4, respectively, staining ofperiodontal ligament. Note that PDL-1 and PDL-10 have similar staining characteristics and strongly stain both cell and matrix materials in the periodontal ligament and epithelium. PDL-4 and PDL-5 stain predominately cellular elements connective tissue. Bar =10 pm. in the periodontal ligament. C alveolar bone; CT cementum; AB
Figure
=
=
=
=
=
=
formed which ertes fluid.
produced copious quantities of high titer as-
Immune-cytochemistry
Adult baboon (Papio cynocephalus) mandibles were obtained fresh at necropsy from the Southwest Foundation for Biomedicai Research, San Antonio. The mandibles were fixed in neutral buffered formalin and decalcified for 3 months in 14% EDTA + 3% DMSO to a radiographie end point.
=
=
Bovine teeth
were
processed in
a
similar fashion. Baboon
teeth, together with alveolar bone and gingiva,
were
embedded in paraffin and sectioned at a thickness of 5 to 7 µ for immunohistochemical analysis. Baboon tissue from other major organ systems were fixed in neutral buffered formalin, paraffin embedded, and sectioned in a similar fashion. Sections were stained with monoclonal antibodies followed by anti-mouse IgG avidin/biotion immunoperoxidase conjugate according to the recommendations of the
Volume 62 Number 3
DUBOIS, EDMONDSON, MILAM, WINBORN, HAY, CARNES, KORNMAN,
Table 3: Localization of Antigens in Baboon Tissues Baboon Tissues
PDL-10
+
+
+ + +
+ + +
+ +
+
+ +
+ +
+
+
+
Skeletal muscle
+ +
+ + +
Urinary Bladder Transitional epithelium
+
+
+
+
glands Excretory cells Myoepithelial cells Hair follicle epithelium Tongue Muscle cells
Epithelium Lip
Muscle Hair follicle
epithelium
+
Salivary glands Acinar cells
Myoepithelial cells Kidney Collecting tubules Thin loops of Henle
-
Testis Seminiferous tubules Uterus Glandular epithelium
Coronary artery
Tunica intima Tunica media Tunic adventitia
Thyroid
-
+
+ +
-
Follicular epithelial cells Colloid Parotid Acini Ductal epithelium
-
—
+
-
+ +
+
+
+ -
The baboon tissues noted above were formalin-fixed and stained with PDL-1 and PDL-10 via an immuno-peroxidase procedure as described in the Methods. + positive reaction; negative reaction. =
=
-
manufacturer.* Negative controls consisted of both 1) deleting the primary antibody and 2) using a chicken specific antibody to fibronectin (B3/D6) in place of the primary antibody.
Photography
Sections were photographed with an Olympus AH-2 photomicroscope at magnifications of 40 to 200 using Kodak TMAX-400 film.
Reagents All chemicals were of reagent grade. Hybridomas and/or monoclonal antibodies to fibronectin, vitronectin, and collagene I and II were obtained from, respectively, the American Type Culture Collection, Telios Pharmaceuticals (San Diego, CA), the Developmental Studies Hybridoma Bank (Baltimore, MD), and Dr. John Stuart (VA Medical Center,
Memphis, TN). #Vecta
Stain,
Vector
Laboratories, Burlingame,
Isolation of Monoclonal Antibodies Reactive with Periodontal Ligament (PDL) Cells As described in the methods section, initial screening via ELISA was carried out with formalin fixed PDL cells such that anti-cell surface monoclonal antibodies would be selected. The initial ELISA screening revealed 41 positive wells out of 720 total (approximately 90% of these wells contained hybrids). In order to confirm the specificity of the anti-PDL hybridomas, a second screening was performed by immunofluorescence staining of bovine PDL cells as well as a series of formalin fixed human cells (HeLla, SV-IMR-90, HepG2, HuTu, OS, RD, A549, and a human chondrosarcoma). Nine hybridomas that secreted monoclonal antibodies reactive only with cultured bovine PDL cells were cloned by limiting dilution and were again retested for reactivity to bovine PDL cells by immunofluorescence. The clones were designated PDL-1, etc.; note that PDL-1 and PDL-2 are subclones. Clone PDL1-10 has been submitted to the American Type Culture Collection, Rockville, MD. Western Blot
CA.
Analysis
tissue was collected, homoTissuemizer** and clarified by centrifugádon. SDS-PAGE of PDL extracts was carried out on 7.5% Polyacrylamide gels which employed a 4.5% stacking gel. Western blotting was carried out with equipment and reagents supplied by Bio-Rad Laboratories, Richmond, CA.19 Gels were electroblotted onto nitrocellulose and incubated overnight with ascites fluid at a dilution of 1:5000. Antibody reactive bands were developed with an alkaline Phosphatase conjugate. While PDL-6 and PDL-7 did not produce distinct bands, Western blotting did reveal bands of antigen reactive with the other monoclonal antibodies studied (Table 1).
