- Email: [email protected]
Role of T cells in the pathogenesis of periapical lesions
Vol. 76 No. 2
ENDODONTICS
August 1993
Editor: Samuel Seltzer
Role of T cells in the pathogenesis of periapical lesions A preliminary
report
John B. Wallstrom, IDDS, MS,” Mahmoud Torabinejad, DMD, MSD,b James Kettering, PhD,C and Paul McMillan, PhD,d Loma Linda, Calif. LOMA LINDA
UNIVERSITY
The pulps of mandibular molars of 15 athymic and 15 conventional rats were surgically exposed and left open to their oral flora. Each group was divided into three subgroups of five animals each. The rats were killed after their pulps were exposed for 2, 4, or 8 weeks. After fixing, decalcifying, and embedding, the specimens were sectioned and stained with hemotoxylin and eosin. They were then examined under a microscopic grid and quantified by percentages of surface areas of bone, connective tissue, bone marrow, intrabony spaces, periapical lesions, and numbers of osteoclasts, with the use of a DataVoice computerized data collection and analysis system. Statistical analysis showed no significant difference between periapical tissue responses of the conventional and athymic groups. The results indicate that the pathogenesis of periapical lesions IS a multifalctorial phenomenon and is not totally dependent on the presence of T-cell lymphocytes. (ORAL SIJRC ORAL MED ORAL PATHOL 1993;76:213-8)
Periapical lesions are areas of inflammatory reactions to various antigens present in infected root canals. Histologic exarnination of these lesions reveals the presence of granulation tissue infiltrated by immunocompetent cells such as lymphocytes, plasma cells, macrophages, polymorphonuclear (PMN) leukocytes, and mast cells. Stern et al.’ examined the cellular composition of human periapical granulomas and found that macrophages were the predominant inflammatory cells, followed in descending order by lymphocytes, plasma Supported in part by a Student Research Support Grant from the Research and Education Foundation of the American Association of Endodontists. The opinions, assertions, materials, and methodologies herein are private ones of the authors and are not to be construed as official or reflecting the views of the American Association of Endodontists or the Research and Education Foundation. This research was also supported in part by a grant from the Office of Research Planning of Loma Linda University School of Dentistry. aEndodontist, Auburn, Wash.; former resident, Department of Endodontics. bProfessor, Department of Endodontics. “Professor, Department of Microbiology. dProfessor, Department of Anatomy. Copyright @ 1993 by Mosby-Year Book, Inc. 0030-4220/93/$1.00 + .lO 7/15/473:24
cells, and neutrophils. Fifty-two percent of the total cells were inflammatory, and of these, 32% were lymphocytes. Nilsen et a1.2studied mononuclear cells in periapical lesions with the use of murine monoclonal antibodies and indirect immunofluorescence. T cells were a major part of the infiltrates and T-helper cells were more numerous than T-cytotoxic/suppressor cells by a ratio of 2: 1. Cymerman et aL3 examined seven periapical lesions for the presence of T-lymphocyte subpopulations with the use of a biotin-avidin-horseradish peroxidase method. Six of the lesion specimens stained positively for the presence of T-cytotoxic/suppressor and T-helper/inducer lymphocytes. The remaining lesion was diagnosed as an apical scar and contained no lymphocytes. Torabinejad and Kettering4 examined human periapical lesions for the presence of B and T lymphocytes by using the indirect immunoper’oxidase technique, They found that all of the lesions stained positive for both types of cells. On the average, the number of T lymphocytes was greater than the number of B cells in the same specimen. Yu and Stashenko5 investigated the composition of the cellular infiltrate of actively developing dental 213
214
Wallstrom et al.
