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Biosynthesis of IgG in periapical lesion explant cultures
0099-2399/91/1704-0143[$03.00/0 JOURNAL OF ENDODONTICS Copyright 9 1991 by The American Association of Endodontists
Printed in U.S.A. VOL. 17, NO. 4, APRIL 1991
SCIENTIFIC ARTICLES Biosynthesis of IgG in Periapical Lesion Explant Cultures J. Craig Baumgartner, DDS, PhD, and William A. Falkler, Jr., MS, PhD
Lally (I 1) have studied explant cultures of periapical lesions. The latter studied five surgically removed periapical lesions from patients that failed to heal following nonsurgical root canal therapy. Jones and Lally (11) cultured periapical lesions in medium containing ~4C-labeled amino acids. They used autoradiography of the immunoelectrophoretic pattern made using samples of the supernatant fluids from the explant cultures to demonstrate the association of 14C-labeled amino acids with IgG and IgA. The purpose of this study was to determine if IgG was synthesized in vitro in explant cultures of periapical inflammatory lesions associated with untreated infected root canals and to determine the level of IgG present in each staphylococcal protein A isolated sample.
The presence of immunoglobulins has been demonstrated in periapical inflammatory lesions associated with endodontic disease. The purpose of this study was to determine if IgG is synthesized in vitro in explant cultures of untreated periapical inflammatory lesions and to determine the level of IgG in isolated samples. Periapical lesions associated with infected root canals were removed from the roots and cultured in tissue culture medium containing tritiated amino acids. Supernatant fluids from the explant tissue cultures were passed through staphylococcal protein A affinity columns to isolate IgG. When the staphylococcal protein A eluents (24-h samples) from six periapical lesions were used in double diffusion in agarose assays, the presence of IgG was demonstrated in all the samples. Radial immunodiffusion assays to quantitate the IgG in staphylococcal protein A eluents showed that the levels of IgG detected in each successive daily supernatant fluid always decreased or else fell below the lower limits of detection. The in vitro biosynthesis of IgG in explant cultures of periapical lesions was demonstrated by the incorporation of tritiated amino acids into isolated IgG.
MATERIALS AND METHODS
Isolation of lgG Staphylococcal protein A is a cell wall component of Staphylococcus aureus which has the unique property of specifically binding to human IgG (12). In this study, affinity columns containing staphylococcal protein A (Pierce, Rockford, IL) were used to isolate IgG from the supernatant fluids of six periapical lesion explants cultured in enriched 3H-radiolabeled RPMI 1640. These six supernatant fluids were chosen from a preliminary study because they yielded two high, two medium, and two low scintillation counts (13). The supernatant fluids from the explant cultures were first centrifuged (Eppendorf model 5414; Springfield, IL) at 12,800 • g for 10 min to remove cellular debris, then diluted with binding buffer (pH 8) (Pierce), and passed through a staphylococcal protein A affinity column. One milliliter of the supernatant fluid collected at 24 h from each of six periapical lesion explants cultured in enriched RPMI 1640 culture medium containing tritium-labeled glutamine and leucine was diluted with 4 ml of binding buffer and passed through the staphylococcal protein A affinity columns. The first 14 ml of binding buffer used to wash the staphylococcal protein A affinity columns contained the tritiated amino acids not incorporated into IgG as indicated by high counts per minute (about 8000 cpm). However, the last 1 ml of binding buffer wash produced cpm similar to background radiation, indicating that the free tritiated amino acids were no longer present in the buffer coming
Microscopic examination of periapical lesions of endodontic origin reveals the presence of granulation tissue infiltrated with cells associated with both specific and nonspecific inflammatory reactions. Periapical granulation tissue contains lymphocyles, plasma cells, macrophages, polymorphonuclear leukocytes, giant cells, and mast cells (1). The presence of numerous plasma cells suggests that biosynthesis may be taking place locally in the inflammatory tissue. Several studies have used immunofluorescence or immunoperoxidase to demonstrate cells in periapical lesions containing IgG, IgA, IgM, and IgE (2-4). Explant culture models have been used to investigate the immune response of periodontal diseased tissues (5-8). Falkler et al. (9) and Hahn (10) investigated immunoglobulins in explant tissue cultures of pulpal tissue, but only Jones and
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through the column. The IgG bound in the affinity column was then eluted with 5 ml of elution buffer (Pierce). The elution buffer containing the IgG was collected in five (1-ml) fractions in 12- x 75-mm tubes (Falcon; Becton Dickinson, Rutherford, N J). The fractions of staphylococcal protein A eluent (SPAE) were immediately frozen at -20~ Before freezing, a scintillation vial was prepared for each of the five fractions of SPAE from each of the six specimens by adding 100 ul from each of the collected fractions of SPAE to a scintillation vial (Wheaton; American Scientific Co., McGraw Park, IL) containing 10 ml of scintillation cocktail (Packard, Scint-A, Downers Grove, IL). The cpm for each fraction of SPAE from the six explant cultures were determined using a scintillation counter (Packard model 3460CD). It was determined that the highest cpm was always in the second fraction. Six additional periapical lesions that had been placed in enriched 3H-radiolabeled RPMI 1640 but immediately frozen after tooth extraction at -20~ to prevent biosynthesis were also used in this experiment. Control specimens cultured in enriched 3H-radiolabeled RPMI 1640 in this experiment consisted of healthy connective tissue from healthy gingiva and bone from the apex of an uninfected tooth. Cocktail blanks, which consisted of a scintillation vial containing only scintillation cocktail, were used to determine the background radiation.