Bovine
periodontal ligament
genized with
-
193
RESULTS
PDL-1
Skin Dermis Sebaceous
KLEBE
a
Localization of Antigens in Periodontal and Other Tissues The decalcified baboon and bovine periodontal tissues were tested with each antibody using the avidin/biotin immunoperoxidase staining procedure. Examination of bovine dental tissues revealed differences in the specificity of the monoclonal antibodies (Table 2, Fig. 1). PDL-3 and PDL8, which originally reacted with formalin fixed cultured bovine PDL cells, did not react with the bovine PDL his-
sections (Table 2, Fig. 1). Only PDL-1, PDL-2, and PDL-10 reacted with baboon tissue and were found to display strong reactivity with the margin of the periodontal ligament (Table 3, Fig. 2). In addition to reaction with the periodontal ligament, the monoclonal antibodies isolated
tological
**Tekmar
Co., Cincinnati, OH.
194
J Periodontol March 1991
PERIODONTAL LIGAMENT CELLS
Figure 2: Distribution of antigen recognized by PDL-10 in various baboon tissues. A: Hair follicle (1 follicle sheath cells; 2 investing connective tissue); B: Thyroid follicles (1 epithelium); C: Lip (1 striated muscle); D: Kidney (I collecting tubules; 2 thin segment of the loop of Henle); E: Minor salivary gland (1 Acinar cells; 2 ducts); F: Coronary artery (1 tunica intima; 2 tunica media). Bar 10 =
=
=
µ .
=
=
=
=
=
=
=
=
Volume 62 Number 3
DUBOIS, EDMONDSON, MILAM, WINBORN, HAY, CARNES, KORNMAN,
KLEBE
195
3: Localization of extracellular matrix molecules. Periodontal tissues were reacted with monoclonal antibodies specific for the following molecules: A and C: vitronectin; and D: fibronectin. A and demonstrate the localization of extracellular matrix molecules in the periodontal ligament while C and D show staining of the functional epithelia. C connective tissue. Note that antialveolar bone; CT cementum; AB vitronectin reacts strongest in the cementoblasts and also stains epithelia. Anti-fibronectin strongly stains both cellular and extracellular components of the periodontal ligament and matrix surrounding cells in the epithelia.
Figure
=
=
=
found to cross-react with several epithelial elements in other tissues (Table 3, Fig. 2). Via the ELISA procedure, all of the anti-PDL monoclonal antibodies were found to be negative for reaction to native bovine fibronectin, bovine vitronectin, and bovine collagen I. were
DISCUSSION This study provides characteristics of a series of monoclonal antibodies that should be useful in the analysis of the periodontal ligament. The monoclonal antibodies reported here recognize cell surface antigens of specific cells in the soft tissues surrounding the tooth (Fig. 1, Table 2); such monoclonal antibodies should be valuable in analyzing cell population dynamics during both the development and pathological alteration of the periodontal ligament. Based on the molecular weights of the antigens recognized and the patterns of cell surface staining revealed by immunofluorescence (Table 1), the monoclonal antibodies studied here appear to recognize different antigens in the periodontal ligament. PDL-1, PDL-2, and PDL-10 stained similar cell types in both bovine and baboon sections. In addition to reaction with cells in the periodontal ligament, the monoclonal antibodies studied here were also
found to react with a variety of structures in other tissues (Figs. 2 and 3, Table 3). For example, both PDL-1 and PDL-10 reacted strongly with the collecting tubules of the kidney, dermis, hair follicle epithelia, thyroid follicular epithelia, etc. (Table 3). Hamburger and co-workers20 have described two monoclonal antibodies prepared against human foreskin keratinocytes that display a pattern of crossreactivity against epithelial derivatives that is similar to that reported in Table 3 of the present report. In the future, it will be important to isolate monoclonal antibodies against many other cell types in the body. Since a great deal of cross-reactivity occurs between cells obtained from different tissues20 (Table 3), it should be possible to obtain monoclonal antibodies to a wide variety of cell types by preparing monoclonal antibodies to only a fraction of the cell types in the body. For example, both our study and that of Hamburger, et al.20 provide monoclonal antibodies to kidney tubules even though kidney cells were not used as an antigen in either study. Since extracellular matrix molecules represent major components of the cell surface,21 we examined the possibility that our monoclonal antibodies reacted with either fibronectin, vitronectin, or collagen by the use of monoclonal antibody based ELISA procedures. Our monoclonal
196
antibodies to bovine periodontal ligament cells were found not to cross-react with fibronectin, vitronectin, and collagen I of bovine origin. Due to the selection for monoclonal antibodies that recognize formalin-fixed cultured cells, the antibodies reported here recognize cell surface antigens which are preserved in formalin-fixed, paraffin embedded specimens. Such antibodies should be useful in clinical diagnosis since they can be used to identify specific cell types in formalin-fixed specimens. In addition, the reactivity of this group of antibodies to cell surface structures should permit their use in isolating living periodontal ligament cells by the use of panning techniques.7 PDL-10 has been submitted to the American Type Culture Collection and should be an important new tool for workers interested in the periodontal
ligament.
