Archs oral Biol. Vol. 35,No. 4,pp.325-328,1990 Printed in Great Britain. ,411rights reserved
0003-9969190 $3.00+O.OO Copyright 0 1990 Pergamon Press plc
SHORT
COMMUNICATION
1:NCREASED INTRACELLULAR LEVELS OF @GLUCURONIDASE IN POLYMORPHONUCLEAR LEUCOCYTES FROM HUMANS WITH RAPIDLY PROGRESSIVE PERIODONTITIS D.J. PIPPIN Department
of Periodontology, University of Missouri at Kansas City School 650 East 25 St, Kansas City, MO 64108, U.S.A.
of Dentistry,
(Accepted 12 October 1989) Summary-The tissue destruction resulting from release of lysosomal enzymes by exocytosis and degranulation of polymorphonuclear leucocytes in host gingiva may contribute significantly to periodontal diseases. In this investigation peripheral blood was obtained from healthy controls and otherwise healthy individuals w:th rapidly progressive periodontitis. Polymorphonuclear leucocytes were isolated and suspended in HBSS for subsequent in vitro FMLP challenge to induce degranulation. The supernatant was tested for p-glucuronidase. Polymorphonuclear leucocytes from patients with rapidly progressive periodontitis contained significantly higher absolute amounts of p-glucuronidase (p < 0.001) and released greater amounts at various molarities of FMLP antigenic challenge (p < 0.01). Such an increase in lysosomal enzyme activity may provide an enhanced potential for tissue destruction in this periodontal disease. Key words:
/?-glucuronidase,
polymorphonuclear
leucocyte,
periodontal
diseases.
lysosomal enzyme released during stimulus-response coupling, was compared to amounts released by polymorphonuclear leucocytes of healthy controls. The control group of 5 medically and periodontally healthy volunteers ranged in age from 27 to 35yr. The experimental group was 5 medically healthy patients between 27 and 35 yr of age with a clear diagnosis of rapidly progressive periodontitis according to the criteria of Page and Schroeder (1982). These patients had early-onset, rapid and severe generalized alveolar bone destruction with no consistent radiographic pattern; amounts of microbial dental plaque were variable. Bleeding upon periodontal probing was a prevalent clinical finding. For each subject, 45 ml of venous blood were aspirated from the antecubital fossa into a 60ml syringe containing 1000 U of heparin in 5 ml of phosphate-buffered saline to yield 20 U/ml. For sedimentation 12.5 ml of 6% Hetastarch (Hespan) were added to produce a 1.5% solution and the syringe placed needle upwards for 1 h. The resulting approx. 36 ml of leucocyte-rich upper serum fraction were expressed into 3 conical centrifuge tubes and centrifuged for 10 min at 150 g. Each cell button was aspirated with a volume of 4ml of serum. Each volume was layered onto a prepared Percoll gradient consisting of 4 ml of 1.092 g/ml, 292 mosmol Percoll and 4 ml of 1.076 g/ml, 292 mosmol Percoll and centrifuged for 25 min at 450 g. Each gradient yielded a virtually pure band of polymorphonuclear leucocytes at the Percoll interface. Each band was aspirated in 3 ml and suspended in 5 vol of HBSS. HBSS had been prepared with 5 mM HEPES buffer and 0.1% glucose and adjusted to 292 mosmol, pH. 7.35.
The neutrophilic polymorphonuclear granulocyte, responding to chemotactic substances elaborated by micro-organisms, provides a formidable defence against the challenge of subgingival microbial plaque. It may also be responsible for considerable host gingival tissue destruction. If dental plaque undergoes ecological succession resulting in colonization by periodontal pathogens that have the capacity to invade tissue (Frank, 1980; Saglie et al., 1982, 1988) then emigrating polymorphonuclear leucocyl.es would encounter invading microbes in the host gingival connective tissues. Lysosomal enzymes and inflammatory mediators would be released in quantity by the receptor-ligand events associated with polymorphonuclear leucocytes (Bang, Cimasoni and Held, 1970; Miller, Lamster and Chasens, 1984; Lamster et al., 1987). In the escalation of inflammation seen in gingivitis and periodontitis, widespread and sustained host tissue degradation would be produced by the release of potent lysosomal enzymes from polymorphonuclear leucocytes by antigen-elicited reverse endocytosis, frustrated phagocylosis, stimulation by antigenantibody complexes and complement, and autolytic death (Hamp and Folke, 1968; Taichman et al., 1977; Genco and Mergenhagen, 1979; Page and Schroeder, 1981). In my study, the secretion by polymorphonuclear leucocytes from patients with rapidly progressive periodontitis of fl-glucuronidase, a characteristic
Abbreviations: FMLP, formyl-methionyl-leucyl-phenylalanine; HBSS, Hank’s balanced salt solution. 325