periapical lesions in rats. They found numerous lymphocytes, PMNs, macrophages, blast cells, plasma cells, and eosinophils. T lymphocytes were the major non-PMN inflammatory cells and outnumbered Iapositive cells including B lymphocytes. In a later study, Stashenkoand Yu6 enumerated the T-helper (TH) cells and T-suppressor (TS) cells in rat periapical lesions. In the early stagesof lesion formation TH cells outnumbered TS cells by a ratio of 1.7. In the later stagesof periapical formation the number of TH cells decreasedand the TS outnumbered the TH cells. They concluded that T-cell mediated phenomena, including delayed type hypersensitivity and secretion of bone resorbing cytokines, play a major role in the formation of periapical lesions. Stabholz and McArthur7 measured the amount of leukocyte migration inhibitor factor in patients with and without periapical lesions. The extract of necrotic pulp tissue triggered a greater degree of inhibition of leukocyte migration in leukocytes from patients with periapical lesions compared with that from patients without periapical lesions. On the basisof theseand other studies,*>9it appears that T lymphocytes and their by-products play an important role in the pathogenesis of periapical lesions. However, there is no published report on the specific role of these cells in the formation of experimentally induced periapical lesions. The purpose of this study was to determine the role of T lymphocytes in the pathogenesis of actively developing periapical lesions in rats. MATERIAL AND METHODS
Fifteen 7 to 9-week-old female athymic nude rats (Taconic Laboratory animals, Germantown, N.Y., and National Cancer Institute, NIH, Bethesda, Md.) were used as experimental animals. These animals, known as mu or Rowett nude, are a genetic mutation of conventional rats with no T-cell function.10 Fifteen 7 to 9-week-old female conventional Sprague-Dawley rats (Taconic Laboratory) were used as controls. A pilot study showedthat the two groups of animals had essentially the same oral flora. At time 0, before the surgical procedure, the animals were anesthetized by intraperitoneal injection of 0.1 ml (5 mg) of sodium pentobarbital per 100 gm of body weight and mounted on a jaw retraction board. Pulpal exposureswere made in the lower first and secondmolars with a No. ‘/zround bur in a highspeedhandpiece. The preparations were made in the occlusal surfaces of the teeth to the depth of the bur head, which exposedthe pulp. Care was taken not to perforate the furcations, and the exposedpulps were left open to the oral environment.
ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY August I993
Five rats from each of the experimental and control groups were killed at 2,4, and 8 weeks after the pulp exposureswith the use of an overdoseof sodium pentobarbital. The mandibles were dissected from their soft tissues and were sectioned with a fissure bur so that only the molar area of the jaw remained. These specimens were decalcified and serially sectioned sagittally at a thickness of 6 pm and stained with hematoxylin and eosin for microscopic observations. Specimens that showed a contiguous connection between the apical foramen and periapical tissues were used for histologic evaluation. The specimenswere examined at X 100 magnification with the use of a microscope equipped with an ocular reticle. The area fractions of bone, connective tissue, tooth, pulp, marrow, and intrabony spaces, periapical lesions, and the number of osteoclasts within the grid were measured. The entire area measured was 1200 ,um per side, centered on the apical foramen. This area was composedof four 600 X 600 pm quadrants defined by the eyepiece reticle. The measurements were done with the use of DataVoice (Support Technology, Grand Terrace, Calif.) computerized data collection and analysis system. The length of intercepts of reticle lines (probe lines) on profiles of features in the section were recorded as coordinates of probe line intersections. Points on the x axis lines of the grid indicated the borders of the structures on the grid. This procedure was done using a Zeiss (Thornwood, N.J.) camera lucida to view, through the microscope, an LED on the cursor of a digitizing tablet. The coordinates of the probe line intersections with the seven tissue components and numbers of osteoclastswere identified by voice. The files created by DataVoice were then analyzed to provide the estimates of area fractions and the osteoclast counts. The results were analyzed statistically with the use of a Kruskal-Wallis one-way analysis of variance to determine differences between the two groups at each fixed time interval. An analysis was then done with a two-way ANOVA to determine whether there were differences between the treatment groups in relation to the group, or to the time intervals, or to some interaction between the treatment groups and time. RESULTS
Histologic examination of periapical tissuesin both treated groups showed the presence of numerous PMN leukocytes. The lesions enlarged as the time of root canal contamination increased (Figs. 1 and 2). The Kruskal-Wallis ANOVA test showed no significant difference in measurementsat eachfixed time interval between the two groups.
Wallstrom
ORAL SURGERY (m MEDICINE ORAL PATHOLOGY Volume 76. Number 2
et al.