Acknowledgments This study was supported, in part, by NIH grants DE09144, AG06872, DE07736, DE00152, and NSF grant DCB8617256. We would like to thank Mr. Ian Lyn for aid in the ELISA determinations.
REFERENCES 1. Blumenthal . The use of collagen membranes to guide regeneration of new connective tissue attachment in dogs. J Periodontol 1988;59:830. 2. Pitaru S, Tal , Soldinger M, Grosskopf A, Noff M. Partial regeneration of periodontal tissues using collagen barriers. J Periodontol
1988;59:380.
3.
4.
J Periodontol March 1991
PERIODONTAL LIGAMENT CELLS
Magnusson I, Batich C, Collins BR. New attachment formation following controlled tissue regeneration using biodegradable membranes. / Periodontol 1988;59:1. Gottlow A, Nyman S, Karring T, Lindhe A. New attachment formation as the result of controlled tissue regeneration. / Clin Periodontol 1986;13:604.
5. Stahl S. Periodontal attachment in health and disease. J West Soc Periodontol Periodontol Abstr 1989;33:147. 6. McCulloch CA, Németh E, Lowenberg , Melcher AH. Paravascular cells in endosteal spaces of alveolar bone contribute to periodontal ligament cell populations. Anat Ree 1987;219:233.
Wood GS, Engleman E Oseroff A. Selective enrichof epidermal cell subpopulations using monoclonal antibodies. J Invest Dermatol 1983;81:1275. 8. Gown AM, Vogel AM, Hoak D, Gough F, McNutt NA. Monoclonal antibodies specific for melanocytic melanocytes. Am J Pathol 7.
Morhenn, VB, ment
1986;123:195.
9.
Bhoopat L, Turner RR, Stathopoulos E,
et al. Immunohistochemical characterization of two new monoclonal antibodies (LN-4, LN-5) reactive with human macrophage subsets and derived malignancies in B5-fixed, paraffin-embedded tissues. Blood 1988; 71:1079. 10. Cordon-Cardo C, Finstad CL, Bander NH, Melamend MR. Immunoanatomic distribution of cytostructural and tissue associated antigens in the human urinary tract. Am J Pathol 1987; 126:269. 11. Bander NH, Cordon-Cardo C, Finstad CL, et al. Immunohistologic dissection of the human kidney using monoclonal antibodies. / Urol
1985;133:502.
12. Lillie RD. H.J. Conn's Biological Stains. 9th ed. Baltimore: Williams & Wilkins; 1977;l-692. 13. Linscott's Directory of Immunological and Biological Reagents. 5th ed. Mill Valley, CA: Linscott's Directory, 1989; 1-200. 14. Knapp W, Rieper , Dorken , Schmidt RE, Stein H, Borne AEG. Towards a better definition of human leucocyte surface molecules. Immunol Today 1990;10:253. 15. Meri A, Winters WD. Cultivation of tissue and cell cultures of human skeletal and soft tissue tumors. TCA Manual 1976;2:327. 16. Schoen RC, Bentley KL, Klebe RJ. Monoclonal antibodies against human fibronectin which inhibit cell attachment. Hybridoma 1982;1:99. 17. Klebe RJ, Hanson DP, Harriss JV, Bentley KL. Uptake by cells of nucleic acids promoted by compounds sharing the pleiotropic effects of poly (ethylene glycol). Terat Mutagen Carcino 1986;6:245. 18. Klebe RJ, Bentley KL. Chemically mediated cell fusion. In: Hybridoma Formation. Bartal AH, Hirshaut Y, eds. Clifton, NJ: Humana Press; 1987:77-96. 19. Protein and Nucleic Acid Blotting and Immunochemical Detection {Bulletin PNA) Richmond, CA: Bio-Rad Laboratories; 1989:1-200. 20. Hamburger AW, Reid YA, Pelle , et al. Isolation and characterization of a monoclonal antibody specific for epithelial cells. Cancer Res 1985;45:783. 21. Ruoslahti E. Fibronectin and its receptors. Ann Rev Biochem
1988;57:375. Send reprint requests to: Dr. Robert J. Klebe, Department of Cellular and Structural Biology, University of Texas Health Science Center, San Antonio, TX 78284-7762. Accepted for publication August 23, 1990.
