Orat Microbiot Itmmttwt 1991: 6: 17-23
Divergent effect of the anaerobic bacteria by-product butyric acid on the immune response: suppression of T-iymphocyte proiiferation and stimuiation of interieul propionate > isobutyrate. T-cell inhibition was partially reversed, at least for propionic and isobutyric acids, by increasing the concentration of macrophages in the assay system. Furthermore, butyric acid displayed an interesting biphasic stimulation of monocyte interleukin-1 beta production, a cytokine with a powerful bone-resorbing activity. Since millimolar concentrations of SCFA are present in gingival fluid from periodontal pockets, the observed results support the role of these byproducts of anaerobic metabolism in the pathogenesis of periodontal diseases.
It is widely recognized that periodontal diseases are infectious in nature and that periodontal tissue breakdown results from the interaction of specific anaerobic bacteria and host immune mechanisms. Among the virulence factors of anaerobic bacteria, our interest has focused on the role of short-chain fatty acids (SCFA) in the pathogenesis of periodontal infections. SCFA are metabolic end-products of anaerobic bacteria metabolism that are released into the microenvironment at the infection site (13), and can diffuse across biological membranes (37). Singer & Buckner (38) originally proposed a possible etiological role of butyric and propionic acids in periodontal diseases. Their suggestion was based on the inhibitory effects of these molecules on gingival fibroblast prohferation. Pre-
vious studies from this and other laboratories have demonstrated that SCFA exert inhibitory effects on human polymorphonuclear leukocyte (PMN) chemotaxis (2, 28), degranulation (10, 30), chemiluminescence (10, 28), phagocytosis (11, 30), and phagocytic killing (28-30), along with inducing ultrastructural modifications (10). These findings are consistent with a pathogenic role of these molecules in periodontal diseases (44), a hypothesis further supported by the observations that SCFA are present in the periodontal pocket, and that the presence of butyric acid correlates with the extent of periodontal breakdown (1, 23). The aim of this investigation was to study the modulatory effects of SCFA on other components of host defenses. Specifically, we evaluated T-cell blasto-
Key words: butyrate: T-celi: macrophage: interleul propionic > iso-butyric acidmacrophage IL-1 beta production. The observed between 1.5 and 2 mM butyrdata show that butyric acid was in fact ate in all the donors. A small amount For all 3 acids, a significant inhibition a sufficient stimulus for IL-1 beta se- of IL-1 beta was produced by macro- vs control was observable at the lowest cretion. Although there was variability phages not stimulated with SCFA. In- concentration tested (0.75 mM). in IL-1 beta synthesis in response to terestingly, higher concentrations reThe significance of these in vitro efbutyrate among the different donors, a sulted in a decline in macrophage IL-1 fects depends upon the actual concenpattern of response common to the 3 beta production. It should be noted trations of SCFA achievable in perioexperimental subjects was observed. In that, in one donor (Fig. 6, bottom), the dontal lesions. Botta et al. (1) reported particular the regression curves (con- amount of IL-1 beta produced in the butyric acid concentrations ranging betinuous lines) clearly show that the presence of 8 mM butyric acid reached tween 0.25 and 1.14 mM in crevicular maximum production of IL-l beta was values lower than basal ones. Taken to- washings (10 //I) from single-site perio-
o
i
•
SCEA and lytnphocytes
8000 —
EQGQ —
4000 —
2000 —
Z000 —
1500 —
I C
1000 —
500 —
2000
1500
1000
sag
_l_l
I I I I L.
