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Lack of lymphocyte stimulation by root canal products
SCIENTIFIC ARTICLES
Lack of l y m p h o c y t e s t i m u l a t i o n by root canal p r o d u c t s Paul D. Eleazer, DDS; Paul A. Farber, DDS, PhD; and Samuel Seltzer, DDS, Philadelphia
The immune system is generally thought to consist of two elements. When foreign material (antigen) is introduced into the body and is recognized as such, antibody formation may be initiated or defense cells may be activated. Antigen may also stimulate both elements. The antibody response is called humoral because of the widespread distribution of these molecules that bind with antigen. Cells that participate in this response are termed Blymphocytes (B-cells) because of their development in bone marrow. In birds, these B-cells mature in a gutassociated organ known as the bursa of FabriciusJ The area of cell maturation in higher animals has not yet been clearly defined. Once activated B-cells divide, they become plasma cells and produce antibody specific for the activating antigen3 The activation of defense cells by antigen is known as the cell-mediated immune response. It is characterized by the production of lymphokines by T-lymphocytes (T-cells), so called because of their maturation in the thymus. 2 Lymphokines are substances that stimulate cell division, inhibit migration of macrophages and other defense cells, attract circulating defense cells, and otherwise help to eliminate the material recognized as foreign. 3 Interaction between humoral im-
388
mune processes and cell-mediated immune processes evidently exists. Claman and associates 4 have shown that in the presence of antigen, T-cells enhance the formation of plasma cells from thymus-independent lymphocytes. The laboratory study of cellmediated immunity has included the use of tissue culture and radioassay techniques. ~ One commonly used method involves measurement of radioactive thymidine uptake by cells to determine the amount of D N A synthesis and, therefore, the amount of cell division. By beginning with cultures containing equal numbers of cells, the stimulation potentials can be determined for the different antigenic substances capable of causing this cell change. 2 Lymphocyte transformation is the term used to describe the in vitro reversible differentiation of small lymphocytes into larger lymphoblasts. This transformation involves nuclear enlargement, appearance of nucleoli, increased cytoplasmic volume, and increased basophilia. 6 Biochemical analyses of lymphoblasts show an increase in R N A metabolism. 7 The increased R N A metabolism was confirmed by the use of the blocking agent, actinomycin D, which stops lymphocyte transformation, s The transformational activities culminate in D N A synthesis and mitosis, produc-
ing more cells to combat the antigen2 A number of different agents are responsible for lymphocyte transformation in vitro, many of which are proved in vitro antigens. Phytohemagglutinin ( P H A ) , the first agent used in vitro, is an extract of the red kidney bean (Phaseolus vulgaris)J ~ This extract produces almost 100% stimulation of small lymphocytes. It is commonly used as a control to determine the potential of a lymphocyte culture to be stimulated. Many other substances also stimulate this lymphocyte reaction. Among these are the allergens pokeweed, 1~ ragweed 14 and grass pollen. 1~ Also, various tissue antigens, such as homologous lymphocytes, 16 macrophages, 17 or platelets TM can initiate the phenomenon. Other tissue antigens, such as red blood cells of sheep can stimulate the change. 19 Heat-killed microorganisms, 2~ cell-free filtrates of Staphylococcus aureaus cultures, -02 and streptolysin S (an antigenic product of group A, hemolytic streptococci) 23 also stimulate lymphocyte cultures. Viruses, such as herpes simplex and Vaccinia also induce blastogenesis of in vitro lymphocytes. 2~ Endotoxin, a substance associated with the cell walls of m a n y gramnegative microorganisms, elicits lymphocyte transformation when complement is present. 25-27 After enzymatic cleavage of the lipopolysaccharide
JOURNAL OF ENDODONTICS ! VOL 1, NO 12, DECEMBER 1975
endotoxin, both the lipid and the polysaccharide fractions stimulate the lymphocytes. 28 Endotoxin has antigenic properties in vivo as well. -~9 On the basis of reported research, there is a reasonable possibility that lymphocyte transformation is at least a part of the natural immune defense mechanism, Tuberculin-induced lymphocyte transformation was first demonstrated in tuberculin-sensitized individuals,a~ A more recent study shows that the process of transformation activates a phosphate metabolism sequence that facilitates globulin secretion. :~1 There are a number of diseases in which a defective in vitro lymphocyte response is associated with a possible in vivo immune disorder. These inelude tuberculosis,31 sarcoidosis, 32 Hodgkin's disease, 3,~ chronic lymphocytic leukemia, zo chronic rubella, 34 and SjSgren's syndrome, a5 Ataxiatelangiectasis, a disease of children that often includes an impaired immune response, also produces a diminished lymphocyte response. ~6-37 Research findings in some dental diseases lead to the postulation that an immunologic phenomenon may be one of the etiologic factors. In patients with high caries indexes, a higher level of circulating antibodies is present against seven individual carlogenie Streptococcus species and Lactobacillus acidophilus. 3s Antibodies against Bacteroides melaninogenicus, a common inhabitant of the oral cavity, were found in all patients tested. 39 Celt-free filtrates of Actinomyces bovis, a frequent oral inhabitant, produced immediate hypersensitivity reactions of the skin in many patients with periodontal disease. 4~ Tissue extracts from oral aphthae produced a significantly greater degree of lymphocyte transformation than did normal controls. 41 Snyderman42 has proposed that the mechanism of periodontal disease in-