215
Fig. 1. .A, Periapical tissues of conventional rat shows moderate inflammatory infiltrate after 4 weeks of pulp exposure. B, Periapical tissues of nude rat shows moderate inflammatory infiltrate after 4 weeks of pulp exposure.
The two-way ANOVA (Table I) showed no significant differences in area measurements related to the two groups when ad.justed for the varied time periods. There was a significant difference in area measurements related to time intervals (p < 0.02) when adjusted for group. The exception was counts of osteoclasts that showed no difference related to group
or time but a possible interaction between group and time (p < 0.05). DISCUSSION
In addition to nonspecific immune responses, the body also has the ability to develop extremely powerful specific immunity against various antigens such as
216
Wallstrom
et al.
ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY August 199.3
Fig. 2. A, Periapical tissues of conventional rat shows heavy infiltration of PMNs after 8 weeks of pulp exposure. B, Periapical tissues of nude rat shows heavy infiltration of PMNs after 8 weeks of pulp exposure. bacteria, viruses, toxins, and foreign tissues. The lymphocytes are important cells in acquired immunity. There are two major types of lymphocytes involved: the T lymphocytes that are responsible for the cell-mediated responses, and the I3 lymphocytes that are involved with the humoral immune reactions. On the basis of the present information, most antigens are T-cell dependent; antibody is not formed without the presence and involvement of T lymphocytes.” Absence of T lymphocytes results in a lack of
antibody formation to T-cell dependent antigens. In addition to their effects on B cells, they also affect other immunocompetent cells such as PMN leukocytes and their own subpopulations.11-‘5 The purpose of the study was to determine what happens when T cells are removed from the cycle of events that occur during the formation of periapical lesions. The accuracy of the conclusions depends on the absence of T cells in the test animals. Vos et al.‘* have shown that nude rats are without T-cell function.
Wallstrom
ORAL SIJRGERY ORAL MEDICINE ORAL PATHOLOGY Volume 76, Number 2 Table
et al.
217
I. Area percentages within the grid for each tissue type at 2, 4, and 8 weeks Area fractions
(percentage) and osteoclast counts Nude rats
‘w Bone Tooth Pulp Lesion Matrix Marrow Spaces Counts
1 30.39 15.82 4.3 0.1 40.02 5.23 3.54 19.8
1.62 16.1 4.33 18.9 52.59 0 1.92 28
8 weeks
2 weeks
14.42 16.04 4.51 12.18 51.2 0 6.9 10.2
30.09 21.23 4.8 1.3 40.58 0 2.0 14.8
It is generally known that there is an age-associated development of someT lymphocytes in older athymic rats. However, it has been postulated that an extrathymic pathway may exist for the maturation of T lymphocytes in these animals.16Yoshie et a1.r7have reported recovery of someT cells from gingival tissues of nude rats thalt implies the presence of some functioning lymphocytes” A recent study by Bell’* showed that nude-derived T cells in nude rats were nonfunctional. Nude anilmals reconstituted with donor T cells developednear normal immune function and a proliferating pool of donor-derived T lymphocytes.‘8 Becauseof direct effects of T cells on other immunocompetent ce:ll~“-‘~~l9 and strong predictions on their participation in pathogenesis of periapical lesions,“9 we expected that the athymic animals would be unlikely to develop periapical lesions after pulpal exposures.The fact that there was no significant difference in the ability to form periapical lesions between the nude rats and the conventional rats suggeststhat the nude rats may have had somefunctional T lymphocytes that affected the other inflammatory cells o’r that neutrophils could still respond via the nonspecific pathways to chemoattractant gradients such as bacterial products, complement component C5a, and leukotriene B4. Further studies a.re in progress to determine the cellular profile of periapical lesions in nude and conventional rats. An examination of blood smears of nude rats in the beginning of the experiment showed an absence of T cells in their blood circulation. To confirm the absence of these cells as the nude rats aged, this test should have been repeated at the end of the experiment. In addition, the cellular profile of periapical lesions should have been determined in both groups as described by Stashenko and Yu.~ However, the results of our study are consistent with those of a periodontal study20 that showed no significant difference in periodontal destruction be-
4 weeks
1
10.0 17.43 2.73 8.88 60.06 0 0.88 12.8
1
8 weeks 18.7 16.5 4.52 8.99 49.16 0 8.1 17.6
tween normal patients and those with suppressed T-cell function. Those investigators concluded that T cells play a minor role in the causeof periodontal disease. On the basis of the results of the latter study and those of our study, it appears that development of periodontal and periapical lesions is not totally dependent on the presence of T lymphocytes. We thank
Dr. Gordon
Rick, Scripps Institute DepartJolla, Calif., for valuable assis-
mentof Oral Pathology,La tance in this study.