10
mM
21
dontal pockets. Since resting crevicular fluid volume has been estimated to be in the 0,5 /zl range (12), the method of sample collection employed, with no correction for the dilution effect, is likely to result in an underestimation of the real concentrations. In fact, it has been reported that butyric acid concentrations in subgingival plaque from periodontitis sites could reach 14,4 mM (23), Nonetheless, both studies agree that butyric acid concentrations correlate with periodontal disease severity (1, 6, 23), In this regard, although butyric acid is produced by several anaerobic species found in the periodontal pocket, production of large amounts of this acid is characteristic of Bacteroides gingivalis (32), which is recognized as one of the most important periodontal pathogens (9, 40), The data presented here indicate that SCFA, and butyric acid in particular, inhibit T-cell prohferation induced by different mitogenic stimuli. This observation is in agreement with an early report on the effect of butyrate on mouse spleen cells (18), Our results also demonstrate that the major inhibitory target of butyric acid is the T-cell and not the macrophage, T-cell inhibition could be partially reversed, at least for propionic and iso-butyric acids, by increasing the concentration of macrophages in the PHA proliferation assay up to a maximum of 40% of the T-cell number (50% in the mixed lymphocyte culture). This relatively high macrophage: T-cell ratio may be necessary to facilitate cell interactions that are critical for induction of the blastogenic response. Although ratios of approximately 10-20% are usually optimal in vitro for macrophage: T-cell induction phenomena, these requirements may be altered in the presence of SCFA that have a differential effect on the 2 cell types, Macrophages, indeed, appear to be stimulated by low concentrations of butyric acid, as demonstrated by the induction of IL1 beta production, IL-1 is an important mediator produced by macrophages (21), B-cells (25) and squamous epithelial cells (20), Moreover, IL-1 beta is the most potent of the bone-resorptive cytokines (41), and most bone-resorbing (OAF) activity in activated peripheral blood mononuclear cell supernatants is attributable to IL-1 beta on the basis of purification (8) and antibody neutralization studies (19). Of interest, IL-1 beta and other bone-resorptive cytokines also inhibit bone formation, an action
22
Eftimiadi et al.
that may contribute to the net loss of bone mass in chronic inflammatory diseases such as periodontitis (42), It is notable that butyric acid induced a biphasic dose response curve of IL-1 beta production. Maximum IL-1 beta production occurred at butyric acid concentrations of 1,5-2 mM, At higher concentrations IL-1 beta production dropped. This pattern of response may be explained as a potential regulatory mechanism exerted by butyrate on macrophage IL-1 beta expression. Since butyrate is known to induce prostaglandin synthetase activity in mammalian cells (16), and prostaglandins inhibit IL1 translation (15), the role of prostaglandins in mediating this effect deserves further study. The relevance, if any, of this mechanism in modulating iti vivo bone resorption associated with periodontal diseases remains to be elucidated. In any case, the results reported above indicate that SCFA are important virulence factors which affect the host defence mechanisms. These findings further support the hypothesis of a role of these bacterial end-products in the pathogenesis of periodontal disease. Acknowledgement
This work was supported in part by grants DE-07378 and DE-04815 from the National Institute of Dental Research.