volves a penetration of the gingival epithelium by microorganisms from plaque colonies. These microorganisms elicited antibody formation in regional lymph nodes and in the local tissue. On introduction of more antigen into the system, an activation of this immune system produced biologically active products capable of increasing vascular permeability, contracting smooth muscle, and eliciting chemotaxis of neutrophils. This inflammation produced tissue-damaging enzymes, which allowed influx of more antigens. A recent study by Horton, Leikin and Oppenheim 4a demonstrated that autologous plaque and saliva induced more lymphocyte transformation in patients with severe periodontal disease than in patients with gingivitis or moderate periodontitis, or in normal controls. The greatest degree of stimulation resulted from the patient's own plaque and saliva. These findings are not in total agreement with those of Ivanyi and Lehner. 44 Their study showed that various bacterial ultrasonicates from patients with moderate periodontitis produced greater stimulation, compared to a depression of stimulation in patients with severe periodontitis. Research of the immunology of the dental pulp and periapical tissues has been mostly concerned with the humoral immune system. Dietz 45 has shown that filtrates of root canal contents caused immunologic reactions when injected intracutaneously. Nishida and associates 46 have noted that the use of Formalin as an adjuvant enhanced the immunologic activity of pulp antigens. Fluorescent antibody techniques have been used to demonstrate the presence and distribution of various immunoglobulins in the pulp. 47 Naidorf 4s also demonstrated pulpat immunoglobulins using Ouchterlony gel-diffusion methods. It has also been demonstrated that bacteria, experimentally inoculated into root canals, can
cause an immune response.49, 50 Rosengren 51 has shown that where bacteria were confined to the root canal the antibody response was minimal, whereas periapical infection elicited a significant response. The presence of foreign protein in the root canal causes hypergammaglobulinemia and other systemic immune manifestationsP2.53 The purpose of the current study was to use lymphoblast transformation analysis to determine if pulp tissues or the soft tissues of periapical lesions from pulpless teeth evoke a cell-mediated immune response. Materials
and
Methods
Pulps or periapical tissues were removed from 23 teeth in 21 patients undergoing treatment in the endodontic clinic at the Temple University School of Dentistry. Medical histories were obtained for all patients. Specimens were obtained from teeth in which there was no detectable communication between the pulp tissue and the oral environment. In two instances, the pulp contents were treated with cresol and formaldehyde (Formocresol) and were not removed at the initial visit. All of the periapical lesions were from teeth that had been previously treated by nonsurgical endodontic therapy. The gross presence or absence of a blood supply was used to classify pulp tissues as vital or necrotic. Tissue specimens were placed in RPMI 1640 tissue culture medium containing a 1% antimicrobial mixture of penicillin, amphotericin B (Fungizone), and streptomycin.* Cells were lysed with either freeze-thaw or ultrasonication techniques. Tissue specimens of surgical patients were also evaluated microscopically. Peripheral venous blood was obtained with a heparinized syringe. Lymphocytes were separated for cell culture with dextran* in five instances. The whole-blood culture technique of 389
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Pauly '~ was used in the remaining cases because of the smaller amount of blood required. Tissue antigen was added to cell culture tubes. Controls consisted of cell cultures with no antigen and cultures with P H A M.t A m i n i m u m of three cultures each was used for possible antigen, PHA, and antigen-free tests. The amount of lymphocyte stimulation was determined by measuring DNA synthesis. One microcurie of tritiated thymidine was added to each of the cultures four hours before harvesting. The D N A was extracted from the cells with use of trichloroacetic acid and ethanol on Millipore filters.~ Seven milliliters of toluene-Liquafluor~ mixed at 23.7:1 was added to each sample to prepare it for scintillation counting. A counting period of one minute in a scintillation spectrometer// was used for each sample. In specimens with low activity, a five-minute counting period was used. Arithmetic mean values were determined for each test group. Anaerobic and aerobic cultures were taken to assess the root canal flora in several of the patients in this study for use in a concurrent project. Different media, including prereduced anaerobically sterilized Brain Heart Infusion Agar supplemented with vitamin K and heme,~, chopped meat-glucose,# thioglycollate with 0.15% agar,# Trypticase soy broth with 0.15% agar, # and Trypticase soy broth without agar# were used to culture any microbes present. In two of the tests involving pulpless teeth, killed microbes from these cultures were checked for lymphoblast transformation potential. In one case, boiled autologous saliva was tested. In eight instances, either a 1:10 or a 1:100 di-
Fig 2--Lymphocyte stimulation by nonvital pulp products.
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Results Five of the 12 pulp specimens were classified as vital (Fig 1, 2), showing at least some evidence of blood supply when viewed by the unaided eye. Five of the 11 periapical tissue samples were found by microscopic examination to be gran~lomas (Fig 3); the others were described as cysts (Fig 4). In all 23 experiments, there was no evidence of increased lymphocyte division. There was no correlation between the results obtained and any part of the medical histories. N o additional stimulation was found when fetal calf serum was substituted for autologous serum. The use of mouse lymphocytes did not result in stimulation above the antigenfree control levels. N o differences were seen related to the freeze-thaw or ultrasonication methods of cell lysis. No significant increases were noted in any of the bacterial samples. In analyzing the data, it is important to note that a positive control ( P H A ) and a negative control (no stimulant) were used for each sample. Thus, results among patients cannot be compared, unless expressed as a percentage of the positive (or negative) control.