REFERENCES 1. Stern M, Dreizen S, Mackler B, et al. Isolation and characterization of inflammatory cells from the human periapical granuloma. J Dent Res 1982;61:1408-12. 2. Nilsen R, Johannessen A, Skaug N, et al. In situ characterization of mononuclear cells in human dental periapical inflammatory lesions using monoclonal antibodies. ORAL SURG ORAL MED ORAL PATHOL 1984;58:160-5. 3. Cymerman J, Cymerman D, Walters J, et al. Human T-lymphocyte subpopulations in chronic periapical lesions. J Endodon 1984;10:9-11. 4. Torabinejad M, Kettering J. Identification and relative concentration of B and T lymphocytes in human chronic periapical lesions. J Endodon 1985;11:122-5. 5. Yu S, Stashenko P. Identification of inflammatory cells in developing rat periapical lesions. J Endodon 1987;13:535-40. 6. Stashenko P, Yu S. T-helper and T-suppressor cell reversal during the development of induced rat periapical lesions. J Dent Res 1989;68:830-4. 7. Stabholz A, McArthur W. Cellular immune response of patients with periapical pathosis to necrotic dental pulp antigens determined by release of LIF. J Endodon 1978;4:282-7. 8. Barkhordar R, Desouza Y. Human T-lymphocyte subpopulations in periapical lesions. ORAL SURG ORAL MED ORAL PATHOL 1988;65:763-6. 9. Kopp W, Schwarting R. Differentiation of T-lymphocytes subpopulations, macrophages, and HLA-DR-restricted cells of apical granulation tissue. J Endodon 1989;15:72-5. 10. Vos J, Berkvens J, Kruijt B. The athymic nude rat. Clin Immunol Immunopathol 1980;15:213-37. 11. Weisbart R, Kwan L, Gold D, et al. Human GM-CSF primes neutrophils for enhanced oxidative metabolism in response to themajor physiologic chemoattractants. Blood 1987;69:18-21. 12. Thurman G, Braude I, Gray P, et al. MIF-like activity of nat-
218
13.
14.
15.
16.
17.
Wallstrom et al.
ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY August I993
ural and recombinant human interferon-gamma and their neutralization by monoclonal antibody. J Immunol 1985; 134:305-9. Weinberg J, Hobbs M, Misukonis M. Phenotypic characterization of gamma interferon-induced human monocyte polykaryons. Blood 198.5;66:1241-6. Fleischmann J, Golde D, Weisbart R, et al. Granulocyte-macrophage colony-stimulating factor enhances phagocytosis of bacteria by human neutrophils. Blood !986;68:708-11. Robb R, Munck A, Smith K. T-cell growth factor receptors: quantification, specificity, and biological relevance. J Exp Med 1981;154:1455-74. Schwinzer R, Hedrick H, Wongeit K. T-cell differentiation in athymic nude rats (mu/mu): demonstration of a distorted T cell subset structure by flow cytometry analysis. Eur J Immuno1 1989;19:1841-7. Yoshie H, Taubman M, Ebersole J, et al. Periodontal bone loss and immune characteristics of congenitally athymic and tby-
mus cell-reconstituted athymic rats. Infect Immtm 1985; 50:403-g. 1X. Bell E, Sparshott S, Drayson M, et al. The origin of T ceils in permanently reconstituted old athymic nude rats: analysis using chromosome or allotype markers. Immunology 1989; 68:547-56. 19. Stobo J. In: Gallin J, Goldstein I, Snyderman R, eds. Inflammation: basic principles and clinical correlates. New York, Raven Press 1988599-612. 20. Oshrain H, Telsey B, Mandel I. A longitudinal study of periodontal disease in patients with reduced immunocapacity. J Periodontol 1983;54:151-4.