References 1, Botta GA, Radin L, Costa A, Schilo GC, Blasi G, Gas-liquid chromatography of gingival fluid as an aid of periodontal diagnosis, J Periodont Res 1985: 20: 450-457, 2, Botta GA, Eftimiadi C, Costa A, Tonetti M, van Steenbergen TJM, de Graaff J, Influence of volatile fatty acids on human granulocyte chemotaxis, FEMS • Mierobiol 1985: 27: 69-72, 3, Colotta F, Peri G, Villa A, Mantovani A, Rapid killing of Actinomycin D-treated tumor cells by human mononuclear cells, I, Effectors belong to the monocyte-macrophage lineage, J Immunol 1984: 132: 936-944, 4, Corte G, Damiani F, Calabi F, Fabbi M, Bargellesi A, Analy,sis of HLA-DR polymorphism by two dimensional peptide mapping, Proe Natl Acad Sci USA 1981:78: 534-538, 5, Csordas A, On the biological role of histone acetylation, Biochem J 1990: 265: 23-38,
6, Curtis MA, Gillett ER. Griffiths GS et al. Detection of high risk groups and individuals for periodontal diseases: laboratory markers from analysis of gingival crevicular fluid, J Clin Periodontol 1989: 16: I-II, 7, deVries JE, Cavites A, Bont WS, Mendelsohn J, The role of monocytes in human lymphocyte activation by mitogens, J Immunol 1979: 122: 1099-1107, 8, Dewhirst FE, Stashenko P, Mole JE, Tsurumachi T, Purification and partial sequence of human osteoclast-activating factor: identity with interleukin 1 beta, J Immunol 1985: 135: 2562-2568, 9, Dzink JL, Soeransky SS, Haffajee AD, The predominant cultivable microbiota of active and inactive lesions of destructive periodontal diseases, J Chn Periodontol 1988: 15: 316-323, 10, Eftimiadi C, Buzzi E. Tonetti M et al. Short chain fatty acids produced by anaerobic bacteria alter the physiological responses of human neutrophils to chemotactic peptide, J Infect 1987: 14: 43-53, 11, Eftimiadi C, Tonetti M, Cavallero A, Saeco O, Rossi GA, Short chain fatty acids produced by anaerobic bacteria inhibit phagocytosis by human lung phagocytes, J Infect Dis 1990: 161: 138-142, 12, Goodson JM, Pharmacokinetic principles controlling efficacy of oral therapy, J Dent Res 1989: 68s: 1625-1632, 13, Gorbach SL, Mayhew .TW, Bartlett JG, Thadepalli H, Onderdonk AB, Rapid diagnosis of anaerobic infections by direct gas liquid chromatography of clinical specimens, J Clin Invest 1976: 57: 478-484, 14, Ivanyi L, Immunosuppression in severe periodontitis. In: Lehner T, Cimasoni G, eds. Borderland between caries and periodontal diseases, Geneve: Editions Medicine et Hygiene, 1986: 223-232, 15, Knudsen PJ, Dinarello CA, Strom TB, Prostaglandins post-transcriptionally inhibit monocyte expression of interleukin I activity by increasing intracellular eyelie adenosine monophosphate, J Immunol 1986: 137: 3189-3194, 16, Koshihara Y, Kawamura M, Senshu T, Murota S, Effeet of sodium n-butyrate on induction of prostaglandin synthetase activity in eloned mastocytoma P-818 2E-6 cells, Biochem J 1981: 194: 111117, 17, Kruh J, Effects of sodium butyrate, a new pharmacological agent, on cells in eulture, Mol Cell Biochem 1982: 42: 65-82, 18, Kyner D, Zabos P, Christman J, Acs G, Effect of sodium butyrate on lymphocyte activation, J Exp Med 1976: 144: 1674-1678, 19, Lorenzo JA, Souza S, Alander C, Raisz L, Dinarello CA, Comparison of the bone resorbing activity in supernatants from phytohemagglutinin-stimulated
human peripheral blood mononuelear cells with that of cytokines through the use of an antiserum to interleukin I, Endocrinology 1987: 121: 1164-1170, 20, Mergenhagen SE, Thymocyte activating factors in human gingival fluids, J Dent Res 1984: 63: 461-4, 21, Mizel SB, Farrar JJ, Revised nomenclature for antigen non-specific T-cell proliferation and helper faetors. Cell Immunol 1979: 48: 433-36, 22, Moretta L, Ferrarini M, Cooper MD, Characterization of human T-cell subpopulations as defined by specific receptors for immunoglobuhns, Contemp Top Immunobiol 1978: 8: 27-54, 23, Naleway C, Chou H, Manos T, Goodman