Double Diffusion in Agarose with Isolated lgG The six SPAEs from periapical tissue explants cultured in enriched 3H-radiolabeled RPMI 1640 were used in a double diffusion in agarose assay for human IgG. Fraction 2 of the five fractions collected was used in this experiment because fraction two of each SPAE always had the highest cpm. In the double diffusion in agarose assay, 45 ul of SPAE (fraction 2) from 24-h supernatant fluids of specimens 52, 58, 70, 78, 79, and 80 were allowed to react with 15 ~1 of goat anti-human 3"chain serum. In addition, 45 #1 of each of the SPAE (fraction 2) from each of the six frozen periapical lesions (F1 to F6) were also allowed to react with 15 ~1 of goat anti-human 3" chain serum. Human serum and enriched -~H-radiolabeled RPMI 1640 were used as control solutions in this experiment.
Radial Immunodiffusion Assay for IgG Radial immunodiffusion was used to detect and quantitate the amount of IgG in the SPAE. For this experiment, lowlevel RID kits (The Binding Site, San Diego, CA) for human IgG, IgA, or IgM were used. Fraction 2 of the SPAE from daily (24-, 48-, 72-, and 96-h) supernatant fluids of specimens 52, 58, 70, 78, 79, and 80 was used in this assay along with standardized human reference sera and enriched 3H radiolabeled RPMI 1640. Five microliters of each sample (in duplicate) were placed in the designated wells and the RID plates incubated at 26~ for 72 h. The diameters of the precipitation tings were then measured using a sidelight (Background Light AutoAssay, New York, NY) and a digital micrometer (Giles Scientific, New York, NY). The average of two readings was squared and the quantity of immunoglobulin (IgG, IgM, or IgA) in the SPAEs was determined by using regression analysis from calibration curves which were produced by plotting the values for the standardized human reference sera in mg/L
Journal of Endodontics
versus the squares of the diameters of the precipitation tings for each reference standard. In addition to the RID assay, average daily cpm were determined for each SPAE. For this experiment, 100 ~1 (in duplicate) from fraction 2 of the SPAE from each of the daily (24-, 48-, 72-, and 96-h) supernatant fluids of specimens 52, 58, 70, 78, 79, and 80 were added to a scintillation vial containing 10 ml of scintillation cocktail. The cpm for each sample was determined using a scintillation counter (Packard model 3460CD). RESULTS IgG in the supernatant fluids from explant cultures of periapical lesions was isolated by passing the supernatant fluids through staphylococcal protein A affinity columns. When the elution buffer from the staphylococcal protein A affinity columns was collected in five (1-ml) fractions, fraction 2 of the eluents always had the highest cpm. In addition, the supernatant fluids from six periapical lesions that had been immediately frozen following extraction at -20~ at the time of tooth extraction were also isolated for IgG using staphylococcal protein A affinity columns as described above. The highest number ofcpm, as with the explant cultures, was in the second 1-ml fraction of the 5 ml of SPAE collected. The cpm determined for fraction 2 of SPAEs using 1 ml of day 1 (24-h) supernatant fluid from each of the six explant cultures, 1 ml from six frozen periapical lesions, and scintillation blanks is shown in Table 1. The average cpm for fraction 2 of the SPAEs from the explant cultures of periapical lesions ranged from 78 to 884 cpm. The average cpm for fraction 2 of the SPAEs from the frozen periapical lesions ranged from 32 to 66 cpm. The cpm for the nine scintillation blanks ranged from 14 to 24 cpm. A one-tailed ANOVA showed a statistically significant (p < 0.05) difference between each of the three groups (nonfrozen periapical lesions, frozen periapical lesions, and scintillation blanks). SPAEs containing isolated IgG were used in a double diffusion in agarose assay for human IgG. In this experiment, 45 ul of SPAEs (fraction 2) from 24-h supernatant fluids of specimens 52, 58, 70, 78, 79, and 80 were allowed to react with 15 ul of goat anti-human 3' chain serum. A line of precipitation developed for each of the above six SPAEs. Photographs of lines of precipitation can be seen in Fig. 1. In addition, 45 ul of SPAE from each of the six frozen periapical lesions (F1 to F6) were also allowed to react with 15 ~1 of goat anti-human 3' chain serum. None of the latter SPAEs produced lines of precipitation. A control used in this experiment that produced a line of precipitation was human serum containing IgG, and a control which did not produce a line of precipitation was RPMI 1640. This experiment demonstrated that at the level of sensitivity of the double diffusion in agarose assay, IgG can be detected in fraction 2 of the SPAEs from supernatant fluids of explant cultures of periapical lesions but not from fraction 2 of the SPAEs from supernatant fluids of explant cultures of periapical lesions that were frozen before culturing. To check for purity and to quantitate the amount of the IgG in the SPAEs, radial immunodiffusion (RID) assays were used to determine the levels of IgG, IgA, and IgM in fraction 2 of the SPAEs from the daily (24-, 48-, 72-, and 96-h) supernatant fluids of specimens 52, 58, 70, 78, 79, and 80.