Monoclonal Antibodies to Periodontal Ligament Cells William T. DuBois, * Jeffery Edmondson,f Stephen B. Milam,T William B. Winborn,f Robert Hay, * David L. Carnes,s Kenneth S. Kornman, * and Robert J. Klebef
Ten mouse monoclonal antibodies were prepared against cultured bovine periodontal ligament cells to be used as reagents for the study of periodontal disease and wound healing. Using standard immunohistochemical methods, these antibodies were found to recognize cell surface antigens in formalin-fixed bovine periodontium. Three of the 10 monoclonal antibodies (i.e., PDL-1, PDL-2, and PDL-10) cross-reacted with cells found in primate periodontium. While the isolated monoclonal antibodies appeared to distinguish subpopulations of cells located in the supporting tissues of teeth, immunohistological examination of other organs (dermis, kidney, skeletal muscle, thyroid, and parotid gland) indicated that a number of cell types of mesenchymal origin share an antigen(s) found on periodontal cells. The monoclonal antibodies described in this report should prove to be useful in studies of periodontal disease and guided tissue regeneration by providing both analytical reagents and immunochemical methods for isolating selected cell populations of the periodontium. J Periodontol, 1991; 62:190-196.
Key Words: Monoclonal antibodies; wound healing; guided tissue regeneration; periodontal diseases; periodontium/anatomy; periodontium/histology.
Periodontal disease results in the destruction of the supporting tissue surrounding the tooth and can lead to loss of function or extraction of the tooth affected. Current therapy is designed to prevent further destruction of normal tissue and enhance the reparative process. One approach, termed guided tissue regeneration, is designed to specifically recruit cells from the periodontal ligament to repair the damaged area with new cementum, ligament, and bone.1"4 The evaluation of this therapy is empirical because of the difficulty in determining the identity of the cell populations which participate in the wound healing process.5'6 Monoclonal antibodies, which are specific for distinct cell types, would be of great benefit in the analysis of periodontal disease. The ability of monoclonal antibodies to react with specific cell types within a mixed population provides a powerful tool to isolate and identify selected cell types.7 Monoclonal antibodies are important reagents in the study of cell population dynamics during developmental and pathological processes.8-" Monoclonal antibodies can now be used to demonstrate cell populations that were not detected by routine histological procedures that depended on chemical dyes.12 In the case of the lymphocytic series, monoclonal antibody methods can detect at least 11 types of T*Department of Periodontics, University of Texas
Health Science TX. ^Department of Cellular and Structural Biology. §Department of Endodontics. 'American Type Culture Collection, Rockville, MD.
San
Antonio,
Center,
cells13
as well as several other white blood cell subgroups13,14 that were only theoretical entities prior to the advent of monoclonal antibody technology. While monoclonal antibodies have been prepared to many cell types found in blood,13-14 a comprehensive series of monocloncal antibodies have not been developed for the analysis of cell population dynamics in most other tissues. The goal of this study has been to produce monoclonal antibodies that recognize cell types of interest in oral biology.
MATERIALS AND METHODS
Cell Cultivation Cell lines were maintained with Dulbecco's medium containing 10% newborn bovine calf serum plus 100 units/ml penicillin and 100 micrograms/ml streptomycin (hereafter referred to as culture medium). During the cell fusion procedure and selection for hybrid clones, medium containing 10% fetal bovine calf serum and/or Hybri-Max CPSR-311 were
employed. mouse myeloma cell line, FOX-NY,
and other percell lines were obtained from the American Type Culture Collection. Bovine periodontal ligament (PDL) cells were adapted to culture by placing minced pieces of bovine periodontal ligament in culture medium. The periodontal ligament specimens were obtained from freshly extracted The
manent
'Sigma
Chemical
Co., St. Louis, MO.
Volume 62 Number 3
DUBOIS, EDMONDSON, MILAM, WINBORN, HAY, CARNES, KORNMAN,
posterior teeth approximately 15 mm apical to the gingival crest. After several days, cells were observed migrating away from attached PDL fragments. Early cultures contained several morphologically distinct cell types while established cultures that were used in monoclonal antibody preparation contained mainly fibroblasts with many branched processes. Immediately prior to use in immunization, the cells were trypsinized and washed with sterile saline. The following human cell lines were obtained from the American Type Culture Collection, HeLa, HepG2, HuTu, RD, and A549. SV40-transformed IMR-90 cells were obtained from the Coriell Institute for Medical Research, Camden, NJ. A chondrosarcoma cell line of human origin15
was
obtained from Dr. Wendell Winters of this institution.