Reprint requests: John B. Wallstrom, DDS, MS 17118 S.E. 328th St. Auburn, WA 98002
CALL FOR LETTERSTO THE EDITOR A separate and distinct space for Letters to the Editor was established by Larry J. Peterson, new editor in chief of ORAL SURGERY, ORAL MEDICINE, ORAL PATHOLOGY in his Editorial in the January 1993 issue. Dr. Peterson also encouraged brief reports on interesting observations and new developments to be submitted to appear in this letters section as well as Letters commenting on earlier published articles. Please submit your letters and brief reports for inclusion in this section. Information for authors for the Journal appears in this issue of ORAL SURGERY, ORAL MEDICI%E, ORAL PATHOLOGY. We look forward to hearing from you.
ENDODONTICS
August 1993
Editor: Samuel Seltzer
Role of T cells in the pathogenesis of periapical lesions A preliminary
report
John B. Wallstrom, IDDS, MS,” Mahmoud Torabinejad, DMD, MSD,b James Kettering, PhD,C and Paul McMillan, PhD,d Loma Linda, Calif. LOMA LINDA
UNIVERSITY
The pulps of mandibular molars of 15 athymic and 15 conventional rats were surgically exposed and left open to their oral flora. Each group was divided into three subgroups of five animals each. The rats were killed after their pulps were exposed for 2, 4, or 8 weeks. After fixing, decalcifying, and embedding, the specimens were sectioned and stained with hemotoxylin and eosin. They were then examined under a microscopic grid and quantified by percentages of surface areas of bone, connective tissue, bone marrow, intrabony spaces, periapical lesions, and numbers of osteoclasts, with the use of a DataVoice computerized data collection and analysis system. Statistical analysis showed no significant difference between periapical tissue responses of the conventional and athymic groups. The results indicate that the pathogenesis of periapical lesions IS a multifalctorial phenomenon and is not totally dependent on the presence of T-cell lymphocytes. (ORAL SIJRC ORAL MED ORAL PATHOL 1993;76:213-8)
Periapical lesions are areas of inflammatory reactions to various antigens present in infected root canals. Histologic exarnination of these lesions reveals the presence of granulation tissue infiltrated by immunocompetent cells such as lymphocytes, plasma cells, macrophages, polymorphonuclear (PMN) leukocytes, and mast cells. Stern et al.’ examined the cellular composition of human periapical granulomas and found that macrophages were the predominant inflammatory cells, followed in descending order by lymphocytes, plasma Supported in part by a Student Research Support Grant from the Research and Education Foundation of the American Association of Endodontists. The opinions, assertions, materials, and methodologies herein are private ones of the authors and are not to be construed as official or reflecting the views of the American Association of Endodontists or the Research and Education Foundation. This research was also supported in part by a grant from the Office of Research Planning of Loma Linda University School of Dentistry. aEndodontist, Auburn, Wash.; former resident, Department of Endodontics. bProfessor, Department of Endodontics. “Professor, Department of Microbiology. dProfessor, Department of Anatomy. Copyright @ 1993 by Mosby-Year Book, Inc. 0030-4220/93/$1.00 + .lO 7/15/473:24
cells, and neutrophils. Fifty-two percent of the total cells were inflammatory, and of these, 32% were lymphocytes. Nilsen et a1.2studied mononuclear cells in periapical lesions with the use of murine monoclonal antibodies and indirect immunofluorescence. T cells were a major part of the infiltrates and T-helper cells were more numerous than T-cytotoxic/suppressor cells by a ratio of 2: 1. Cymerman et aL3 examined seven periapical lesions for the presence of T-lymphocyte subpopulations with the use of a biotin-avidin-horseradish peroxidase method. Six of the lesion specimens stained positively for the presence of T-cytotoxic/suppressor and T-helper/inducer lymphocytes. The remaining lesion was diagnosed as an apical scar and contained no lymphocytes. Torabinejad and Kettering4 examined human periapical lesions for the presence of B and T lymphocytes by using the indirect immunoper’oxidase technique, They found that all of the lesions stained positive for both types of cells. On the average, the number of T lymphocytes was greater than the number of B cells in the same specimen. Yu and Stashenko5 investigated the composition of the cellular infiltrate of actively developing dental 213
214
Wallstrom et al.