Vol. 17, No. 4, April 1991
Biosynthesis of IgG
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TABLE 1. Results* of scintillation counts for fraction 2 of SPAE using either 1 ml of 24-h supernatant fluids from six periapical lesions or 1 ml of 24-h supernatant fluids from six periapical lesions that had been frozent before incubation cpm
Periapical specimen (24-h incubation) SPAE 52 SPAE 58 SPAE 70 SPAE 78 SPAE 79 SPAE 80 Frozen periapical specimen (24-h incubation) SPAE F1 SPAE F2 SPAE F3 SPAE F4 SPAE F5 SPAE F6 Scintillation blanks:[: 1 2 3 4 5 6 7 8 9
Trial 1
Trial 2
Average
760 231 102 81 730 252
1008 236 109 76 726 251
884 234 106 78 728 252
53 32 55 47 66 56
54 31 53 48 67 58
54 32 54 48 66 57
14 12 13 14 14 16 16 13 23
13 17 14 13 14 18 13 15 24
14 14 14 14 14 17 14 14 24
* One-way ANOVA showed that the difference in the cpm for each of the above groups is statistically significant at p < 0.05. i" At the time of tooth extraction, each tooth with the attached periapical lesion was frozen at -20~ :~ Scintillation blanks consisted of the vial with 10 ml of scintillation cocktail only.
Calibration curves were produced by plotting the values for the reference standards of immunoglobulin in mg/L versus the squares of the diameters of the precipitation rings. The calibration standards for IgG, IgA, or IgM all produced precipitation rings and were considered the positive controls. The staphylococcal protein A elution buffer which produced no precipitation ring was considered the negative control. Neither IgA nor lgM was detected in any of the SPAEs from the supernatant fluids used in this experiment. The results of using RID to detect and quantitate the amount of IgG are presented in Table 2 along with the scintillation cpm for each SPAE. The amount of IgG detected in each successive daily (24-, 48-, 72-, and 96-h) supernatant fluid from each sample always decreased or fell below the lower level of detection. The daily decrease in the amount of IgG detected using RID was significant (ANOVA; F = 6.45, p = 0.005). In addition to the RID assay, the average number ofcpm in each successive daily (24-, 48-, 72-, and 96-h) supernatant fluid from each of the above six specimens was also determined and shown to always decrease (Table 1). The daily decrease in cpm was also significant (ANOVA: F = 5.62, p = 0.009). DISCUSSION To demonstrate in vitro biosynthesis, periapical lesions were cultured in enriched RPMI 1640 containing tritiated
FIG 1. Lines of precipitation demonstrating the presence of IgG were produced when elution samples (52, 58, 60, 64, 79, and 80) of isolated IgG from affinity columns containing staphylococcal protein A were placed in the peripheral wells and allowed to react by double diffusion in agarose with goat anti-human 3' chain serum in the center wells.
leucine and glutamine. Using fraction 2 of the SPAEs, which contains the majority of the isolated IgG, a significant difference in the cpm was shown between the periapical lesions cultured in RPMI 1640 containing tritiated amino acids, lesions frozen in RPMI 1640 containing tritiated amino acids, and scintillation blanks. The detection of tritiated amino acids in isolated IgG proves that IgG was being synthesized in vitro in explant cultures and strongly suggest that the local production of IgG takes place in vivo in periapical lesions. This is the first time that tritiated amino acids have been used to demonstrate the biosynthesis of IgG in explant cultures of periapical lesions associated with untreated root canal infections. This study supports the finding of IgG containing mononuclear cells in periapical lesions by other investigators using other methods (2, 3, 14, 15). It is interesting that the frozen lesions produced some tritium-labeled IgG at a level significantly lower than the unfrozen specimens, but significantly greater than the scintillation blanks. Plasma cells are capable of releasing thousands of molecules of immunoglobulin in a second (16). Apparently enough labeled IgG accumulated in the length of time it took to freeze the cells to yield a significant scintillation count from background radiation. It is also possible that some of the plasma cells or B ceils (lymphocytes) were able to survive the one-time freezing, or perhaps molecules of IgG in the
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Baumgartner and Falkler
Journal of Endodontics
1,035 101 83 17
supernatant fluids was being diluted when the culture medium was replenished each day. It is likely that both the amount of biosynthesis and the amount of serum IgG decrease with each successive day. The concentrations of IgG as determined by RID do not directly correspond from specimen to specimen with the scintillation cpm. This suggests that the rate of in vitro biosynthesis is different with each specimen. The latter would probably be related to the number and activity of B lymphocytes and plasma cells.
48 ND ---
235 130 22 18
The informed consent of all human subjects who participated in the experimental investigation reported or described in this manuscript was obtained after the nature of the procedure and possible discomforts and risks had been fully explained.