C, Robinson P, Singer R, Assessment of the potential relationship between levels of SCFA found in subgingival plaque and periodontal health, J Dent Res 1989: 68s: 121, 24, Okada H, Tomonobu K, Yainagami H, Identification and distribution of iinmunocompetent cells in inflamed gingiva of human chronic periodontitis. Infect Immun 1983: 41: 365-374, 25, Pistoia V, Cozzolino F, Rubartelli A, Toreia M, Roncella S, Ferrarini M, /" vitro production of IL-1 by normal and malignant human B lymphocytes, J Immunol 1986: 135: 1688-1692, 26, Pistoia V, Cozzolino F, Toreia M, Castigli E, Ferrarini M, Production of B cell growth factor by Leu-7 -I-, OKM1 + nonT cell with the feature of large granular lymphocytes (LGL), J Immunol 1985: 134: 3179-3184, 27, Prasad KN, Butyric acid: a small fatty acid with diverse biological functions. Life Sei 1980: 27: 1351-1358, 28, Rotstein OD, Pruett TL, Fiegel VD, Nelson RD, Simmons RL, Succinic acid: a metabolic by-product of Bacteroides species inhibits polymorphonuclear leukocyte function. Infect Immun 1985: 48: 402-408, 29, Rotstein OD, Nasmith PE, Grinstein S, The Bacteroides by-product succinic acid inhibits neutrophil respiratory burst by reducing intracellular pH, Infect Immun 1987: 55: 864-870, 30, Rotstein OD, Vittorini T, Kao J, Me Buiney ME, Nasmith PE, Grinstein S, A soluble Bacteroides by-product impairs phagoeytic killing of Escherichia coli by neutrophils. Infect Iminun 1989: 57: 745-753, 31, Seymour GJ, Possible mechanisms involved in the immune regulation of chronic inllammatory periodontal diseases, J Dent Res 1987: 66: 2-9, 32, Shah HN, Collins MD, Proposal for reclassification of Bacteroides asaccharolyticus, Bacteroides gingivalis, and Bacteroides endodontalis in a new genus, Porphyromonas. Int J Syst Bacteriol 1988: 38: 128-131, 33, Shenker BJ, Listgarten MA, Taichman NS, Suppression of human lymphocyte
SCEA atid lymphocvtes
34.
35.
36.
37.
responses by oral spirochetes: a raonocyte dependent phenomenon. J Immunol 1984: 132: 2039-2045. Shenker BJ, McArthur WP, Tsai CC. Immune suppression induced by Actitwbacillus actinomycetetncomitatis. I. Effects on human peripheral blood lymphocyte responses to mitogens and antigens. J Immunol 1982: 128: 148-154. Shenker BJ, DiRienzo JM. Suppression of human peripheral lymphocytes by Fusobacteriwn nucleatum. J Immunol 1984: 132: 2357-2362. Shenker BJ, Tsai CC, Taichman NS. Suppression of lymphocyte responses by Actinobacillus actinomvcetemcomitans. J Periodont Res 1982: 17: 462-465. Siegel IA. Permeability of oral mucosa
38.
39.
40.
41.
to organic acids. J Dent Res 1977: 56B: 64s. Singer RE, Buckner BA. Butyrate and propionate: important components of toxic dental plaque extracts. Infect Immun 1981: 32: 458-463. Singer RE, Buckner BA, Meckel AH, Leonard GJ. Propionate and butyrate induction of gingival inflammation in the beagle. J Dent Res 1980: 59: 435s. Slots J, Genco RJ. Black pigmented Bacteroides species, Captiocytophaga species and Actitiobacillus actitiomycctetneotnitans in human periodontal disease: virulence factors in colonization, survival, and tissue distruction. J Dent Res 1984: 63: 412-421. Stashenko P, Dewhirst FE, Peros WJ,
23
Kent RL, Ago JM. Synergistic interaction between interleukin I, tumor necrosis factor and lymphotoxin in bone resorption. J Immunol 1987: 138: 1464-1468. 42. Stashenko P, Obernesser MS, Dewhirst FE. Effect of immune cytokines on bone. Immunol Invest 1989: 18: 239-249. 43. Taubman MA, Stoufi ED, Seymour GJ, Smith DJ, Ebersole JL. Immunoregulatory aspects of periodontal disease. Adv Dent Res 1988: 2: 328-333. 44. Tonetti M, Eftimiadi C, Damiani G, Buffa P, Buffa D, Botta GA. Short chain fatty acids present in periodontal pockets may play a role in human periodontal diseases. J Periodont Res 1987: 22190-191.