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Discussion The fact that no evidence of lymo
Fig 4--Lymphocyte stimulation by periapica! cystic lesions.
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JOURNAL OF ENDODONTICS ] VOL 1, NO 12, DECEMBER 1975
phocyte stimulation was found in this study is not conclusive evidence that a cell-mediated immune response is absent in all cases. Kantz and Henry 5r' have detected microorganisms in 92% of intact pulpless teeth: however, there was no way to determine in the current study how long the microbes were present or what effect they may have had on necrotic tissue. There was also no information available on the numbers of organisms in the canals. The presence of necrotic tissue that may be inaccessible to the vascular system might prevent interaction of some necessary factor or factors in the immune system. A cell mediated immune reaction may occur in a patient with periodontal disease where a hypersensitivity to the microorganism(s) has already been established. It is also possible that a cell-mediated immune response could result from repeated exposure of the damaged tissue to microorganisms, such as in patients with several teeth with pulpal infection. These possibilities have not been investigated. Another possibility is suggested by a study of Kiger, Wright and Creamer ~6 in which there was no correlation between severity, or even presence of periodontal disease, and lymphocyte stimulation reactions to various oral microorganisms. Perhaps oral bacteria are present in concentrations too low to cause a clinically significant hypersensitivity. It is also possible that some as yet undefined factors in the test system are responsible for the differences in response. Nishida and co-workers 46 have demonstrated that Formalin acts on proteins in dental pulp extracts to enhance their antigenicity. It was postulated that cross-linking between molecules occurred because of an interaction of the Formalin with free amino groups on the dental pulp proteins. Others 57.58 have also noted a similar change in protein due to f o r m a l d e 392
hyde. Therefore, from an immunologic standpoint, it would seem unwise to use Formalin-containing compounds in the body. It has been shown that intracytoplasmic deposition of irritants is required for the perpetuation of a granuloma. 59,n~Spector and Heesom, m using labeled antigen-antibody complexes, found that these complexes were sequestered in macrophages present in granulomas for relatively long periods. Killed microbes that were radio labeled were also retained by the macrophages. Microorganisms that clinically do not cause granulomatous reactions, such as S albus, were digested to a significantly greater degree than were organisms such as Mycobacterium tuberculosis, which was only 40% digested at 14 days. 6'' Thus, it is likely that intracytoplasmic deposits of foreign material, including antigenantibody complexes, partially degraded microorganisms, and endodontic filling materials, may be responsible for the perpetuation of the periapical granuloma. In such cases, surgical curettage of the area to eliminate the lesion may be well advised. This seems especially important if an antigen-host interaction is being perpetuated by the sequestered material. Burleson G3 has discovered another possible justification for removal of granulomas. He noted that leukocytes in infected granulation tissue produced a factor that inhibits phagocytosis. He produced sterile granulation tissue that lacked this antiphagocytic factor. It is not known if this factor is present in the dental granuloma in situ. An absence of lymphocyte transformation does not rule out a functioning cell-mediated immune system. Curtis and Hersh 64 recently demonstrated that one antigen (keyhole limpet hemocyanin) causes production of a significant amount of migration inhibition factor in h u m a n lymphocytes in vitro, while no other cell-mediated
immune response parameters were increased. Summary
Pulp or periapical tissues removed from 23 teeth in patients undergoing dental treatment were placed in tissue culture media. The cells were lysed by freeze-thaw and ultrasonication techniques. Lymphocytes were obtained by adding some of the patients' peripheral venous blood to the cultures. The amount of lymphocyte stimulation was determined by measuring the amount of tritiated thymidine taken up in D N A synthesis. In all 23 experiments, there was no evidence of increased lymphocyte division. These results suggest that a cell-mediated immune response is lacking in human dental pulp and periapical tissue. However, these results do not preclude the possibility of this aspect of immunity being present. '::Sigma Chemical Co., St. Louis, Mo. tGrand Island Biologicals, Grand Island, NY. ,+Millipore Corp., Bedford, Mass. w England Nuclear, Boston, Mass. //Packard Tri-Carb Spectrometer, Model 3375, Downers Grove, Ill. 82 Biologicals, Chapel Hill, NC. #Baltimore Biological Laboratory, Baltimore, Md. From a thesis submitted by Dr. Eleazer in partial fulfillment for the degree of a Master of Science at Temple University. Dr. Eleazer, formerly a graduate student in endodontics, is in private practice, Albany, Ga; Dr. Farber is associate professor of pathology; and Dr. Seltzer is professor and chairman of endodontology, Temple University School of Dentistry. Requests for reprints should be directed to Dr. Samuel Seltzer, Temple University, School of Dentistry, 3223 N Broad St, Philadelphia, Pa 19140. References