26.6 16.6 13.8 9.1
47 19 ND ND
117 24 27 16
7.28 5.89 4.74 4.12
53.0 34.7 22.5 17.0
122 70 35 19
85 30 28 17
9.20 7.95 4.66 ND
84.6 63.2 21.7 ND
525 82 150 32 --
744 330 146 66
8.16 4.78 ND ND
66.6 22.8 ND ND
159 35 ---
267 176 34 20
8.92 6.95 3.93 ND
79.6 48.3 15.4 --
200 120 20 --
-----
TABLE 2. Results of radial immunodiffusion and scintillation counts using fraction 2 of daily (24 to 96 h)* SPAE SPAE
NO. 52 (h) 24 48 72 96 NO. 58 (h) 24 48 72 96
Diameter of Average Precipitation Diameter Mg:~ Average Ring (ram)l" Squared IgG/L cpmw
8.47 4.81 4.05 3.46
71.7 23.1 16.4 12.0
438 82 37 37 NDII
5.18 3.87 ND ND
26.8 15.0 ND ND
5.16 4.08 3.72 3.02
No. 7O (h) 24 48 72 96
No. 78 (h) 24 48 72 96 No. 79 (h) 24 48 72 96
No. 80 (h) 24 48 72 96 Standards used for calibration curve A B C SPAE Buffer
* A significantdaily decreaseof IgG (ANOVA:F = 6,45, p = 0.002) and of cpm (ANOVA: F = 5.62, p = 0.009) was detected. 1 Averageof two readings. :1:mg IgG/L determined from calibration curve using three standard concentrationsof IgG versussquareof the diameterof the precipitationrings. wAveragescintillationcpm for 100-ul samplesof SPAEused in RID assay. Data correctedbecauseSPAE52 (24 h) and 79 (24 h) were diluted 2:5. IIND, no determinationof the quantityof immunoglobulinbecausea lineof precipitation was not detectedor the extrapolatedvalueusing regressionanalysiswas negative.
latter stages of biosynthesis were able to bind some of the labeled amino acids. The specificity of the staphylococcal protein A affinity columns for IgG was supported because neither IgA nor IgM could be detected in fraction two of the SPAEs from the 24h supernatant fluids of specimens 52, 58, 70, 78, 79, and 80 by using low-level IgA or IgM RID assay kits. The presence of IgG in SPAE was first demonstrated by double diffusion in agarose. RID was then used to detect and quantitate the level of IgG in fraction 2 of the SPAEs from daily (24-, 48-, 72-, and 96-h) supernatant fluids of specimens 52, 58, 70, 78, 79, and 80 and the scintillation cpm for each corresponding SPAE both showed significant decreases with each successive day. This supports the premise that either the amount of biosynthesis of the immunoglobulins was decreasing with time or that the amount of serum IgG in the
The opinions and assertions herein are the private views of the authors and are not to be construed as official or reflecting the views of the Department of the Army or the Department of Defense. The authors express their thanks to the American Association of Endodontists Endowment and Memorial Foundation for funding the purchase of the tritiated amino acids used in this study. We express our appreciation to Mr. Robert Burge, Division of Biometrics, Walter Reed Army Center, Washington, DC, for his assistance with the statistical analysis of the data. Dr. Baumgartner is chief, Microbiology Branch, United States Army Institute of Dental Research, Walter Reed Army Medical Center, Washington, DC. Dr. Falkler is professor and chairman, Microbiology Department, University of Maryland Dental School, Baltimore, MD. Address requests for reprints to Dr. J. Craig Baumgartner, 10376 Currycomb Ct., Columbia, MD 21044. This study was part of a dissertation submitted in partial fulfillment of the requirements for the degree of PhD at the University of Maryland Dental School, Baltimore, MD.