326
D. J.
Cells were pelleted at 70 g for 10 min, the supernatant was removed and the cells resuspended in 5 vol HBSS. Cells were again pelleted at 70g for 10 min and the supernatant removed. The washed polymorphonuclear leucocytes were then suspended in HBSS plus calcium, pooled and counted in a haemocytometer. Cell viability was judged to be above 99% by trypan blue exclusion. Cell density was corrected to an interim concentration of 5 x IO6 polymorphonuclear leucocytes per 1.8 ml in HBSS plus calcium (CaCl, to yield 0.6 mM Ca in the final incubation volume) for subsequent incubation. All manipulations were performed at room temperature. Volumes of 1.8 ml of cell suspension were placed into 2.2 ml Eppendorf microtubes by Pasteur pipette and the experiments performed in duplicate (final concentration of 5 x lo6 cells in 2.0 ml). The polymorphonuclear leucocyte suspensions were preincubated at 37°C for 10min with cytochalasin B (Sigma Chemical Co., St Louis, MO, U.S.A.); 0.1 ml of stock was added to give a final concentration of 5 pg/ml. The induction of polymorphonuclear leucocyte lysosomal enzyme release was produced in vitro as a selective, non-cytotoxic receptor-ligand by challenge with FMLP (Sigma interaction Chemical Co.) in the presence of cytochalasin B and calcium (Smith, Wierenga and Iden, 1980). Portions of 0.1 ml FMLP were added (final molarities of 1 x 10-6, 5 x IO-’ and 1 x lo-‘M) and incubated for 5 min. The microtubes were then centrifuged at 750 g for 10 min and 1 ml of clear supernatant isolated and stored at -70°C for analysis the next day. Supernatants from unchallenged polymorphonuclear leucocytes (0.1 ml HBSS added instead of FMLP to give 5 x lo6 polymorphonuclear leucocytes in 2.0 ml) were used to determine non-specific enzyme release, which was subtracted from the FMLP challenges. Total enzyme content of 5 x lo6 polymorphonuclear leucocytes suspended in 2.0 ml of HBSS plus calcium was determined by sonication at 4°C in the presence of 0.3% Triton X-100. Exocytosis was monitored by a standard assay for /I-glucuronidase (Metcalf, 1986) consisting of cleavage of 0.03 M phenolphthalein glucuronic acid substrate (Sigma Chemical Co.) liberating free phenolphthalein, whose red colour intensity at alkaline pH is proportional to enzyme activity. After a 9 h incubation at 37°C pH 4.5, the reaction was stopped with glycine buffer at pH 10.5 and spectrophotometric analysis performed at 540 nm. Spectrophotometric values were compared to a previously prepared standard absorption curve of known phenolphthalein concentrations to determine pmoles of phenolphthalein present in the sample. Enzyme amounts are expressed as per cent of total activity (per cent of total cellular enzyme content) (Fig. 1) and as absolute amounts represented by pmoles of phenolphthalein (Fig. 2). When expressed as a per cent of total activity, degranulation of periodontitis and control polymorphonuclear leucocytes was not statistically significantly different (t-test) at any of the 3 FMLP concentrations (Fig. 1). Also, at decreasing molarities of FMLP incubation there was a corresponding decrease in response, as expected, for both groups of
PIPPIN
70 65 60 55 50I Q
45.
;
40.
1
35.
s
30.