periapical lesions in rats. They found numerous lymphocytes, PMNs, macrophages, blast cells, plasma cells, and eosinophils. T lymphocytes were the major non-PMN inflammatory cells and outnumbered Iapositive cells including B lymphocytes. In a later study, Stashenkoand Yu6 enumerated the T-helper (TH) cells and T-suppressor (TS) cells in rat periapical lesions. In the early stagesof lesion formation TH cells outnumbered TS cells by a ratio of 1.7. In the later stagesof periapical formation the number of TH cells decreasedand the TS outnumbered the TH cells. They concluded that T-cell mediated phenomena, including delayed type hypersensitivity and secretion of bone resorbing cytokines, play a major role in the formation of periapical lesions. Stabholz and McArthur7 measured the amount of leukocyte migration inhibitor factor in patients with and without periapical lesions. The extract of necrotic pulp tissue triggered a greater degree of inhibition of leukocyte migration in leukocytes from patients with periapical lesions compared with that from patients without periapical lesions. On the basisof theseand other studies,*>9it appears that T lymphocytes and their by-products play an important role in the pathogenesis of periapical lesions. However, there is no published report on the specific role of these cells in the formation of experimentally induced periapical lesions. The purpose of this study was to determine the role of T lymphocytes in the pathogenesis of actively developing periapical lesions in rats. MATERIAL AND METHODS
Fifteen 7 to 9-week-old female athymic nude rats (Taconic Laboratory animals, Germantown, N.Y., and National Cancer Institute, NIH, Bethesda, Md.) were used as experimental animals. These animals, known as mu or Rowett nude, are a genetic mutation of conventional rats with no T-cell function.10 Fifteen 7 to 9-week-old female conventional Sprague-Dawley rats (Taconic Laboratory) were used as controls. A pilot study showedthat the two groups of animals had essentially the same oral flora. At time 0, before the surgical procedure, the animals were anesthetized by intraperitoneal injection of 0.1 ml (5 mg) of sodium pentobarbital per 100 gm of body weight and mounted on a jaw retraction board. Pulpal exposureswere made in the lower first and secondmolars with a No. ‘/zround bur in a highspeedhandpiece. The preparations were made in the occlusal surfaces of the teeth to the depth of the bur head, which exposedthe pulp. Care was taken not to perforate the furcations, and the exposedpulps were left open to the oral environment.
ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY August I993
Five rats from each of the experimental and control groups were killed at 2,4, and 8 weeks after the pulp exposureswith the use of an overdoseof sodium pentobarbital. The mandibles were dissected from their soft tissues and were sectioned with a fissure bur so that only the molar area of the jaw remained. These specimens were decalcified and serially sectioned sagittally at a thickness of 6 pm and stained with hematoxylin and eosin for microscopic observations. Specimens that showed a contiguous connection between the apical foramen and periapical tissues were used for histologic evaluation. The specimenswere examined at X 100 magnification with the use of a microscope equipped with an ocular reticle. The area fractions of bone, connective tissue, tooth, pulp, marrow, and intrabony spaces, periapical lesions, and the number of osteoclasts within the grid were measured. The entire area measured was 1200 ,um per side, centered on the apical foramen. This area was composedof four 600 X 600 pm quadrants defined by the eyepiece reticle. The measurements were done with the use of DataVoice (Support Technology, Grand Terrace, Calif.) computerized data collection and analysis system. The length of intercepts of reticle lines (probe lines) on profiles of features in the section were recorded as coordinates of probe line intersections. Points on the x axis lines of the grid indicated the borders of the structures on the grid. This procedure was done using a Zeiss (Thornwood, N.J.) camera lucida to view, through the microscope, an LED on the cursor of a digitizing tablet. The coordinates of the probe line intersections with the seven tissue components and numbers of osteoclastswere identified by voice. The files created by DataVoice were then analyzed to provide the estimates of area fractions and the osteoclast counts. The results were analyzed statistically with the use of a Kruskal-Wallis one-way analysis of variance to determine differences between the two groups at each fixed time interval. An analysis was then done with a two-way ANOVA to determine whether there were differences between the treatment groups in relation to the group, or to the time intervals, or to some interaction between the treatment groups and time. RESULTS
Histologic examination of periapical tissuesin both treated groups showed the presence of numerous PMN leukocytes. The lesions enlarged as the time of root canal contamination increased (Figs. 1 and 2). The Kruskal-Wallis ANOVA test showed no significant difference in measurementsat eachfixed time interval between the two groups.