Monoclonal Antibody Production Balb-C mice were immunized by intraperitoneal injection with 107 trypsinized cultured periodontal ligament cells in Freund's complete adjuvant each week for 4 to 6 weeks. The animals were sacrificed and their spleens harvested for the fusion procedure. Spleen cells were fused with FOXNY myeloma cells in the presence of 50% polyethylene glycol" as described previously.16 Hybridoma clones were selected with hypoxanthine:aminopterin:thymidine (HAT) media.17'18 Ten to 14 days post-fusion, culture supernatants were initially screened by ELISA for antibody activity to formalin fixed bovine PDL cells. Cells in positive wells were cloned by limiting dilution and the clones isolated were re-screened by immunofluorescence. Hybridoma cell lines in culture media containing 10% DMSO were frozen in liquid nitrogen. PDL-10 has been submitted to the American Type Culture Collection.
ELISA Procedure The ELISA procedure employed at this stage of hybridoma selection has been described in detail previously.16 In brief, bovine PDL cells were grown to confluence in 96-well plates and fixed with neutral-buffered-formalin. Wells were blocked with 1% bovine serum albumin in Tween-PBS (0.05% Tween-20 in 0.15 M NaCl + 0.05 M sodium phosphate, pH 7.2) for 15 minutes and washed. One hundred microliters of culture supernatant were added to each test well and incubated at 37° for 30 minutes. Positive wells were identified following reaction with rabbit anti-mouse-IgG peroxidase conjugate and o-dianisidine peroxidase substrate. As a positive control, polyclonal serum collected from the immunized mouse at the time of sacrifice was employed; the negative control was media conditioned by the FOX-NY parental cell line. Immunofluorescence
Assay
Following cloning by limiting dilution, hybridomas were retested by immunofluorescence as follows. Bovine PDL cells were grown on acid washed glass slides and fixed ^Gibco, Grand Island,
NY.
Table 1:
Antigenic Recognition by
Monoclonal PDL1 PDL2 PDL3 PDL4 PDL5 PDL8 PDL9 PDL10
191
PDL Monoclonal Antibodies
Immunofluorescence Pattern Fibrillar Fibrillar diffuse
Antibody
KLEBE
Punctate, diffuse
Molecular Weight Estimation (kD) 63 63 163; 108; 73 63
Diffuse fibrillar
63; 57 73
109; 63; 57 100; 73
Perinuclear; punctate Strong fibrillar; punctate -
Immunofluorescence patterns were obtained by reacting cultured bovine PDL cells with ascites fluid at a dilution of 1:500 in PBS followed by FITC anti-mouse IgG conjugate at a 1:20 dilution. The appearance of antigen on the cell surface is given above. Note that different patterns are generated by each of the antibodies indicating that a different antigenic determinant is recognized (note that PDL1 and PDL2 are subclones). Molecular weight estimation was carried out by Western blotting as described in text.
Table 2: Localization of Antigens in the PDL Monoclonal Cementum/Cemento- CementoExtracellular Dentin blasts Fibroblasts Matrix cytes Antibody PDL1 PDL2 PDL4 PDL5 PDL9 PDL10
-
-
+ + +
+
+ +
(±) (±) (±)
+ +
-++(+)+ + (±) -
+
+
+
Gingival
Connective Junctional Osteoblasts Osteocytes Tissue Epithelium PDL1 PDL2 PDL4 PDL5 PDL9 PDL10
+ +
+ +
+
+ + + +
+ + +
-
Gingival Epithelium
+ +
+ +
+
+
-
Bovine periodontal tissue was decalcified, embedded, sectioned, and reacted with an avidin-biotin immunoperoxidase reagent as described in the text. Note the similarity between the distribution of the antigens recognized by PDL1, PDL2, and PDL10 with vitronectin. PDL5 and PDL9 reacted with discrete cells within and immediately surrounding the periodontal
ligament. ( + positive reaction; negative reaction; ( ± ) cells positive while other cells are negative). =
=
=
some
-
with neutral buffered formalin. The fixed cells were incubated with hybridoma culture supernatant, washed, and stained with a fluorescein-conjugated goat anti-mouse IgG. Epifluorescence microscopy revealed several hybridomas that produced antibodies which reacted with cell surface antigens on the formalin fixed bovine PDL cells that did not react with a panel of human cell lines (HeLà, HepG2, HuTu, RD, A549, SV-IMR-90, and a human
chondrosarcoma). Ascites Fluid Production Those hybridomas that were
positive by immunofluoresselected for further study. One to ten million hybridoma cells were injected intraperitoneally into pristane-primed Balb-C mice. One to 2 weeks later tumors had cence were
192
J Periodontol March 1991
PERIODONTAL LIGAMENT CELLS
1: Reaction ofperiodontal tissues with anti-periodontal ligament monoclonal antibodies. Bovine periodontal tissues were reacted with monoclonal antibodies conjugated with avidinlbiotin as described in the text. PDL-1 stain of periodontal Sites of positive antibody reaction are indicated by arrows (unlabeled). A PDLPDL-10 stain of periodontal ligament; D PDL-1 stain of gingival epithelium; C ligament; 10 stain of gingival epithelium; E and F PDL-5 and PDL-4, respectively, staining ofperiodontal ligament. Note that PDL-1 and PDL-10 have similar staining characteristics and strongly stain both cell and matrix materials in the periodontal ligament and epithelium. PDL-4 and PDL-5 stain predominately cellular elements connective tissue. Bar =10 pm. in the periodontal ligament. C alveolar bone; CT cementum; AB
Figure
=
=
=
=
=
=
formed which ertes fluid.