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2. Cooper. M.D., and Lawton, A.R. The development of the immune system. Sci Am 231:58 Nov 1974. 3. Gordon, B.L., and Ford, D.K. Essentials of immunology. Philadelphia, F. A. Davis Co., 1972, p 17. 4. Claman, H.N.; Chaperon, E.A.; and Triplett, R.F. Thymus-marrow cell combinations. Synergism in antibody production. Proc Soc Exp Biol Med 122:1167, 1966. 5. Ling, N.R. Lymphocyte stimulation. Amsterdam, North-Holland Co., 1968, pp 37-68. 6. Oppenheim, J.J. Immunological relevance of antigen and antigen antibody complex induced lymphocyte transformation. Ann Allergy 27:305 July 1969. 7. Cooper, H.L., and Rubin, A.D. R N A metabolism in lymphocytes stimulated by phytohemagglutinin: initial responses to phytohemagglutinin. Blood 25: 1014 June 1965. 8. Hirshhorn, K.; Bach, F.; Kolodny, R.L.; and others. Immune response and mitosis of human peripheral blood lymphocytes in vitro. Science 142:1185 Nov 23, 1963. 9. MacKinney, A.A., Jr.; Stohlman, F., Jr.; and Brecher, G. The kinetics of cell proliferation in cultures of human peripheral blood. Blood 19:349 March 1962. 10. Hungerford, D.A.; Donnelly, A.J.; Nowell, P.C.; and Beck, S. The chromosome constitution of a human phenotypic intersex. Am I Hum Genet 11:215 Sept 1959. I1. Nowell, P.C. Phytohemagglutinin: an initiator of mitosis in cultures of normal human leukocytes. Cancer Res 20: 462 May 1960. 12. Cooper, E.H., and Barkhan, P. Mitogenic activity of phytohemagglutinin. Lancet 2:210, 1961. 13. Farnes, P.; Barker, B.E.; Brownhill, L.E.; and Fanger, H. Mitogenic activity in Phytolacca americana (pokeweed). Lancet 2:1100 Nov 21, 1964. 14. Young, S.H.; Zimmerman, R.E.; and Smithwick, E.M. The in vitro response of human lymphoeytes challenged by ragweed antigen. Pediatrics 42:976 Dec 1968. 15. Weiner, S., and Brasch, J. The mitogenic effect of grass pollen extract. Med J Aust Suppl 52:148, 1965. 16. Bain, B., and Lowenstein, L. Genetic studies on the mixed leukocyte reaction. Science 145:1315 Sept 18, 1964. 17. Marshall, W.H.; Rigo, S.J.; and Melman, S. Lymphocyte transformation and mitosis in vitro initiated by homologous macrophages. An improved method
for histocompatibility testing. Lancet 1: 730 April 2, 1966. 18. Dausset, J., and Rapaport, F.T. Transplantation antigen activity of human blood platelets. Transplantation 4: 182 March 1966. 19. Johnson, G.J., and Russell, P.S. Reaction of human lymphocytes in culture to components of the medium. Nature 208:343 Oct 23, 1965. 20. Oppenheim, J.J.; Wbang, J.; and Frei, E., 3rd. Immunologic and cytogenic studies of chronic lymphocytic leukemia cells. Blood 26:121 Aug 1965. 21. Oppenheim, J J . ; Blaese, R.M.; and Waldmann, T.A. The transformation of normal and Wiskott-Aldrich lymphocytes by nonspecific leucocyte bacterial, protein, and carbohydrate stimulants, abstracted. Clin Res 17:357, 1969. 22. Ling, N.R., and Husband, E.M. Specific and nonspecific stimulation of peripheral lymphocytes. Lancet 1:363 Feb 15, 1964. 23. Hirshhorn, K.; Schreibman, R.R.; Verbo, S.; and Gruskin, R.H. The action of streptolysin S on peripheral lymphocytes of normal subjects and patients with acute rheumatic fever. Proc Natl Acad Sci 52:1151 Nov 1964. 24. Rosenberg, G.L.; Farber, P.A.; and Notkins, A.L. In vitro stimulation of sensitized lymphocytes by herpes simplex virus and vaccinia virus. Proc Natl Acad Sci 69:756 March 1972. 25. Oppenheim, J.J., and Perry, S. Effects of endotoxins on cultured leukocytes. Proc Soc Exp Biol Med 118:1014 April 1965. 26. Mergenhagen, S.E.; Synderman, R.; Gewurz, H.; and Shin, H.S. Significance of complement to the mechanism of action of endotoxin. Curr Top Microbiol Immunol 50:37, 1969. 27. Mergenhagen, S.E.; Tempel, T.R.; and Synderman, R. Immunologic reactions and periodontal inflammation. J Dent Res 49:256 March-April 1970. 28. Mergenhagen, S.E. Nature and significance of somatic antigens of oral bacteria. J Dent Res 46:46 Jan-Feb 1967. 29. Berglund, S.E.; Rizzo, A.A.; and Mergenhagen, S.E. The immune response in rabbits to bacterial somatic antigen administered via the oral mucosa. Arch Oral Biol 14:7 Jan 1969. 30. Pearmain, G.; Lycette, R.R.; and Fitzgerald, P.H. Tuberculin-induced mitosis in peripheral blood leucocytes. Lancet 1:637 March 23, 1963. 31. Heilman, D.H., and McFarland, W. Inhibition of tuberculin-induced mitogenesis in cultures of lymphocytes from