Divergent effect of the anaerobic bacteria by-product butyric acid on the immune response: suppression of T-iymphocyte proiiferation and stimuiation of interieul propionate > isobutyrate. T-cell inhibition was partially reversed, at least for propionic and isobutyric acids, by increasing the concentration of macrophages in the assay system. Furthermore, butyric acid displayed an interesting biphasic stimulation of monocyte interleukin-1 beta production, a cytokine with a powerful bone-resorbing activity. Since millimolar concentrations of SCFA are present in gingival fluid from periodontal pockets, the observed results support the role of these byproducts of anaerobic metabolism in the pathogenesis of periodontal diseases.
It is widely recognized that periodontal diseases are infectious in nature and that periodontal tissue breakdown results from the interaction of specific anaerobic bacteria and host immune mechanisms. Among the virulence factors of anaerobic bacteria, our interest has focused on the role of short-chain fatty acids (SCFA) in the pathogenesis of periodontal infections. SCFA are metabolic end-products of anaerobic bacteria metabolism that are released into the microenvironment at the infection site (13), and can diffuse across biological membranes (37). Singer & Buckner (38) originally proposed a possible etiological role of butyric and propionic acids in periodontal diseases. Their suggestion was based on the inhibitory effects of these molecules on gingival fibroblast prohferation. Pre-
vious studies from this and other laboratories have demonstrated that SCFA exert inhibitory effects on human polymorphonuclear leukocyte (PMN) chemotaxis (2, 28), degranulation (10, 30), chemiluminescence (10, 28), phagocytosis (11, 30), and phagocytic killing (28-30), along with inducing ultrastructural modifications (10). These findings are consistent with a pathogenic role of these molecules in periodontal diseases (44), a hypothesis further supported by the observations that SCFA are present in the periodontal pocket, and that the presence of butyric acid correlates with the extent of periodontal breakdown (1, 23). The aim of this investigation was to study the modulatory effects of SCFA on other components of host defenses. Specifically, we evaluated T-cell blasto-
Key words: butyrate: T-celi: macrophage: interleul propionic > iso-butyric acidmacrophage IL-1 beta production. The observed between 1.5 and 2 mM butyrdata show that butyric acid was in fact ate in all the donors. A small amount For all 3 acids, a significant inhibition a sufficient stimulus for IL-1 beta se- of IL-1 beta was produced by macro- vs control was observable at the lowest cretion. Although there was variability phages not stimulated with SCFA. In- concentration tested (0.75 mM). in IL-1 beta synthesis in response to terestingly, higher concentrations reThe significance of these in vitro efbutyrate among the different donors, a sulted in a decline in macrophage IL-1 fects depends upon the actual concenpattern of response common to the 3 beta production. It should be noted trations of SCFA achievable in perioexperimental subjects was observed. In that, in one donor (Fig. 6, bottom), the dontal lesions. Botta et al. (1) reported particular the regression curves (con- amount of IL-1 beta produced in the butyric acid concentrations ranging betinuous lines) clearly show that the presence of 8 mM butyric acid reached tween 0.25 and 1.14 mM in crevicular maximum production of IL-l beta was values lower than basal ones. Taken to- washings (10 //I) from single-site perio-
o
i
•
SCEA and lytnphocytes
8000 —
EQGQ —
4000 —
2000 —
Z000 —
1500 —
I C
1000 —
500 —
2000
1500
1000
sag
_l_l
I I I I L.