produced copious quantities of high titer as-
Immune-cytochemistry
Adult baboon (Papio cynocephalus) mandibles were obtained fresh at necropsy from the Southwest Foundation for Biomedicai Research, San Antonio. The mandibles were fixed in neutral buffered formalin and decalcified for 3 months in 14% EDTA + 3% DMSO to a radiographie end point.
=
=
Bovine teeth
were
processed in
a
similar fashion. Baboon
teeth, together with alveolar bone and gingiva,
were
embedded in paraffin and sectioned at a thickness of 5 to 7 µ for immunohistochemical analysis. Baboon tissue from other major organ systems were fixed in neutral buffered formalin, paraffin embedded, and sectioned in a similar fashion. Sections were stained with monoclonal antibodies followed by anti-mouse IgG avidin/biotion immunoperoxidase conjugate according to the recommendations of the
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DUBOIS, EDMONDSON, MILAM, WINBORN, HAY, CARNES, KORNMAN,
Table 3: Localization of Antigens in Baboon Tissues Baboon Tissues
PDL-10
+
+
+ + +
+ + +
+ +
+
+ +
+ +
+
+
+
Skeletal muscle
+ +
+ + +
Urinary Bladder Transitional epithelium
+
+
+
+
glands Excretory cells Myoepithelial cells Hair follicle epithelium Tongue Muscle cells
Epithelium Lip
Muscle Hair follicle
epithelium
+
Salivary glands Acinar cells
Myoepithelial cells Kidney Collecting tubules Thin loops of Henle
-
Testis Seminiferous tubules Uterus Glandular epithelium
Coronary artery
Tunica intima Tunica media Tunic adventitia
Thyroid
-
+
+ +
-
Follicular epithelial cells Colloid Parotid Acini Ductal epithelium
-
—
+
-
+ +
+
+
+ -
The baboon tissues noted above were formalin-fixed and stained with PDL-1 and PDL-10 via an immuno-peroxidase procedure as described in the Methods. + positive reaction; negative reaction. =
=
-
manufacturer.* Negative controls consisted of both 1) deleting the primary antibody and 2) using a chicken specific antibody to fibronectin (B3/D6) in place of the primary antibody.
Photography
Sections were photographed with an Olympus AH-2 photomicroscope at magnifications of 40 to 200 using Kodak TMAX-400 film.
Reagents All chemicals were of reagent grade. Hybridomas and/or monoclonal antibodies to fibronectin, vitronectin, and collagene I and II were obtained from, respectively, the American Type Culture Collection, Telios Pharmaceuticals (San Diego, CA), the Developmental Studies Hybridoma Bank (Baltimore, MD), and Dr. John Stuart (VA Medical Center,
Memphis, TN). #Vecta
Stain,
Vector
Laboratories, Burlingame,
Isolation of Monoclonal Antibodies Reactive with Periodontal Ligament (PDL) Cells As described in the methods section, initial screening via ELISA was carried out with formalin fixed PDL cells such that anti-cell surface monoclonal antibodies would be selected. The initial ELISA screening revealed 41 positive wells out of 720 total (approximately 90% of these wells contained hybrids). In order to confirm the specificity of the anti-PDL hybridomas, a second screening was performed by immunofluorescence staining of bovine PDL cells as well as a series of formalin fixed human cells (HeLla, SV-IMR-90, HepG2, HuTu, OS, RD, A549, and a human chondrosarcoma). Nine hybridomas that secreted monoclonal antibodies reactive only with cultured bovine PDL cells were cloned by limiting dilution and were again retested for reactivity to bovine PDL cells by immunofluorescence. The clones were designated PDL-1, etc.; note that PDL-1 and PDL-2 are subclones. Clone PDL1-10 has been submitted to the American Type Culture Collection, Rockville, MD. Western Blot
CA.