tuberculous donors. Int Arch Allergy 30: 58, 1966. 32. Hirscbhorn, K.; Schreibman, R.R.; Bach, F.H.; and Siltzbach, L.E. In-vitro studies of lymphocytes from patients with sarcoidosis and lymphoproliferative diseases. Lancet 2:842 Oct 17, 1964. 33. Hersh, E.M., and Oppenheim, J.J. Impaired in vitro lymphocyte transformation in Hodgkin's disease. New Eng J Med 273:1006 Nov 4, 1965. 34. Olson, G.B.; South, M.A.; and Good, R.A. Phytohemagglutinin unresponsiveness of lymphocytes from babies with congenital rubella. Nature 214:695, 1967. 35. Leventhal, B.G.; Waldorf, D.S.; and Talal, N. Impaired lymphocyte transformation and delayed hypersensitivity in Sj6gren's syndrome. J Clin Invest 46:1338 Aug 1967. 36. Leikin, S.L.; Bazelon, M.; and Park, K.H. In vitro lymphocyte transformation in ataxia-telangiectasia. J Pediat 68:477 March 1966. 37. Oppenheim, J.J.; Barlow, M.; Waldmann, T.A.; and Block, J.B. Impaired in vitro lymphocyte transformation in patients with ataxia-telangiectasis. Br Med J 2:330 Sept 1966. 38. Lehner, T.; Wilton, J.M.; and Ward, R.G. Serum antibodies in dental caries in man. Arch Oral Biol 15:481, 1970. 39. Courant, P.R., and Gibbons, R.J. Biochemical and immunological heterogeneity of Baeteroides melaninogenicus. Arch Oral Biol 12:1605 Dec 1967. 40. Nisengard, R.; Beutner, E.H.; and Hazen, S.P. Immunologic studies of periodontal disease. Bacterial hypersensitivity and periodontal disease. J Periodontol 39:329 Nov 1968. 41. Lehner, T. Stimulation of lymphocyte transformation by tissue homogenares in recurrent oral ulceration. Immunology i3:159 Aug 1967. 42. Snyderman, R. Periodontal disease: a model for the study of inflammation. J Infect Dis 123:676 June 1971. 43. Horton, J.E.; Leikin, S.; and Oppenheim, J.J. Human lymphoproliferative reaction to saliva and dental plaquedeposits: an in vitro correlation with periodontal disease. J Periodontol 43: 522 Sept 1972. 44. Ivanyi, L., and Lehner, T. Stimulation of lymphocyte transformation by bacterial antigens in patients with periodontal disease. Arch Oral Biol 15:1089 Nov 1970. 45. Dietz, V.H. Intracutaneous tests using filtrates prepared from pathologic
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pulps of human teeth, with special reference to rheumatoid arthritis. Oral Surg 5:877 Aug 1952. 46. Nishida, O.; Okada, H.; Kawagoe, K.; Tokunaga, A.; Tanihata, H.; Aono, M.; and Yokomizo, I. Investigation of homologous antibodies to an extract of rabbit dental pulp. Arch Oral Biol 16: 733 July 1971. 47. Honjo, H.; Tsubakimoto, K.; Utsumi, N.; and Tsutsui, M. Localization of plasma proteins in the human dental pulp. J Dent Res 49:888 July-Aug 1970. 48. Naidorf, I.J. Immunoglobulins in periapical granulomas: a preliminary report. J Endod 1:15 Jan 1975. 49. Kennedy, D.R.; Hamilton, T.R.; and Syverton, J.T. Effects on monkeys of introduction of hemolytic streptococci into root canals. J Dent Res 36:494 Aug 1957. 50. Rosengren, L. Inoculation of Streptococcus mutans and other bacteria into dental pulps of rats. Isr J Dent Med 19:1 Dec 1970. 51. Rosengren, L. The antibody response to experimental streptococcal infection of the dental pulp of the cat.
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Odont Tskr 70:261, 1962. 52. Okada, H.; Aono, M.; Yoshida, M.; Munemoto, K.; Nishida, O.; and Yokomizo, 1. Experimental study on focal infection in rabbits by prolonged sensitization through dental pulp canals. Arch Oral Biol 12:1017 Sept 1967. 53. Barnes, G.W., and Langland, K. Antibody formation in primates following introduction of antigens into the root canal. J Dent Res 45:1111 July-Aug 1966. 54. Pauly, J.L. Whole blood culture technique for functional studies of lymphocytes. Proc Soc Exp Biol Med 140: 40, 1972. 55. Kantz, W.E., and Henry, C.A. Isolation and classification of anaerobic bacteria from intact pulp chambers of nonvital teeth in man. Arch Oral Biol 19:91 Jan 1974. 56. Kiger, R.D.; Wright, W.H.; and Creamer, H.R. The significance of lymphocyte transformation responses to various microbial stimulants. J Periodontol 45:780 Nov 1974. 57. Horsfall, F.L., Jr. Formaldehyde and serum proteins; their immunologic
characteristics. J lmmunol 27:553 Dec 1934. 58. Jacobs, J.L.; and Sommers, S.C. Specificity of formolized proteins. J Immunol 36:531 June 1939. 59. Spector, W.G.; Heesom, N.; and Stevens, J.E. Factors influencing chronicity in inflammation of rat skin. J Pathol Bact 96:203 July 1968. 60. Ryan, G.B., and Spector, W.G. Natural selection of long-lived macrophages in experimental granulomata. J Pathol 99:139 Oct 1969. 61. Spector, W.G., and Heesom, N. The production of granulomata by antigen-antibody complexes. J Pathol 98:31 May 1969. 62. Spector, W.G.; Reichhold, N.; and Ryan, G.B. Degradation of granulomainducing micro-organisms by macrophages. J Pathol 101:339 Aug 1970. 63. Burleson, R.L. Antiphagocytosis-a new variable in the pathophysiology of tissue infection. Surgery 74:14 July 1973. 64. Curtis, J.E., and Hersh, E.M. Cellular immunity in man: correlation of leukocyte migration inhibition factor formation and delayed hypersensitivity. Cell Immunol 8:55 July 1973.