3
25. 26,
5 0
p
non-ohrllmp
1 x 10%
5 x 10m7M
1 x 10-71
Fig. 1. Per cent of total activity at receptor-ligand degranulation induced by FMLP challenge. A = Rapidly progressive periodontitis subjects, x = control. Data expressed as mean + 1SD; values offset for clarity. polymorphonuclear leucocytes with no statistically significant difference between the slopes. The values for the non-challenge groups, used to assess spontaneous or non-specific degranulation, were low. When total intracellular absolute amounts were compared (Fig. 2), a statistically significant difference (r-test, p < 0.001) was noted between the periodontitis polymorphonuclear leucocytes and the control polymorphonuclear leucocytes with the periodontitis mean nearly twice that of the controls. This was reflected in the amounts of /I-glucuronidase released when challenged by FMLP incubation concentrations of 1 x 10m6, 5 x IO-’ and 1 x lo-‘M (Fig. 2). There was a statistically significant difference (t-test, p < 0.01) at each FMLP molarity with higher absolute amounts secreted by periodontitis leucocytes. Control values consistently exhibited a tighter cluster than periodontitis values, which showed greater standard deviations throughout the groups (Figs 1 and 2). Degranulation of periodontitis and control polymorphonuclear leucocytes was similar when expressed as per cent of total activity (Fig. 1). This is interpreted as periodontitis polymorphonuclear leucocytes having an unaltered capacity to degranulate and not being excessively irritable to stimulus. However, total intracellular enzyme content was significantly higher in periodontitis polymorphonuclear leucocytes and, thus, an equal percentage response resulted in the secretion of much higher absolute amounts of P-glucuronidase at all FMLP molarities (Fig. 2). Are the increased absolute amounts of lysosomal enzymes contained in the polymorphonuclear leucocytes from rapidly progressive periodontitis a reactive phenomenon or an a priori genetically determined polymorphonuclear leucocyte characteristic of the individual? The mature polymorphonuclear leucocyte contains 200-300 lysosomes per cell, with a ratio of 2 or 3 specific granules per azurophilic granule (Bainton, Ullyot and Farquhar, 1971; Bainton, Nichols and Farquhar, 1976; Murphy, 1976). Thus, intracellular amounts of lysosomal enzymes may vary
Increased
leucocyte
/I-glucuronidase
in periodontitis
327
500,
T
.318.0
1 288.2 284.4 222.8
1 lo
31.8
OL
f Non-chllennpe1 x 10m8M
5 x W7M
1 x 1O-7M
TotalActlrlty
Fig. 2. Absolute amounts of /I-glucuronidase released at receptor-ligand degranulation induced by FMLP challenge. A = Rapidly progressive periodontitis subjects, x = control. p-Glucuronidase activities are represented by pmoles phenolphthalein/9 h per 5 x lo6 cells. Data expressed as mean ) 1 SD.
But the assessment of genetic influences on such variance, especially if the expression is subtle, is difficult. Thlere is limited information on the individual phenotypic expression of the genome for numbers of lysosomes, the limits of synthesis of component enzymes or the number of mitotic events after the promyelocyte stage. However, it is well known that polymorphonuclear leucocyte granulopoiesis and maturation are regulated by a series of feedback loops determining rates of production and rates of release (Williams et al., 1983). As such, polymorphonuclear lieucocyte precursors are certainly sensitive to infection .and microbial products (ZuckerFranklin, 1975). An individual with rapidly progressive periodontitis does indeed have a significant on-going infection and one may speculate on the propensity for that process to induce increased amounts of lysosomal enzymes. Whether genetically determined or induced, such an alteration in the cytochemistry of the polymorphonuclear leucocyte may play a part in the multifactorial pathogenesis of periodontal disease. If the quantity of deleterious extracellular lysosomal enzymes and immunoreactive substances is substantially increased during the inflammatory response to microbial plaque in the host gingival connective tissue, then there is the potential for a significantly enhanced tissue destruction as well. physiologically.
AcknowledPements-I counsel of-Professor Dr Jim Faed.
am Alan
erateful for the advice Laws, Dr Max Shepherd
and and
REFERENCES
Bainton D. F. Ullyot J. L. and Farquhar M. G. (1971) The development of neutrophilic polymorphonuclear leukocytes in human bone marrow. J. exp. Med. 134,907-934.