References 1. Stern MH, Dreizen S, Mackler BF, Selbst AG, Levy BM. Quantitative analysis of cellular composition of human periapical granuloma. J Endodon 1981 ;7:117-22. 2. Matthews JB, Mason GI. Immunoglobulin producing cells in human periapical granulomas. Br J Oral Surg 1983;21:192-7. 3, Pulver WH, Taubman MA, Smith DJ. Immune components in human dental periapical lesions. Arch Oral Bio11978;23:435-43. 4. Torabinejad M, Kettering J, Bakland L. Localization of IgE immunoglobulins in human dental periapical lesions by the peroxidase-antiperoxidase method. Arch Oral Biol 1981 ;26:677-81. 5. Lally ET, Baehni PC, McArthur WP. Local immunoglobulin synthesis in periodontal disease. J Periodont Res 1980;15:159-64. 6. Martin SA, Falkler WA Jr., Suzuki JB, Hawley CB, Mackler BF. Local and systemic immunoglobulins reactive to Bacteroides gingivalis in rapidly progressive and adult periodontitis. J Periodont Res 1986;21:351-64. 7. Martin SA, Falkler JWA, Vincent JW, Mackler BF, Suzuki JB. A comparison of reactivity of Eubacterium species with localized and serum immunoglobulins from rapidly progressive and adult periodontitis patients. J Periodont 1988;59:32-9. 8. Hall E. Study of the Focalized humoral immune response to impricated microorganisms in juvenile periodontitis [PhD Dissertation]. Baltimore: University of Maryland, 1988. 9, Falkler WA Jr, Martin SA, Tolba M, Siegel MA, Mackler BF. Reaction of pulpal immunoglobulins to oral microorganisms by an enzyme-linked immunosorbent assay. J Endodon 1987;13:260-6. 10. Hahn CI. Immunohistologic, immunologic, and microbiologic studies of putpal pathosis [PhD Dissertation]. Baltimore: University of Maryland, 1989. 11. Jones OJ, Lally ET. Biosynthesis of immunoglobulin isotypes in human periapical lesion. J Endodon 1980;6:672-7. 12. Bjork I. Some physiochemical properties of protein A from Staphylococcus aureus. Eur J Biochem 1972;29:579-84. 13. Baumgartner J. Microbiological and immunological investigation of periapical pathosis [PhD Dissertation]. Baltimore: University of Maryland, 1990. 14. Toiler P. Immunoglobulins and immunoglobulin-containing cells in cysts of the jaws. Lancet 1969;2:178-81. 15. Skaug N. Proteins in fluid from non-keratinizing jaw cysts. J Oral Pathol 1974;3:47-61. 16. Stites DP, Stobo JD, Wells JV. Basic and clinical immunology. 6th ed. Norwalk, CT: Appleton and Lange, 1987.
Printed in U.S.A. VOL. 17, NO. 4, APRIL 1991
SCIENTIFIC ARTICLES Biosynthesis of IgG in Periapical Lesion Explant Cultures J. Craig Baumgartner, DDS, PhD, and William A. Falkler, Jr., MS, PhD
Lally (I 1) have studied explant cultures of periapical lesions. The latter studied five surgically removed periapical lesions from patients that failed to heal following nonsurgical root canal therapy. Jones and Lally (11) cultured periapical lesions in medium containing ~4C-labeled amino acids. They used autoradiography of the immunoelectrophoretic pattern made using samples of the supernatant fluids from the explant cultures to demonstrate the association of 14C-labeled amino acids with IgG and IgA. The purpose of this study was to determine if IgG was synthesized in vitro in explant cultures of periapical inflammatory lesions associated with untreated infected root canals and to determine the level of IgG present in each staphylococcal protein A isolated sample.
The presence of immunoglobulins has been demonstrated in periapical inflammatory lesions associated with endodontic disease. The purpose of this study was to determine if IgG is synthesized in vitro in explant cultures of untreated periapical inflammatory lesions and to determine the level of IgG in isolated samples. Periapical lesions associated with infected root canals were removed from the roots and cultured in tissue culture medium containing tritiated amino acids. Supernatant fluids from the explant tissue cultures were passed through staphylococcal protein A affinity columns to isolate IgG. When the staphylococcal protein A eluents (24-h samples) from six periapical lesions were used in double diffusion in agarose assays, the presence of IgG was demonstrated in all the samples. Radial immunodiffusion assays to quantitate the IgG in staphylococcal protein A eluents showed that the levels of IgG detected in each successive daily supernatant fluid always decreased or else fell below the lower limits of detection. The in vitro biosynthesis of IgG in explant cultures of periapical lesions was demonstrated by the incorporation of tritiated amino acids into isolated IgG.
MATERIALS AND METHODS
Isolation of lgG Staphylococcal protein A is a cell wall component of Staphylococcus aureus which has the unique property of specifically binding to human IgG (12). In this study, affinity columns containing staphylococcal protein A (Pierce, Rockford, IL) were used to isolate IgG from the supernatant fluids of six periapical lesion explants cultured in enriched 3H-radiolabeled RPMI 1640. These six supernatant fluids were chosen from a preliminary study because they yielded two high, two medium, and two low scintillation counts (13). The supernatant fluids from the explant cultures were first centrifuged (Eppendorf model 5414; Springfield, IL) at 12,800 • g for 10 min to remove cellular debris, then diluted with binding buffer (pH 8) (Pierce), and passed through a staphylococcal protein A affinity column. One milliliter of the supernatant fluid collected at 24 h from each of six periapical lesion explants cultured in enriched RPMI 1640 culture medium containing tritium-labeled glutamine and leucine was diluted with 4 ml of binding buffer and passed through the staphylococcal protein A affinity columns. The first 14 ml of binding buffer used to wash the staphylococcal protein A affinity columns contained the tritiated amino acids not incorporated into IgG as indicated by high counts per minute (about 8000 cpm). However, the last 1 ml of binding buffer wash produced cpm similar to background radiation, indicating that the free tritiated amino acids were no longer present in the buffer coming