Lack of l y m p h o c y t e s t i m u l a t i o n by root canal p r o d u c t s Paul D. Eleazer, DDS; Paul A. Farber, DDS, PhD; and Samuel Seltzer, DDS, Philadelphia
The immune system is generally thought to consist of two elements. When foreign material (antigen) is introduced into the body and is recognized as such, antibody formation may be initiated or defense cells may be activated. Antigen may also stimulate both elements. The antibody response is called humoral because of the widespread distribution of these molecules that bind with antigen. Cells that participate in this response are termed Blymphocytes (B-cells) because of their development in bone marrow. In birds, these B-cells mature in a gutassociated organ known as the bursa of FabriciusJ The area of cell maturation in higher animals has not yet been clearly defined. Once activated B-cells divide, they become plasma cells and produce antibody specific for the activating antigen3 The activation of defense cells by antigen is known as the cell-mediated immune response. It is characterized by the production of lymphokines by T-lymphocytes (T-cells), so called because of their maturation in the thymus. 2 Lymphokines are substances that stimulate cell division, inhibit migration of macrophages and other defense cells, attract circulating defense cells, and otherwise help to eliminate the material recognized as foreign. 3 Interaction between humoral im-
388
mune processes and cell-mediated immune processes evidently exists. Claman and associates 4 have shown that in the presence of antigen, T-cells enhance the formation of plasma cells from thymus-independent lymphocytes. The laboratory study of cellmediated immunity has included the use of tissue culture and radioassay techniques. ~ One commonly used method involves measurement of radioactive thymidine uptake by cells to determine the amount of D N A synthesis and, therefore, the amount of cell division. By beginning with cultures containing equal numbers of cells, the stimulation potentials can be determined for the different antigenic substances capable of causing this cell change. 2 Lymphocyte transformation is the term used to describe the in vitro reversible differentiation of small lymphocytes into larger lymphoblasts. This transformation involves nuclear enlargement, appearance of nucleoli, increased cytoplasmic volume, and increased basophilia. 6 Biochemical analyses of lymphoblasts show an increase in R N A metabolism. 7 The increased R N A metabolism was confirmed by the use of the blocking agent, actinomycin D, which stops lymphocyte transformation, s The transformational activities culminate in D N A synthesis and mitosis, produc-
ing more cells to combat the antigen2 A number of different agents are responsible for lymphocyte transformation in vitro, many of which are proved in vitro antigens. Phytohemagglutinin ( P H A ) , the first agent used in vitro, is an extract of the red kidney bean (Phaseolus vulgaris)J ~ This extract produces almost 100% stimulation of small lymphocytes. It is commonly used as a control to determine the potential of a lymphocyte culture to be stimulated. Many other substances also stimulate this lymphocyte reaction. Among these are the allergens pokeweed, 1~ ragweed 14 and grass pollen. 1~ Also, various tissue antigens, such as homologous lymphocytes, 16 macrophages, 17 or platelets TM can initiate the phenomenon. Other tissue antigens, such as red blood cells of sheep can stimulate the change. 19 Heat-killed microorganisms, 2~ cell-free filtrates of Staphylococcus aureaus cultures, -02 and streptolysin S (an antigenic product of group A, hemolytic streptococci) 23 also stimulate lymphocyte cultures. Viruses, such as herpes simplex and Vaccinia also induce blastogenesis of in vitro lymphocytes. 2~ Endotoxin, a substance associated with the cell walls of m a n y gramnegative microorganisms, elicits lymphocyte transformation when complement is present. 25-27 After enzymatic cleavage of the lipopolysaccharide
JOURNAL OF ENDODONTICS ! VOL 1, NO 12, DECEMBER 1975
endotoxin, both the lipid and the polysaccharide fractions stimulate the lymphocytes. 28 Endotoxin has antigenic properties in vivo as well. -~9 On the basis of reported research, there is a reasonable possibility that lymphocyte transformation is at least a part of the natural immune defense mechanism, Tuberculin-induced lymphocyte transformation was first demonstrated in tuberculin-sensitized individuals,a~ A more recent study shows that the process of transformation activates a phosphate metabolism sequence that facilitates globulin secretion. :~1 There are a number of diseases in which a defective in vitro lymphocyte response is associated with a possible in vivo immune disorder. These inelude tuberculosis,31 sarcoidosis, 32 Hodgkin's disease, 3,~ chronic lymphocytic leukemia, zo chronic rubella, 34 and SjSgren's syndrome, a5 Ataxiatelangiectasis, a disease of children that often includes an impaired immune response, also produces a diminished lymphocyte response. ~6-37 Research findings in some dental diseases lead to the postulation that an immunologic phenomenon may be one of the etiologic factors. In patients with high caries indexes, a higher level of circulating antibodies is present against seven individual carlogenie Streptococcus species and Lactobacillus acidophilus. 3s Antibodies against Bacteroides melaninogenicus, a common inhabitant of the oral cavity, were found in all patients tested. 39 Celt-free filtrates of Actinomyces bovis, a frequent oral inhabitant, produced immediate hypersensitivity reactions of the skin in many patients with periodontal disease. 4~ Tissue extracts from oral aphthae produced a significantly greater degree of lymphocyte transformation than did normal controls. 41 Snyderman42 has proposed that the mechanism of periodontal disease in-