Bainton D. F., Nichols B. A. and Farquhar M. G. (1976) Primary lysosomes of blood leukocytes. In: Lysosomes in Biology and Pathology (Edited by Dingle J. T. and Dean R. T.) Vol. 5, Chap. 1, pp. 3-32. North-Holland, Amsterdam. Bang J., Cimasoni G. and Held A. J. (1970) Betaglucuronidase correlated with inflammation in the exudate from human gingiva. Archs oral Biol. 15, 44545 1. Frank R. M. (1980) Bacterial penetration in the apical pocket wall of advanced human periodontitis. J. periodont. Rex 15, 563-573. Genco R. J. and Mergenhagen S. E. (1979) Summary of a workshop on leukocyte function in bacterial diseases with an emphasis on periodontal disease. J. infect. Dis. 139, 604612. Hamp S. E. and Folke L. E. (I 968) The lysosomes and their possible role in periodontal disease. Odonf. Tidsk. 76, 353-375. Lamster I. B., Harper D. S., Fiorello L. A., Oshrain R. L., Celenti R. S. and Gordon J. M. (1987) Lysosomal and cytoplasmic enzyme activity, crevicular fluid volume and clinical parameters characterizing gingival sites with shallow to intermediate probing depths. J. Periodont. 58, 614-621. Metcalf J. A. (1986) Laborarory Manual of Neutrophil Funcfion, pp. 147-148. Raven Press, New York. Miller D. R., Lamster I. B. and Chasens A. I. (1984) Role of the polymorphonuclear leukocyte in periodontal health and disease. J. clin. Periodont. 11, I-15. Murphy P. (1976) The Neutrophil, p. 40. Plenum Medical Book Co., New York. Page R. C. and Schroeder H. E. (1981) Current status of the host response in chronic marginal periodontitis. J. Periodont. 52, 47749 1. Page R. C. and Schroeder H. E. (1982) Periodontiris in Man and Other Animals, p. 227. Karger, New York. Saglie R., Newman M. G., Carranza F. A. and Pattison G. C. (1982) Bacterial invasion of gingiva in advanced periodontitis in humans. J. Periodont. 53, 217-222. Saglie F. R., Pertuiset J., Rezende M. T., Nestor M., Marfany A. and Cheng J. (1988) In situ correlative
328
D. J.
immuno-identification of mononuclear infiltrates and invasive bacteria in diseased gingiva. J. Periodont. 59, 688496.
Smith R. J., Wierenga W. and Iden S. S. (1980) Characteristics of N-formyl-methionyl-leucyl-phenylalanine as an inducer of lysosomal enzyme release from human neutrophils. Inflammation 4, 73-88. Taichman N. S., Tsai C.-C., Baehni P. C., Stoller N. and McArthur W. P. (1977) Interaction of inflammatory cells and oral microorganisms. IV. In vitro release of lysosomal constituents from polymorphonuclear leukocytes exposed
PIPPIN
to supragingival and subgingival bacterial plaque. Infect. Immun. 16, 1013-1023.
Williams W. J., Beutler E., Erslev A. J. and Lichtman M. A. (1983) Hematology, 3rd edn, pp. 759-763, 794799. McGraw-Hill, New York. Zucker-Franklin D. (1975) Physiological and pathological variations in the ultrastructure of neutrophils and monocytes. In: Clinics in Haematology. Vol. 4~3. Granulocyre and Monocyte Abnormalities (Edited by Lichtman M. A.) Chap. I, p. 493. Saunders, Philadelphia, PA.
0003-9969190 $3.00+O.OO Copyright 0 1990 Pergamon Press plc
SHORT
COMMUNICATION
1:NCREASED INTRACELLULAR LEVELS OF @GLUCURONIDASE IN POLYMORPHONUCLEAR LEUCOCYTES FROM HUMANS WITH RAPIDLY PROGRESSIVE PERIODONTITIS D.J. PIPPIN Department
of Periodontology, University of Missouri at Kansas City School 650 East 25 St, Kansas City, MO 64108, U.S.A.