Microscopic examination of periapical lesions of endodontic origin reveals the presence of granulation tissue infiltrated with cells associated with both specific and nonspecific inflammatory reactions. Periapical granulation tissue contains lymphocyles, plasma cells, macrophages, polymorphonuclear leukocytes, giant cells, and mast cells (1). The presence of numerous plasma cells suggests that biosynthesis may be taking place locally in the inflammatory tissue. Several studies have used immunofluorescence or immunoperoxidase to demonstrate cells in periapical lesions containing IgG, IgA, IgM, and IgE (2-4). Explant culture models have been used to investigate the immune response of periodontal diseased tissues (5-8). Falkler et al. (9) and Hahn (10) investigated immunoglobulins in explant tissue cultures of pulpal tissue, but only Jones and
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through the column. The IgG bound in the affinity column was then eluted with 5 ml of elution buffer (Pierce). The elution buffer containing the IgG was collected in five (1-ml) fractions in 12- x 75-mm tubes (Falcon; Becton Dickinson, Rutherford, N J). The fractions of staphylococcal protein A eluent (SPAE) were immediately frozen at -20~ Before freezing, a scintillation vial was prepared for each of the five fractions of SPAE from each of the six specimens by adding 100 ul from each of the collected fractions of SPAE to a scintillation vial (Wheaton; American Scientific Co., McGraw Park, IL) containing 10 ml of scintillation cocktail (Packard, Scint-A, Downers Grove, IL). The cpm for each fraction of SPAE from the six explant cultures were determined using a scintillation counter (Packard model 3460CD). It was determined that the highest cpm was always in the second fraction. Six additional periapical lesions that had been placed in enriched 3H-radiolabeled RPMI 1640 but immediately frozen after tooth extraction at -20~ to prevent biosynthesis were also used in this experiment. Control specimens cultured in enriched 3H-radiolabeled RPMI 1640 in this experiment consisted of healthy connective tissue from healthy gingiva and bone from the apex of an uninfected tooth. Cocktail blanks, which consisted of a scintillation vial containing only scintillation cocktail, were used to determine the background radiation.
Double Diffusion in Agarose with Isolated lgG The six SPAEs from periapical tissue explants cultured in enriched 3H-radiolabeled RPMI 1640 were used in a double diffusion in agarose assay for human IgG. Fraction 2 of the five fractions collected was used in this experiment because fraction two of each SPAE always had the highest cpm. In the double diffusion in agarose assay, 45 ul of SPAE (fraction 2) from 24-h supernatant fluids of specimens 52, 58, 70, 78, 79, and 80 were allowed to react with 15 ~1 of goat anti-human 3"chain serum. In addition, 45 #1 of each of the SPAE (fraction 2) from each of the six frozen periapical lesions (F1 to F6) were also allowed to react with 15 ~1 of goat anti-human 3" chain serum. Human serum and enriched -~H-radiolabeled RPMI 1640 were used as control solutions in this experiment.
Radial Immunodiffusion Assay for IgG Radial immunodiffusion was used to detect and quantitate the amount of IgG in the SPAE. For this experiment, lowlevel RID kits (The Binding Site, San Diego, CA) for human IgG, IgA, or IgM were used. Fraction 2 of the SPAE from daily (24-, 48-, 72-, and 96-h) supernatant fluids of specimens 52, 58, 70, 78, 79, and 80 was used in this assay along with standardized human reference sera and enriched 3H radiolabeled RPMI 1640. Five microliters of each sample (in duplicate) were placed in the designated wells and the RID plates incubated at 26~ for 72 h. The diameters of the precipitation tings were then measured using a sidelight (Background Light AutoAssay, New York, NY) and a digital micrometer (Giles Scientific, New York, NY). The average of two readings was squared and the quantity of immunoglobulin (IgG, IgM, or IgA) in the SPAEs was determined by using regression analysis from calibration curves which were produced by plotting the values for the standardized human reference sera in mg/L
Journal of Endodontics