Wallstrom
ORAL SURGERY (m MEDICINE ORAL PATHOLOGY Volume 76. Number 2
et al.
215
Fig. 1. .A, Periapical tissues of conventional rat shows moderate inflammatory infiltrate after 4 weeks of pulp exposure. B, Periapical tissues of nude rat shows moderate inflammatory infiltrate after 4 weeks of pulp exposure.
The two-way ANOVA (Table I) showed no significant differences in area measurements related to the two groups when ad.justed for the varied time periods. There was a significant difference in area measurements related to time intervals (p < 0.02) when adjusted for group. The exception was counts of osteoclasts that showed no difference related to group
or time but a possible interaction between group and time (p < 0.05). DISCUSSION
In addition to nonspecific immune responses, the body also has the ability to develop extremely powerful specific immunity against various antigens such as
216
Wallstrom
et al.
ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY August 199.3
Fig. 2. A, Periapical tissues of conventional rat shows heavy infiltration of PMNs after 8 weeks of pulp exposure. B, Periapical tissues of nude rat shows heavy infiltration of PMNs after 8 weeks of pulp exposure. bacteria, viruses, toxins, and foreign tissues. The lymphocytes are important cells in acquired immunity. There are two major types of lymphocytes involved: the T lymphocytes that are responsible for the cell-mediated responses, and the I3 lymphocytes that are involved with the humoral immune reactions. On the basis of the present information, most antigens are T-cell dependent; antibody is not formed without the presence and involvement of T lymphocytes.” Absence of T lymphocytes results in a lack of
antibody formation to T-cell dependent antigens. In addition to their effects on B cells, they also affect other immunocompetent cells such as PMN leukocytes and their own subpopulations.11-‘5 The purpose of the study was to determine what happens when T cells are removed from the cycle of events that occur during the formation of periapical lesions. The accuracy of the conclusions depends on the absence of T cells in the test animals. Vos et al.‘* have shown that nude rats are without T-cell function.
Wallstrom
ORAL SIJRGERY ORAL MEDICINE ORAL PATHOLOGY Volume 76, Number 2 Table
et al.
217
I. Area percentages within the grid for each tissue type at 2, 4, and 8 weeks Area fractions
(percentage) and osteoclast counts Nude rats
‘w Bone Tooth Pulp Lesion Matrix Marrow Spaces Counts
1 30.39 15.82 4.3 0.1 40.02 5.23 3.54 19.8
1.62 16.1 4.33 18.9 52.59 0 1.92 28
8 weeks
2 weeks
14.42 16.04 4.51 12.18 51.2 0 6.9 10.2
30.09 21.23 4.8 1.3 40.58 0 2.0 14.8
It is generally known that there is an age-associated development of someT lymphocytes in older athymic rats. However, it has been postulated that an extrathymic pathway may exist for the maturation of T lymphocytes in these animals.16Yoshie et a1.r7have reported recovery of someT cells from gingival tissues of nude rats thalt implies the presence of some functioning lymphocytes” A recent study by Bell’* showed that nude-derived T cells in nude rats were nonfunctional. Nude anilmals reconstituted with donor T cells developednear normal immune function and a proliferating pool of donor-derived T lymphocytes.‘8 Becauseof direct effects of T cells on other immunocompetent ce:ll~“-‘~~l9 and strong predictions on their participation in pathogenesis of periapical lesions,“9 we expected that the athymic animals would be unlikely to develop periapical lesions after pulpal exposures.The fact that there was no significant difference in the ability to form periapical lesions between the nude rats and the conventional rats suggeststhat the nude rats may have had somefunctional T lymphocytes that affected the other inflammatory cells o’r that neutrophils could still respond via the nonspecific pathways to chemoattractant gradients such as bacterial products, complement component C5a, and leukotriene B4. Further studies a.re in progress to determine the cellular profile of periapical lesions in nude and conventional rats. An examination of blood smears of nude rats in the beginning of the experiment showed an absence of T cells in their blood circulation. To confirm the absence of these cells as the nude rats aged, this test should have been repeated at the end of the experiment. In addition, the cellular profile of periapical lesions should have been determined in both groups as described by Stashenko and Yu.