of Dentistry,
(Accepted 12 October 1989) Summary-The tissue destruction resulting from release of lysosomal enzymes by exocytosis and degranulation of polymorphonuclear leucocytes in host gingiva may contribute significantly to periodontal diseases. In this investigation peripheral blood was obtained from healthy controls and otherwise healthy individuals w:th rapidly progressive periodontitis. Polymorphonuclear leucocytes were isolated and suspended in HBSS for subsequent in vitro FMLP challenge to induce degranulation. The supernatant was tested for p-glucuronidase. Polymorphonuclear leucocytes from patients with rapidly progressive periodontitis contained significantly higher absolute amounts of p-glucuronidase (p < 0.001) and released greater amounts at various molarities of FMLP antigenic challenge (p < 0.01). Such an increase in lysosomal enzyme activity may provide an enhanced potential for tissue destruction in this periodontal disease. Key words:
/?-glucuronidase,
polymorphonuclear
leucocyte,
periodontal
diseases.
lysosomal enzyme released during stimulus-response coupling, was compared to amounts released by polymorphonuclear leucocytes of healthy controls. The control group of 5 medically and periodontally healthy volunteers ranged in age from 27 to 35yr. The experimental group was 5 medically healthy patients between 27 and 35 yr of age with a clear diagnosis of rapidly progressive periodontitis according to the criteria of Page and Schroeder (1982). These patients had early-onset, rapid and severe generalized alveolar bone destruction with no consistent radiographic pattern; amounts of microbial dental plaque were variable. Bleeding upon periodontal probing was a prevalent clinical finding. For each subject, 45 ml of venous blood were aspirated from the antecubital fossa into a 60ml syringe containing 1000 U of heparin in 5 ml of phosphate-buffered saline to yield 20 U/ml. For sedimentation 12.5 ml of 6% Hetastarch (Hespan) were added to produce a 1.5% solution and the syringe placed needle upwards for 1 h. The resulting approx. 36 ml of leucocyte-rich upper serum fraction were expressed into 3 conical centrifuge tubes and centrifuged for 10 min at 150 g. Each cell button was aspirated with a volume of 4ml of serum. Each volume was layered onto a prepared Percoll gradient consisting of 4 ml of 1.092 g/ml, 292 mosmol Percoll and 4 ml of 1.076 g/ml, 292 mosmol Percoll and centrifuged for 25 min at 450 g. Each gradient yielded a virtually pure band of polymorphonuclear leucocytes at the Percoll interface. Each band was aspirated in 3 ml and suspended in 5 vol of HBSS. HBSS had been prepared with 5 mM HEPES buffer and 0.1% glucose and adjusted to 292 mosmol, pH. 7.35.
The neutrophilic polymorphonuclear granulocyte, responding to chemotactic substances elaborated by micro-organisms, provides a formidable defence against the challenge of subgingival microbial plaque. It may also be responsible for considerable host gingival tissue destruction. If dental plaque undergoes ecological succession resulting in colonization by periodontal pathogens that have the capacity to invade tissue (Frank, 1980; Saglie et al., 1982, 1988) then emigrating polymorphonuclear leucocyl.es would encounter invading microbes in the host gingival connective tissues. Lysosomal enzymes and inflammatory mediators would be released in quantity by the receptor-ligand events associated with polymorphonuclear leucocytes (Bang, Cimasoni and Held, 1970; Miller, Lamster and Chasens, 1984; Lamster et al., 1987). In the escalation of inflammation seen in gingivitis and periodontitis, widespread and sustained host tissue degradation would be produced by the release of potent lysosomal enzymes from polymorphonuclear leucocytes by antigen-elicited reverse endocytosis, frustrated phagocylosis, stimulation by antigenantibody complexes and complement, and autolytic death (Hamp and Folke, 1968; Taichman et al., 1977; Genco and Mergenhagen, 1979; Page and Schroeder, 1981). In my study, the secretion by polymorphonuclear leucocytes from patients with rapidly progressive periodontitis of fl-glucuronidase, a characteristic
Abbreviations: FMLP, formyl-methionyl-leucyl-phenylalanine; HBSS, Hank’s balanced salt solution. 325