Analysis
tissue was collected, homoTissuemizer** and clarified by centrifugádon. SDS-PAGE of PDL extracts was carried out on 7.5% Polyacrylamide gels which employed a 4.5% stacking gel. Western blotting was carried out with equipment and reagents supplied by Bio-Rad Laboratories, Richmond, CA.19 Gels were electroblotted onto nitrocellulose and incubated overnight with ascites fluid at a dilution of 1:5000. Antibody reactive bands were developed with an alkaline Phosphatase conjugate. While PDL-6 and PDL-7 did not produce distinct bands, Western blotting did reveal bands of antigen reactive with the other monoclonal antibodies studied (Table 1).
Bovine
periodontal ligament
genized with
-
193
RESULTS
PDL-1
Skin Dermis Sebaceous
KLEBE
a
Localization of Antigens in Periodontal and Other Tissues The decalcified baboon and bovine periodontal tissues were tested with each antibody using the avidin/biotin immunoperoxidase staining procedure. Examination of bovine dental tissues revealed differences in the specificity of the monoclonal antibodies (Table 2, Fig. 1). PDL-3 and PDL8, which originally reacted with formalin fixed cultured bovine PDL cells, did not react with the bovine PDL his-
sections (Table 2, Fig. 1). Only PDL-1, PDL-2, and PDL-10 reacted with baboon tissue and were found to display strong reactivity with the margin of the periodontal ligament (Table 3, Fig. 2). In addition to reaction with the periodontal ligament, the monoclonal antibodies isolated
tological
**Tekmar
Co., Cincinnati, OH.
194
J Periodontol March 1991
PERIODONTAL LIGAMENT CELLS
Figure 2: Distribution of antigen recognized by PDL-10 in various baboon tissues. A: Hair follicle (1 follicle sheath cells; 2 investing connective tissue); B: Thyroid follicles (1 epithelium); C: Lip (1 striated muscle); D: Kidney (I collecting tubules; 2 thin segment of the loop of Henle); E: Minor salivary gland (1 Acinar cells; 2 ducts); F: Coronary artery (1 tunica intima; 2 tunica media). Bar 10 =
=
=
µ .
=
=
=
=
=
=
=
=
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DUBOIS, EDMONDSON, MILAM, WINBORN, HAY, CARNES, KORNMAN,
KLEBE
195
3: Localization of extracellular matrix molecules. Periodontal tissues were reacted with monoclonal antibodies specific for the following molecules: A and C: vitronectin; and D: fibronectin. A and demonstrate the localization of extracellular matrix molecules in the periodontal ligament while C and D show staining of the functional epithelia. C connective tissue. Note that antialveolar bone; CT cementum; AB vitronectin reacts strongest in the cementoblasts and also stains epithelia. Anti-fibronectin strongly stains both cellular and extracellular components of the periodontal ligament and matrix surrounding cells in the epithelia.
Figure
=
=
=
found to cross-react with several epithelial elements in other tissues (Table 3, Fig. 2). Via the ELISA procedure, all of the anti-PDL monoclonal antibodies were found to be negative for reaction to native bovine fibronectin, bovine vitronectin, and bovine collagen I. were
DISCUSSION This study provides characteristics of a series of monoclonal antibodies that should be useful in the analysis of the periodontal ligament. The monoclonal antibodies reported here recognize cell surface antigens of specific cells in the soft tissues surrounding the tooth (Fig. 1, Table 2); such monoclonal antibodies should be valuable in analyzing cell population dynamics during both the development and pathological alteration of the periodontal ligament. Based on the molecular weights of the antigens recognized and the patterns of cell surface staining revealed by immunofluorescence (Table 1), the monoclonal antibodies studied here appear to recognize different antigens in the periodontal ligament. PDL-1, PDL-2, and PDL-10 stained similar cell types in both bovine and baboon sections. In addition to reaction with cells in the periodontal ligament, the monoclonal antibodies studied here were also
found to react with a variety of structures in other tissues (Figs. 2 and 3, Table 3). For example, both PDL-1 and PDL-10 reacted strongly with the collecting tubules of the kidney, dermis, hair follicle epithelia, thyroid follicular epithelia, etc. (Table 3). Hamburger and co-workers20 have described two monoclonal antibodies prepared against human foreskin keratinocytes that display a pattern of crossreactivity against epithelial derivatives that is similar to that reported in Table 3 of the present report. In the future, it will be important to isolate monoclonal antibodies against many other cell types in the body. Since a great deal of cross-reactivity occurs between cells obtained from different tissues20 (Table 3), it should be possible to obtain monoclonal antibodies to a wide variety of cell types by preparing monoclonal antibodies to only a fraction of the cell types in the body. For example, both our study and that of Hamburger, et al.20 provide monoclonal antibodies to kidney tubules even though kidney cells were not used as an antigen in either study. Since extracellular matrix molecules represent major components of the cell surface,21 we examined the possibility that our monoclonal antibodies reacted with either fibronectin, vitronectin, or collagen by the use of monoclonal antibody based ELISA procedures. Our monoclonal
196
antibodies to bovine periodontal ligament cells were found not to cross-react with fibronectin, vitronectin, and collagen I of bovine origin. Due to the selection for monoclonal antibodies that recognize formalin-fixed cultured cells, the antibodies reported here recognize cell surface antigens which are preserved in formalin-fixed, paraffin embedded specimens. Such antibodies should be useful in clinical diagnosis since they can be used to identify specific cell types in formalin-fixed specimens. In addition, the reactivity of this group of antibodies to cell surface structures should permit their use in isolating living periodontal ligament cells by the use of panning techniques.7 PDL-10 has been submitted to the American Type Culture Collection and should be an important new tool for workers interested in the periodontal
ligament.