~ However, the results of our study are consistent with those of a periodontal study20 that showed no significant difference in periodontal destruction be-
4 weeks
1
10.0 17.43 2.73 8.88 60.06 0 0.88 12.8
1
8 weeks 18.7 16.5 4.52 8.99 49.16 0 8.1 17.6
tween normal patients and those with suppressed T-cell function. Those investigators concluded that T cells play a minor role in the causeof periodontal disease. On the basis of the results of the latter study and those of our study, it appears that development of periodontal and periapical lesions is not totally dependent on the presence of T lymphocytes. We thank
Dr. Gordon
Rick, Scripps Institute DepartJolla, Calif., for valuable assis-
mentof Oral Pathology,La tance in this study.
REFERENCES 1. Stern M, Dreizen S, Mackler B, et al. Isolation and characterization of inflammatory cells from the human periapical granuloma. J Dent Res 1982;61:1408-12. 2. Nilsen R, Johannessen A, Skaug N, et al. In situ characterization of mononuclear cells in human dental periapical inflammatory lesions using monoclonal antibodies. ORAL SURG ORAL MED ORAL PATHOL 1984;58:160-5. 3. Cymerman J, Cymerman D, Walters J, et al. Human T-lymphocyte subpopulations in chronic periapical lesions. J Endodon 1984;10:9-11. 4. Torabinejad M, Kettering J. Identification and relative concentration of B and T lymphocytes in human chronic periapical lesions. J Endodon 1985;11:122-5. 5. Yu S, Stashenko P. Identification of inflammatory cells in developing rat periapical lesions. J Endodon 1987;13:535-40. 6. Stashenko P, Yu S. T-helper and T-suppressor cell reversal during the development of induced rat periapical lesions. J Dent Res 1989;68:830-4. 7. Stabholz A, McArthur W. Cellular immune response of patients with periapical pathosis to necrotic dental pulp antigens determined by release of LIF. J Endodon 1978;4:282-7. 8. Barkhordar R, Desouza Y. Human T-lymphocyte subpopulations in periapical lesions. ORAL SURG ORAL MED ORAL PATHOL 1988;65:763-6. 9. Kopp W, Schwarting R. Differentiation of T-lymphocytes subpopulations, macrophages, and HLA-DR-restricted cells of apical granulation tissue. J Endodon 1989;15:72-5. 10. Vos J, Berkvens J, Kruijt B. The athymic nude rat. Clin Immunol Immunopathol 1980;15:213-37. 11. Weisbart R, Kwan L, Gold D, et al. Human GM-CSF primes neutrophils for enhanced oxidative metabolism in response to themajor physiologic chemoattractants. Blood 1987;69:18-21. 12. Thurman G, Braude I, Gray P, et al. MIF-like activity of nat-
218
13.
14.
15.
16.
17.
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Reprint requests: John B. Wallstrom, DDS, MS 17118 S.E. 328th St. Auburn, WA 98002
CALL FOR LETTERSTO THE EDITOR A separate and distinct space for Letters to the Editor was established by Larry J. Peterson, new editor in chief of ORAL SURGERY, ORAL MEDICINE, ORAL PATHOLOGY in his Editorial in the January 1993 issue. Dr. Peterson also encouraged brief reports on interesting observations and new developments to be submitted to appear in this letters section as well as Letters commenting on earlier published articles. Please submit your letters and brief reports for inclusion in this section. Information for authors for the Journal appears in this issue of ORAL SURGERY, ORAL MEDICI%E, ORAL PATHOLOGY. We look forward to hearing from you.