326
D. J.
Cells were pelleted at 70 g for 10 min, the supernatant was removed and the cells resuspended in 5 vol HBSS. Cells were again pelleted at 70g for 10 min and the supernatant removed. The washed polymorphonuclear leucocytes were then suspended in HBSS plus calcium, pooled and counted in a haemocytometer. Cell viability was judged to be above 99% by trypan blue exclusion. Cell density was corrected to an interim concentration of 5 x IO6 polymorphonuclear leucocytes per 1.8 ml in HBSS plus calcium (CaCl, to yield 0.6 mM Ca in the final incubation volume) for subsequent incubation. All manipulations were performed at room temperature. Volumes of 1.8 ml of cell suspension were placed into 2.2 ml Eppendorf microtubes by Pasteur pipette and the experiments performed in duplicate (final concentration of 5 x lo6 cells in 2.0 ml). The polymorphonuclear leucocyte suspensions were preincubated at 37°C for 10min with cytochalasin B (Sigma Chemical Co., St Louis, MO, U.S.A.); 0.1 ml of stock was added to give a final concentration of 5 pg/ml. The induction of polymorphonuclear leucocyte lysosomal enzyme release was produced in vitro as a selective, non-cytotoxic receptor-ligand by challenge with FMLP (Sigma interaction Chemical Co.) in the presence of cytochalasin B and calcium (Smith, Wierenga and Iden, 1980). Portions of 0.1 ml FMLP were added (final molarities of 1 x 10-6, 5 x IO-’ and 1 x lo-‘M) and incubated for 5 min. The microtubes were then centrifuged at 750 g for 10 min and 1 ml of clear supernatant isolated and stored at -70°C for analysis the next day. Supernatants from unchallenged polymorphonuclear leucocytes (0.1 ml HBSS added instead of FMLP to give 5 x lo6 polymorphonuclear leucocytes in 2.0 ml) were used to determine non-specific enzyme release, which was subtracted from the FMLP challenges. Total enzyme content of 5 x lo6 polymorphonuclear leucocytes suspended in 2.0 ml of HBSS plus calcium was determined by sonication at 4°C in the presence of 0.3% Triton X-100. Exocytosis was monitored by a standard assay for /I-glucuronidase (Metcalf, 1986) consisting of cleavage of 0.03 M phenolphthalein glucuronic acid substrate (Sigma Chemical Co.) liberating free phenolphthalein, whose red colour intensity at alkaline pH is proportional to enzyme activity. After a 9 h incubation at 37°C pH 4.5, the reaction was stopped with glycine buffer at pH 10.5 and spectrophotometric analysis performed at 540 nm. Spectrophotometric values were compared to a previously prepared standard absorption curve of known phenolphthalein concentrations to determine pmoles of phenolphthalein present in the sample. Enzyme amounts are expressed as per cent of total activity (per cent of total cellular enzyme content) (Fig. 1) and as absolute amounts represented by pmoles of phenolphthalein (Fig. 2). When expressed as a per cent of total activity, degranulation of periodontitis and control polymorphonuclear leucocytes was not statistically significantly different (t-test) at any of the 3 FMLP concentrations (Fig. 1). Also, at decreasing molarities of FMLP incubation there was a corresponding decrease in response, as expected, for both groups of
PIPPIN
70 65 60 55 50I Q
45.
;
40.
1
35.
s
30.
3
25. 26,
5 0
p
non-ohrllmp
1 x 10%
5 x 10m7M
1 x 10-71
Fig. 1. Per cent of total activity at receptor-ligand degranulation induced by FMLP challenge. A = Rapidly progressive periodontitis subjects, x = control. Data expressed as mean + 1SD; values offset for clarity. polymorphonuclear leucocytes with no statistically significant difference between the slopes. The values for the non-challenge groups, used to assess spontaneous or non-specific degranulation, were low. When total intracellular absolute amounts were compared (Fig. 2), a statistically significant difference (r-test, p < 0.001) was noted between the periodontitis polymorphonuclear leucocytes and the control polymorphonuclear leucocytes with the periodontitis mean nearly twice that of the controls. This was reflected in the amounts of /I-glucuronidase released when challenged by FMLP incubation concentrations of 1 x 10m6, 5 x IO-’ and 1 x lo-‘M (Fig. 2). There was a statistically significant difference (t-test, p < 0.01) at each FMLP molarity with higher absolute amounts secreted by periodontitis leucocytes. Control values consistently exhibited a tighter cluster than periodontitis