10
mM
21
dontal pockets. Since resting crevicular fluid volume has been estimated to be in the 0,5 /zl range (12), the method of sample collection employed, with no correction for the dilution effect, is likely to result in an underestimation of the real concentrations. In fact, it has been reported that butyric acid concentrations in subgingival plaque from periodontitis sites could reach 14,4 mM (23), Nonetheless, both studies agree that butyric acid concentrations correlate with periodontal disease severity (1, 6, 23), In this regard, although butyric acid is produced by several anaerobic species found in the periodontal pocket, production of large amounts of this acid is characteristic of Bacteroides gingivalis (32), which is recognized as one of the most important periodontal pathogens (9, 40), The data presented here indicate that SCFA, and butyric acid in particular, inhibit T-cell prohferation induced by different mitogenic stimuli. This observation is in agreement with an early report on the effect of butyrate on mouse spleen cells (18), Our results also demonstrate that the major inhibitory target of butyric acid is the T-cell and not the macrophage, T-cell inhibition could be partially reversed, at least for propionic and iso-butyric acids, by increasing the concentration of macrophages in the PHA proliferation assay up to a maximum of 40% of the T-cell number (50% in the mixed lymphocyte culture). This relatively high macrophage: T-cell ratio may be necessary to facilitate cell interactions that are critical for induction of the blastogenic response. Although ratios of approximately 10-20% are usually optimal in vitro for macrophage: T-cell induction phenomena, these requirements may be altered in the presence of SCFA that have a differential effect on the 2 cell types, Macrophages, indeed, appear to be stimulated by low concentrations of butyric acid, as demonstrated by the induction of IL1 beta production, IL-1 is an important mediator produced by macrophages (21), B-cells (25) and squamous epithelial cells (20), Moreover, IL-1 beta is the most potent of the bone-resorptive cytokines (41), and most bone-resorbing (OAF) activity in activated peripheral blood mononuclear cell supernatants is attributable to IL-1 beta on the basis of purification (8) and antibody neutralization studies (19). Of interest, IL-1 beta and other bone-resorptive cytokines also inhibit bone formation, an action
22
Eftimiadi et al.
that may contribute to the net loss of bone mass in chronic inflammatory diseases such as periodontitis (42), It is notable that butyric acid induced a biphasic dose response curve of IL-1 beta production. Maximum IL-1 beta production occurred at butyric acid concentrations of 1,5-2 mM, At higher concentrations IL-1 beta production dropped. This pattern of response may be explained as a potential regulatory mechanism exerted by butyrate on macrophage IL-1 beta expression. Since butyrate is known to induce prostaglandin synthetase activity in mammalian cells (16), and prostaglandins inhibit IL1 translation (15), the role of prostaglandins in mediating this effect deserves further study. The relevance, if any, of this mechanism in modulating iti vivo bone resorption associated with periodontal diseases remains to be elucidated. In any case, the results reported above indicate that SCFA are important virulence factors which affect the host defence mechanisms. These findings further support the hypothesis of a role of these bacterial end-products in the pathogenesis of periodontal disease. Acknowledgement
This work was supported in part by grants DE-07378 and DE-04815 from the National Institute of Dental Research.