versus the squares of the diameters of the precipitation tings for each reference standard. In addition to the RID assay, average daily cpm were determined for each SPAE. For this experiment, 100 ~1 (in duplicate) from fraction 2 of the SPAE from each of the daily (24-, 48-, 72-, and 96-h) supernatant fluids of specimens 52, 58, 70, 78, 79, and 80 were added to a scintillation vial containing 10 ml of scintillation cocktail. The cpm for each sample was determined using a scintillation counter (Packard model 3460CD). RESULTS IgG in the supernatant fluids from explant cultures of periapical lesions was isolated by passing the supernatant fluids through staphylococcal protein A affinity columns. When the elution buffer from the staphylococcal protein A affinity columns was collected in five (1-ml) fractions, fraction 2 of the eluents always had the highest cpm. In addition, the supernatant fluids from six periapical lesions that had been immediately frozen following extraction at -20~ at the time of tooth extraction were also isolated for IgG using staphylococcal protein A affinity columns as described above. The highest number ofcpm, as with the explant cultures, was in the second 1-ml fraction of the 5 ml of SPAE collected. The cpm determined for fraction 2 of SPAEs using 1 ml of day 1 (24-h) supernatant fluid from each of the six explant cultures, 1 ml from six frozen periapical lesions, and scintillation blanks is shown in Table 1. The average cpm for fraction 2 of the SPAEs from the explant cultures of periapical lesions ranged from 78 to 884 cpm. The average cpm for fraction 2 of the SPAEs from the frozen periapical lesions ranged from 32 to 66 cpm. The cpm for the nine scintillation blanks ranged from 14 to 24 cpm. A one-tailed ANOVA showed a statistically significant (p < 0.05) difference between each of the three groups (nonfrozen periapical lesions, frozen periapical lesions, and scintillation blanks). SPAEs containing isolated IgG were used in a double diffusion in agarose assay for human IgG. In this experiment, 45 ul of SPAEs (fraction 2) from 24-h supernatant fluids of specimens 52, 58, 70, 78, 79, and 80 were allowed to react with 15 ul of goat anti-human 3' chain serum. A line of precipitation developed for each of the above six SPAEs. Photographs of lines of precipitation can be seen in Fig. 1. In addition, 45 ul of SPAE from each of the six frozen periapical lesions (F1 to F6) were also allowed to react with 15 ~1 of goat anti-human 3' chain serum. None of the latter SPAEs produced lines of precipitation. A control used in this experiment that produced a line of precipitation was human serum containing IgG, and a control which did not produce a line of precipitation was RPMI 1640. This experiment demonstrated that at the level of sensitivity of the double diffusion in agarose assay, IgG can be detected in fraction 2 of the SPAEs from supernatant fluids of explant cultures of periapical lesions but not from fraction 2 of the SPAEs from supernatant fluids of explant cultures of periapical lesions that were frozen before culturing. To check for purity and to quantitate the amount of the IgG in the SPAEs, radial immunodiffusion (RID) assays were used to determine the levels of IgG, IgA, and IgM in fraction 2 of the SPAEs from the daily (24-, 48-, 72-, and 96-h) supernatant fluids of specimens 52, 58, 70, 78, 79, and 80.
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TABLE 1. Results* of scintillation counts for fraction 2 of SPAE using either 1 ml of 24-h supernatant fluids from six periapical lesions or 1 ml of 24-h supernatant fluids from six periapical lesions that had been frozent before incubation cpm
Periapical specimen (24-h incubation) SPAE 52 SPAE 58 SPAE 70 SPAE 78 SPAE 79 SPAE 80 Frozen periapical specimen (24-h incubation) SPAE F1 SPAE F2 SPAE F3 SPAE F4 SPAE F5 SPAE F6 Scintillation blanks:[: 1 2 3 4 5 6 7 8 9
Trial 1
Trial 2
Average
760 231 102 81 730 252
1008 236 109 76 726 251
884 234 106 78 728 252
53 32 55 47 66 56
54 31 53 48 67 58
54 32 54 48 66 57
14 12 13 14 14 16 16 13 23
13 17 14 13 14 18 13 15 24
14 14 14 14 14 17 14 14 24
* One-way ANOVA showed that the difference in the cpm for each of the above groups is statistically significant at p < 0.05. i" At the time of tooth extraction, each tooth with the attached periapical lesion was frozen at -20~ :~ Scintillation blanks consisted of the vial with 10 ml of scintillation cocktail only.
Calibration curves were produced by plotting the values for the reference standards of immunoglobulin in mg/L versus the squares of the diameters of the precipitation rings. The calibration standards for IgG, IgA, or IgM all produced precipitation rings and were considered the positive controls. The staphylococcal protein A elution buffer which produced no precipitation ring was considered the negative control. Neither IgA nor lgM was detected in any of the SPAEs from the supernatant fluids used in this experiment. The results of using RID to detect and quantitate the amount of IgG are presented in Table 2 along with the scintillation cpm for each SPAE. The amount of IgG detected in each successive daily (24-, 48-, 72-, and 96-h) supernatant fluid from each sample always decreased or fell below the lower level of detection. The daily decrease in the amount of IgG detected using RID was significant (ANOVA; F = 6.45, p = 0.005). In addition to the RID assay, the average number ofcpm in each successive daily (24-, 48-, 72-, and 96-h) supernatant fluid from each of the above six specimens was also determined and shown to always decrease (Table 1). The daily decrease in cpm was also significant (ANOVA: F = 5.62, p = 0.009). DISCUSSION To demonstrate in vitro biosynthesis, periapical lesions were cultured in enriched RPMI 1640 containing tritiated
FIG 1. Lines of precipitation demonstrating the presence of IgG were produced when elution samples (52, 58, 60, 64, 79, and 80) of isolated IgG from affinity columns containing staphylococcal protein A were placed in the peripheral wells and allowed to react by double diffusion in agarose with goat anti-human 3' chain serum in the center wells.