volves a penetration of the gingival epithelium by microorganisms from plaque colonies. These microorganisms elicited antibody formation in regional lymph nodes and in the local tissue. On introduction of more antigen into the system, an activation of this immune system produced biologically active products capable of increasing vascular permeability, contracting smooth muscle, and eliciting chemotaxis of neutrophils. This inflammation produced tissue-damaging enzymes, which allowed influx of more antigens. A recent study by Horton, Leikin and Oppenheim 4a demonstrated that autologous plaque and saliva induced more lymphocyte transformation in patients with severe periodontal disease than in patients with gingivitis or moderate periodontitis, or in normal controls. The greatest degree of stimulation resulted from the patient's own plaque and saliva. These findings are not in total agreement with those of Ivanyi and Lehner. 44 Their study showed that various bacterial ultrasonicates from patients with moderate periodontitis produced greater stimulation, compared to a depression of stimulation in patients with severe periodontitis. Research of the immunology of the dental pulp and periapical tissues has been mostly concerned with the humoral immune system. Dietz 45 has shown that filtrates of root canal contents caused immunologic reactions when injected intracutaneously. Nishida and associates 46 have noted that the use of Formalin as an adjuvant enhanced the immunologic activity of pulp antigens. Fluorescent antibody techniques have been used to demonstrate the presence and distribution of various immunoglobulins in the pulp. 47 Naidorf 4s also demonstrated pulpat immunoglobulins using Ouchterlony gel-diffusion methods. It has also been demonstrated that bacteria, experimentally inoculated into root canals, can
cause an immune response.49, 50 Rosengren 51 has shown that where bacteria were confined to the root canal the antibody response was minimal, whereas periapical infection elicited a significant response. The presence of foreign protein in the root canal causes hypergammaglobulinemia and other systemic immune manifestationsP2.53 The purpose of the current study was to use lymphoblast transformation analysis to determine if pulp tissues or the soft tissues of periapical lesions from pulpless teeth evoke a cell-mediated immune response. Materials
and
Methods
Pulps or periapical tissues were removed from 23 teeth in 21 patients undergoing treatment in the endodontic clinic at the Temple University School of Dentistry. Medical histories were obtained for all patients. Specimens were obtained from teeth in which there was no detectable communication between the pulp tissue and the oral environment. In two instances, the pulp contents were treated with cresol and formaldehyde (Formocresol) and were not removed at the initial visit. All of the periapical lesions were from teeth that had been previously treated by nonsurgical endodontic therapy. The gross presence or absence of a blood supply was used to classify pulp tissues as vital or necrotic. Tissue specimens were placed in RPMI 1640 tissue culture medium containing a 1% antimicrobial mixture of penicillin, amphotericin B (Fungizone), and streptomycin.* Cells were lysed with either freeze-thaw or ultrasonication techniques. Tissue specimens of surgical patients were also evaluated microscopically. Peripheral venous blood was obtained with a heparinized syringe. Lymphocytes were separated for cell culture with dextran* in five instances. The whole-blood culture technique of 389
| Mouse Blood
Control T:ssue P H A
FCB
: Fetal calf serum used
Fig 1--Lymphocyte stimulation by vital pulp tissues.
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Pauly '~ was used in the remaining cases because of the smaller amount of blood required. Tissue antigen was added to cell culture tubes. Controls consisted of cell cultures with no antigen and cultures with P H A M.t A m i n i m u m of three cultures each was used for possible antigen, PHA, and antigen-free tests. The amount of lymphocyte stimulation was determined by measuring DNA synthesis. One microcurie of tritiated thymidine was added to each of the cultures four hours before harvesting. The D N A was extracted from the cells with use of trichloroacetic acid and ethanol on Millipore filters.~ Seven milliliters of toluene-Liquafluor~ mixed at 23.7:1 was added to each sample to prepare it for scintillation counting. A counting period of one minute in a scintillation spectrometer// was used for each sample. In specimens with low activity, a five-minute counting period was used. Arithmetic mean values were determined for each test group. Anaerobic and aerobic cultures were taken to assess the root canal flora in several of the patients in this study for use in a concurrent project. Different media, including prereduced anaerobically sterilized Brain Heart Infusion Agar supplemented with vitamin K and heme,~, chopped meat-glucose,# thioglycollate with 0.15% agar,# Trypticase soy broth with 0.15% agar, # and Trypticase soy broth without agar# were used to culture any microbes present. In two of the tests involving pulpless teeth, killed microbes from these cultures were checked for lymphoblast transformation potential. In one case, boiled autologous saliva was tested. In eight instances, either a 1:10 or a 1:100 di-
Fig 2--Lymphocyte stimulation by nonvital pulp products.
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Fig 3--Lymphocyte stimulation by periapical granulomas.
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Corynebacterium acnes was used. In all tests involving cultured microorganisms, sterile medium was used as a control. Fetal calf serum was used with the patient's lymphocytes in six cases to determine if factors in the patient's serum were suppressing a response. Mouse blood was used in two test series to see if heterologous lymphocytes would react.
m
I0 C
Results Five of the 12 pulp specimens were classified as vital (Fig 1, 2), showing at least some evidence of blood supply when viewed by the unaided eye. Five of the 11 periapical tissue samples were found by microscopic examination to be gran~lomas (Fig 3); the others were described as cysts (Fig 4). In all 23 experiments, there was no evidence of increased lymphocyte division. There was no correlation between the results obtained and any part of the medical histories. N o additional stimulation was found when fetal calf serum was substituted for autologous serum. The use of mouse lymphocytes did not result in stimulation above the antigenfree control levels. N o differences were seen related to the freeze-thaw or ultrasonication methods of cell lysis. No significant increases were noted in any of the bacterial samples. In analyzing the data, it is important to note that a positive control ( P H A ) and a negative control (no stimulant) were used for each sample. Thus, results among patients cannot be compared, unless expressed as a percentage of the positive (or negative) control.