References 1, Botta GA, Radin L, Costa A, Schilo GC, Blasi G, Gas-liquid chromatography of gingival fluid as an aid of periodontal diagnosis, J Periodont Res 1985: 20: 450-457, 2, Botta GA, Eftimiadi C, Costa A, Tonetti M, van Steenbergen TJM, de Graaff J, Influence of volatile fatty acids on human granulocyte chemotaxis, FEMS • Mierobiol 1985: 27: 69-72, 3, Colotta F, Peri G, Villa A, Mantovani A, Rapid killing of Actinomycin D-treated tumor cells by human mononuclear cells, I, Effectors belong to the monocyte-macrophage lineage, J Immunol 1984: 132: 936-944, 4, Corte G, Damiani F, Calabi F, Fabbi M, Bargellesi A, Analy,sis of HLA-DR polymorphism by two dimensional peptide mapping, Proe Natl Acad Sci USA 1981:78: 534-538, 5, Csordas A, On the biological role of histone acetylation, Biochem J 1990: 265: 23-38,
6, Curtis MA, Gillett ER. Griffiths GS et al. Detection of high risk groups and individuals for periodontal diseases: laboratory markers from analysis of gingival crevicular fluid, J Clin Periodontol 1989: 16: I-II, 7, deVries JE, Cavites A, Bont WS, Mendelsohn J, The role of monocytes in human lymphocyte activation by mitogens, J Immunol 1979: 122: 1099-1107, 8, Dewhirst FE, Stashenko P, Mole JE, Tsurumachi T, Purification and partial sequence of human osteoclast-activating factor: identity with interleukin 1 beta, J Immunol 1985: 135: 2562-2568, 9, Dzink JL, Soeransky SS, Haffajee AD, The predominant cultivable microbiota of active and inactive lesions of destructive periodontal diseases, J Chn Periodontol 1988: 15: 316-323, 10, Eftimiadi C, Buzzi E. Tonetti M et al. Short chain fatty acids produced by anaerobic bacteria alter the physiological responses of human neutrophils to chemotactic peptide, J Infect 1987: 14: 43-53, 11, Eftimiadi C, Tonetti M, Cavallero A, Saeco O, Rossi GA, Short chain fatty acids produced by anaerobic bacteria inhibit phagocytosis by human lung phagocytes, J Infect Dis 1990: 161: 138-142, 12, Goodson JM, Pharmacokinetic principles controlling efficacy of oral therapy, J Dent Res 1989: 68s: 1625-1632, 13, Gorbach SL, Mayhew .TW, Bartlett JG, Thadepalli H, Onderdonk AB, Rapid diagnosis of anaerobic infections by direct gas liquid chromatography of clinical specimens, J Clin Invest 1976: 57: 478-484, 14, Ivanyi L, Immunosuppression in severe periodontitis. In: Lehner T, Cimasoni G, eds. Borderland between caries and periodontal diseases, Geneve: Editions Medicine et Hygiene, 1986: 223-232, 15, Knudsen PJ, Dinarello CA, Strom TB, Prostaglandins post-transcriptionally inhibit monocyte expression of interleukin I activity by increasing intracellular eyelie adenosine monophosphate, J Immunol 1986: 137: 3189-3194, 16, Koshihara Y, Kawamura M, Senshu T, Murota S, Effeet of sodium n-butyrate on induction of prostaglandin synthetase activity in eloned mastocytoma P-818 2E-6 cells, Biochem J 1981: 194: 111117, 17, Kruh J, Effects of sodium butyrate, a new pharmacological agent, on cells in eulture, Mol Cell Biochem 1982: 42: 65-82, 18, Kyner D, Zabos P, Christman J, Acs G, Effect of sodium butyrate on lymphocyte activation, J Exp Med 1976: 144: 1674-1678, 19, Lorenzo JA, Souza S, Alander C, Raisz L, Dinarello CA, Comparison of the bone resorbing activity in supernatants from phytohemagglutinin-stimulated
human peripheral blood mononuelear cells with that of cytokines through the use of an antiserum to interleukin I, Endocrinology 1987: 121: 1164-1170, 20, Mergenhagen SE, Thymocyte activating factors in human gingival fluids, J Dent Res 1984: 63: 461-4, 21, Mizel SB, Farrar JJ, Revised nomenclature for antigen non-specific T-cell proliferation and helper faetors. Cell Immunol 1979: 48: 433-36, 22, Moretta L, Ferrarini M, Cooper MD, Characterization of human T-cell subpopulations as defined by specific receptors for immunoglobuhns, Contemp Top Immunobiol 1978: 8: 27-54, 23, Naleway C, Chou H, Manos T, Goodman