leucine and glutamine. Using fraction 2 of the SPAEs, which contains the majority of the isolated IgG, a significant difference in the cpm was shown between the periapical lesions cultured in RPMI 1640 containing tritiated amino acids, lesions frozen in RPMI 1640 containing tritiated amino acids, and scintillation blanks. The detection of tritiated amino acids in isolated IgG proves that IgG was being synthesized in vitro in explant cultures and strongly suggest that the local production of IgG takes place in vivo in periapical lesions. This is the first time that tritiated amino acids have been used to demonstrate the biosynthesis of IgG in explant cultures of periapical lesions associated with untreated root canal infections. This study supports the finding of IgG containing mononuclear cells in periapical lesions by other investigators using other methods (2, 3, 14, 15). It is interesting that the frozen lesions produced some tritium-labeled IgG at a level significantly lower than the unfrozen specimens, but significantly greater than the scintillation blanks. Plasma cells are capable of releasing thousands of molecules of immunoglobulin in a second (16). Apparently enough labeled IgG accumulated in the length of time it took to freeze the cells to yield a significant scintillation count from background radiation. It is also possible that some of the plasma cells or B ceils (lymphocytes) were able to survive the one-time freezing, or perhaps molecules of IgG in the
146
Baumgartner and Falkler
Journal of Endodontics
1,035 101 83 17
supernatant fluids was being diluted when the culture medium was replenished each day. It is likely that both the amount of biosynthesis and the amount of serum IgG decrease with each successive day. The concentrations of IgG as determined by RID do not directly correspond from specimen to specimen with the scintillation cpm. This suggests that the rate of in vitro biosynthesis is different with each specimen. The latter would probably be related to the number and activity of B lymphocytes and plasma cells.
48 ND ---
235 130 22 18
The informed consent of all human subjects who participated in the experimental investigation reported or described in this manuscript was obtained after the nature of the procedure and possible discomforts and risks had been fully explained.
26.6 16.6 13.8 9.1
47 19 ND ND
117 24 27 16
7.28 5.89 4.74 4.12
53.0 34.7 22.5 17.0
122 70 35 19
85 30 28 17
9.20 7.95 4.66 ND
84.6 63.2 21.7 ND
525 82 150 32 --
744 330 146 66
8.16 4.78 ND ND
66.6 22.8 ND ND
159 35 ---
267 176 34 20
8.92 6.95 3.93 ND
79.6 48.3 15.4 --
200 120 20 --
-----
TABLE 2. Results of radial immunodiffusion and scintillation counts using fraction 2 of daily (24 to 96 h)* SPAE SPAE
NO. 52 (h) 24 48 72 96 NO. 58 (h) 24 48 72 96
Diameter of Average Precipitation Diameter Mg:~ Average Ring (ram)l" Squared IgG/L cpmw
8.47 4.81 4.05 3.46
71.7 23.1 16.4 12.0
438 82 37 37 NDII
5.18 3.87 ND ND
26.8 15.0 ND ND
5.16 4.08 3.72 3.02
No. 7O (h) 24 48 72 96
No. 78 (h) 24 48 72 96 No. 79 (h) 24 48 72 96
No. 80 (h) 24 48 72 96 Standards used for calibration curve A B C SPAE Buffer
* A significantdaily decreaseof IgG (ANOVA:F = 6,45, p = 0.002) and of cpm (ANOVA: F = 5.62, p = 0.009) was detected. 1 Averageof two readings. :1:mg IgG/L determined from calibration curve using three standard concentrationsof IgG versussquareof the diameterof the precipitationrings. wAveragescintillationcpm for 100-ul samplesof SPAEused in RID assay. Data correctedbecauseSPAE52 (24 h) and 79 (24 h) were diluted 2:5. IIND, no determinationof the quantityof immunoglobulinbecausea lineof precipitation was not detectedor the extrapolatedvalueusing regressionanalysiswas negative.
latter stages of biosynthesis were able to bind some of the labeled amino acids. The specificity of the staphylococcal protein A affinity columns for IgG was supported because neither IgA nor IgM could be detected in fraction two of the SPAEs from the 24h supernatant fluids of specimens 52, 58, 70, 78, 79, and 80 by using low-level IgA or IgM RID assay kits. The presence of IgG in SPAE was first demonstrated by double diffusion in agarose. RID was then used to detect and quantitate the level of IgG in fraction 2 of the SPAEs from daily (24-, 48-, 72-, and 96-h) supernatant fluids of specimens 52, 58, 70, 78, 79, and 80 and the scintillation cpm for each corresponding SPAE both showed significant decreases with each successive day. This supports the premise that either the amount of biosynthesis of the immunoglobulins was decreasing with time or that the amount of serum IgG in the
The opinions and assertions herein are the private views of the authors and are not to be construed as official or reflecting the views of the Department of the Army or the Department of Defense. The authors express their thanks to the American Association of Endodontists Endowment and Memorial Foundation for funding the purchase of the tritiated amino acids used in this study. We express our appreciation to Mr. Robert Burge, Division of Biometrics, Walter Reed Army Center, Washington, DC, for his assistance with the statistical analysis of the data. Dr. Baumgartner is chief, Microbiology Branch, United States Army Institute of Dental Research, Walter Reed Army Medical Center, Washington, DC. Dr. Falkler is professor and chairman, Microbiology Department, University of Maryland Dental School, Baltimore, MD. Address requests for reprints to Dr. J. Craig Baumgartner, 10376 Currycomb Ct., Columbia, MD 21044. This study was part of a dissertation submitted in partial fulfillment of the requirements for the degree of PhD at the University of Maryland Dental School, Baltimore, MD.
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