Acknowledgments This study was supported, in part, by NIH grants DE09144, AG06872, DE07736, DE00152, and NSF grant DCB8617256. We would like to thank Mr. Ian Lyn for aid in the ELISA determinations.
REFERENCES 1. Blumenthal . The use of collagen membranes to guide regeneration of new connective tissue attachment in dogs. J Periodontol 1988;59:830. 2. Pitaru S, Tal , Soldinger M, Grosskopf A, Noff M. Partial regeneration of periodontal tissues using collagen barriers. J Periodontol
1988;59:380.
3.
4.
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PERIODONTAL LIGAMENT CELLS
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Wood GS, Engleman E Oseroff A. Selective enrichof epidermal cell subpopulations using monoclonal antibodies. J Invest Dermatol 1983;81:1275. 8. Gown AM, Vogel AM, Hoak D, Gough F, McNutt NA. Monoclonal antibodies specific for melanocytic melanocytes. Am J Pathol 7.
Morhenn, VB, ment
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9.
Bhoopat L, Turner RR, Stathopoulos E,
et al. Immunohistochemical characterization of two new monoclonal antibodies (LN-4, LN-5) reactive with human macrophage subsets and derived malignancies in B5-fixed, paraffin-embedded tissues. Blood 1988; 71:1079. 10. Cordon-Cardo C, Finstad CL, Bander NH, Melamend MR. Immunoanatomic distribution of cytostructural and tissue associated antigens in the human urinary tract. Am J Pathol 1987; 126:269. 11. Bander NH, Cordon-Cardo C, Finstad CL, et al. Immunohistologic dissection of the human kidney using monoclonal antibodies. / Urol
1985;133:502.
12. Lillie RD. H.J. Conn's Biological Stains. 9th ed. Baltimore: Williams & Wilkins; 1977;l-692. 13. Linscott's Directory of Immunological and Biological Reagents. 5th ed. Mill Valley, CA: Linscott's Directory, 1989; 1-200. 14. Knapp W, Rieper , Dorken , Schmidt RE, Stein H, Borne AEG. Towards a better definition of human leucocyte surface molecules. Immunol Today 1990;10:253. 15. Meri A, Winters WD. Cultivation of tissue and cell cultures of human skeletal and soft tissue tumors. TCA Manual 1976;2:327. 16. Schoen RC, Bentley KL, Klebe RJ. Monoclonal antibodies against human fibronectin which inhibit cell attachment. Hybridoma 1982;1:99. 17. Klebe RJ, Hanson DP, Harriss JV, Bentley KL. Uptake by cells of nucleic acids promoted by compounds sharing the pleiotropic effects of poly (ethylene glycol). Terat Mutagen Carcino 1986;6:245. 18. Klebe RJ, Bentley KL. Chemically mediated cell fusion. In: Hybridoma Formation. Bartal AH, Hirshaut Y, eds. Clifton, NJ: Humana Press; 1987:77-96. 19. Protein and Nucleic Acid Blotting and Immunochemical Detection {Bulletin PNA) Richmond, CA: Bio-Rad Laboratories; 1989:1-200. 20. Hamburger AW, Reid YA, Pelle , et al. Isolation and characterization of a monoclonal antibody specific for epithelial cells. Cancer Res 1985;45:783. 21. Ruoslahti E. Fibronectin and its receptors. Ann Rev Biochem
1988;57:375. Send reprint requests to: Dr. Robert J. Klebe, Department of Cellular and Structural Biology, University of Texas Health Science Center, San Antonio, TX 78284-7762. Accepted for publication August 23, 1990.