C, Robinson P, Singer R, Assessment of the potential relationship between levels of SCFA found in subgingival plaque and periodontal health, J Dent Res 1989: 68s: 121, 24, Okada H, Tomonobu K, Yainagami H, Identification and distribution of iinmunocompetent cells in inflamed gingiva of human chronic periodontitis. Infect Immun 1983: 41: 365-374, 25, Pistoia V, Cozzolino F, Rubartelli A, Toreia M, Roncella S, Ferrarini M, /" vitro production of IL-1 by normal and malignant human B lymphocytes, J Immunol 1986: 135: 1688-1692, 26, Pistoia V, Cozzolino F, Toreia M, Castigli E, Ferrarini M, Production of B cell growth factor by Leu-7 -I-, OKM1 + nonT cell with the feature of large granular lymphocytes (LGL), J Immunol 1985: 134: 3179-3184, 27, Prasad KN, Butyric acid: a small fatty acid with diverse biological functions. Life Sei 1980: 27: 1351-1358, 28, Rotstein OD, Pruett TL, Fiegel VD, Nelson RD, Simmons RL, Succinic acid: a metabolic by-product of Bacteroides species inhibits polymorphonuclear leukocyte function. Infect Immun 1985: 48: 402-408, 29, Rotstein OD, Nasmith PE, Grinstein S, The Bacteroides by-product succinic acid inhibits neutrophil respiratory burst by reducing intracellular pH, Infect Immun 1987: 55: 864-870, 30, Rotstein OD, Vittorini T, Kao J, Me Buiney ME, Nasmith PE, Grinstein S, A soluble Bacteroides by-product impairs phagoeytic killing of Escherichia coli by neutrophils. Infect Iminun 1989: 57: 745-753, 31, Seymour GJ, Possible mechanisms involved in the immune regulation of chronic inllammatory periodontal diseases, J Dent Res 1987: 66: 2-9, 32, Shah HN, Collins MD, Proposal for reclassification of Bacteroides asaccharolyticus, Bacteroides gingivalis, and Bacteroides endodontalis in a new genus, Porphyromonas. Int J Syst Bacteriol 1988: 38: 128-131, 33, Shenker BJ, Listgarten MA, Taichman NS, Suppression of human lymphocyte
SCEA atid lymphocvtes
34.
35.
36.
37.
responses by oral spirochetes: a raonocyte dependent phenomenon. J Immunol 1984: 132: 2039-2045. Shenker BJ, McArthur WP, Tsai CC. Immune suppression induced by Actitwbacillus actinomycetetncomitatis. I. Effects on human peripheral blood lymphocyte responses to mitogens and antigens. J Immunol 1982: 128: 148-154. Shenker BJ, DiRienzo JM. Suppression of human peripheral lymphocytes by Fusobacteriwn nucleatum. J Immunol 1984: 132: 2357-2362. Shenker BJ, Tsai CC, Taichman NS. Suppression of lymphocyte responses by Actinobacillus actinomvcetemcomitans. J Periodont Res 1982: 17: 462-465. Siegel IA. Permeability of oral mucosa
38.
39.
40.
41.
to organic acids. J Dent Res 1977: 56B: 64s. Singer RE, Buckner BA. Butyrate and propionate: important components of toxic dental plaque extracts. Infect Immun 1981: 32: 458-463. Singer RE, Buckner BA, Meckel AH, Leonard GJ. Propionate and butyrate induction of gingival inflammation in the beagle. J Dent Res 1980: 59: 435s. Slots J, Genco RJ. Black pigmented Bacteroides species, Captiocytophaga species and Actitiobacillus actitiomycctetneotnitans in human periodontal disease: virulence factors in colonization, survival, and tissue distruction. J Dent Res 1984: 63: 412-421. Stashenko P, Dewhirst FE, Peros WJ,
23
Kent RL, Ago JM. Synergistic interaction between interleukin I, tumor necrosis factor and lymphotoxin in bone resorption. J Immunol 1987: 138: 1464-1468. 42. Stashenko P, Obernesser MS, Dewhirst FE. Effect of immune cytokines on bone. Immunol Invest 1989: 18: 239-249. 43. Taubman MA, Stoufi ED, Seymour GJ, Smith DJ, Ebersole JL. Immunoregulatory aspects of periodontal disease. Adv Dent Res 1988: 2: 328-333. 44. Tonetti M, Eftimiadi C, Damiani G, Buffa P, Buffa D, Botta GA. Short chain fatty acids present in periodontal pockets may play a role in human periodontal diseases. J Periodont Res 1987: 22190-191.