_
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o
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Discussion The fact that no evidence of lymo
Fig 4--Lymphocyte stimulation by periapica! cystic lesions.
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JOURNAL OF ENDODONTICS ] VOL 1, NO 12, DECEMBER 1975
phocyte stimulation was found in this study is not conclusive evidence that a cell-mediated immune response is absent in all cases. Kantz and Henry 5r' have detected microorganisms in 92% of intact pulpless teeth: however, there was no way to determine in the current study how long the microbes were present or what effect they may have had on necrotic tissue. There was also no information available on the numbers of organisms in the canals. The presence of necrotic tissue that may be inaccessible to the vascular system might prevent interaction of some necessary factor or factors in the immune system. A cell mediated immune reaction may occur in a patient with periodontal disease where a hypersensitivity to the microorganism(s) has already been established. It is also possible that a cell-mediated immune response could result from repeated exposure of the damaged tissue to microorganisms, such as in patients with several teeth with pulpal infection. These possibilities have not been investigated. Another possibility is suggested by a study of Kiger, Wright and Creamer ~6 in which there was no correlation between severity, or even presence of periodontal disease, and lymphocyte stimulation reactions to various oral microorganisms. Perhaps oral bacteria are present in concentrations too low to cause a clinically significant hypersensitivity. It is also possible that some as yet undefined factors in the test system are responsible for the differences in response. Nishida and co-workers 46 have demonstrated that Formalin acts on proteins in dental pulp extracts to enhance their antigenicity. It was postulated that cross-linking between molecules occurred because of an interaction of the Formalin with free amino groups on the dental pulp proteins. Others 57.58 have also noted a similar change in protein due to f o r m a l d e 392
hyde. Therefore, from an immunologic standpoint, it would seem unwise to use Formalin-containing compounds in the body. It has been shown that intracytoplasmic deposition of irritants is required for the perpetuation of a granuloma. 59,n~Spector and Heesom, m using labeled antigen-antibody complexes, found that these complexes were sequestered in macrophages present in granulomas for relatively long periods. Killed microbes that were radio labeled were also retained by the macrophages. Microorganisms that clinically do not cause granulomatous reactions, such as S albus, were digested to a significantly greater degree than were organisms such as Mycobacterium tuberculosis, which was only 40% digested at 14 days. 6'' Thus, it is likely that intracytoplasmic deposits of foreign material, including antigenantibody complexes, partially degraded microorganisms, and endodontic filling materials, may be responsible for the perpetuation of the periapical granuloma. In such cases, surgical curettage of the area to eliminate the lesion may be well advised. This seems especially important if an antigen-host interaction is being perpetuated by the sequestered material. Burleson G3 has discovered another possible justification for removal of granulomas. He noted that leukocytes in infected granulation tissue produced a factor that inhibits phagocytosis. He produced sterile granulation tissue that lacked this antiphagocytic factor. It is not known if this factor is present in the dental granuloma in situ. An absence of lymphocyte transformation does not rule out a functioning cell-mediated immune system. Curtis and Hersh 64 recently demonstrated that one antigen (keyhole limpet hemocyanin) causes production of a significant amount of migration inhibition factor in h u m a n lymphocytes in vitro, while no other cell-mediated
immune response parameters were increased. Summary
Pulp or periapical tissues removed from 23 teeth in patients undergoing dental treatment were placed in tissue culture media. The cells were lysed by freeze-thaw and ultrasonication techniques. Lymphocytes were obtained by adding some of the patients' peripheral venous blood to the cultures. The amount of lymphocyte stimulation was determined by measuring the amount of tritiated thymidine taken up in D N A synthesis. In all 23 experiments, there was no evidence of increased lymphocyte division. These results suggest that a cell-mediated immune response is lacking in human dental pulp and periapical tissue. However, these results do not preclude the possibility of this aspect of immunity being present. '::Sigma Chemical Co., St. Louis, Mo. tGrand Island Biologicals, Grand Island, NY. ,+Millipore Corp., Bedford, Mass. w England Nuclear, Boston, Mass. //Packard Tri-Carb Spectrometer, Model 3375, Downers Grove, Ill. 82 Biologicals, Chapel Hill, NC. #Baltimore Biological Laboratory, Baltimore, Md. From a thesis submitted by Dr. Eleazer in partial fulfillment for the degree of a Master of Science at Temple University. Dr. Eleazer, formerly a graduate student in endodontics, is in private practice, Albany, Ga; Dr. Farber is associate professor of pathology; and Dr. Seltzer is professor and chairman of endodontology, Temple University School of Dentistry. Requests for reprints should be directed to Dr. Samuel Seltzer, Temple University, School of Dentistry, 3223 N Broad St, Philadelphia, Pa 19140. References
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JOURNAL OF ENDODONTICS I VOL 1, NO 12, DECEMBER 1975
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