values, which showed greater standard deviations throughout the groups (Figs 1 and 2). Degranulation of periodontitis and control polymorphonuclear leucocytes was similar when expressed as per cent of total activity (Fig. 1). This is interpreted as periodontitis polymorphonuclear leucocytes having an unaltered capacity to degranulate and not being excessively irritable to stimulus. However, total intracellular enzyme content was significantly higher in periodontitis polymorphonuclear leucocytes and, thus, an equal percentage response resulted in the secretion of much higher absolute amounts of P-glucuronidase at all FMLP molarities (Fig. 2). Are the increased absolute amounts of lysosomal enzymes contained in the polymorphonuclear leucocytes from rapidly progressive periodontitis a reactive phenomenon or an a priori genetically determined polymorphonuclear leucocyte characteristic of the individual? The mature polymorphonuclear leucocyte contains 200-300 lysosomes per cell, with a ratio of 2 or 3 specific granules per azurophilic granule (Bainton, Ullyot and Farquhar, 1971; Bainton, Nichols and Farquhar, 1976; Murphy, 1976). Thus, intracellular amounts of lysosomal enzymes may vary
Increased
leucocyte
/I-glucuronidase
in periodontitis
327
500,
T
.318.0
1 288.2 284.4 222.8
1 lo
31.8
OL
f Non-chllennpe1 x 10m8M
5 x W7M
1 x 1O-7M
TotalActlrlty
Fig. 2. Absolute amounts of /I-glucuronidase released at receptor-ligand degranulation induced by FMLP challenge. A = Rapidly progressive periodontitis subjects, x = control. p-Glucuronidase activities are represented by pmoles phenolphthalein/9 h per 5 x lo6 cells. Data expressed as mean ) 1 SD.
But the assessment of genetic influences on such variance, especially if the expression is subtle, is difficult. Thlere is limited information on the individual phenotypic expression of the genome for numbers of lysosomes, the limits of synthesis of component enzymes or the number of mitotic events after the promyelocyte stage. However, it is well known that polymorphonuclear leucocyte granulopoiesis and maturation are regulated by a series of feedback loops determining rates of production and rates of release (Williams et al., 1983). As such, polymorphonuclear lieucocyte precursors are certainly sensitive to infection .and microbial products (ZuckerFranklin, 1975). An individual with rapidly progressive periodontitis does indeed have a significant on-going infection and one may speculate on the propensity for that process to induce increased amounts of lysosomal enzymes. Whether genetically determined or induced, such an alteration in the cytochemistry of the polymorphonuclear leucocyte may play a part in the multifactorial pathogenesis of periodontal disease. If the quantity of deleterious extracellular lysosomal enzymes and immunoreactive substances is substantially increased during the inflammatory response to microbial plaque in the host gingival connective tissue, then there is the potential for a significantly enhanced tissue destruction as well. physiologically.
AcknowledPements-I counsel of-Professor Dr Jim Faed.
am Alan
erateful for the advice Laws, Dr Max Shepherd
and and
REFERENCES
Bainton D. F. Ullyot J. L. and Farquhar M. G. (1971) The development of neutrophilic polymorphonuclear leukocytes in human bone marrow. J. exp. Med. 134,907-934.
Bainton D. F., Nichols B. A. and Farquhar M. G. (1976) Primary lysosomes of blood leukocytes. In: Lysosomes in Biology and Pathology (Edited by Dingle J. T. and Dean R. T.) Vol. 5, Chap. 1, pp. 3-32. North-Holland, Amsterdam. Bang J., Cimasoni G. and Held A. J. (1970) Betaglucuronidase correlated with inflammation in the exudate from human gingiva. Archs oral Biol. 15, 44545 1. Frank R. M. (1980) Bacterial penetration in the apical pocket wall of advanced human periodontitis. J. periodont. Rex 15, 563-573. Genco R. J. and Mergenhagen S. E. (1979) Summary of a workshop on leukocyte function in bacterial diseases with an emphasis on periodontal disease. J. infect. Dis. 139, 604612. Hamp S. E. and Folke L. E. (I 968) The lysosomes and their possible role in periodontal disease. Odonf. Tidsk. 76, 353-375. Lamster I. B., Harper D. S., Fiorello L. A., Oshrain R. L., Celenti R. S. and Gordon J. M. (1987) Lysosomal and cytoplasmic enzyme activity, crevicular fluid volume and clinical parameters characterizing gingival sites with shallow to intermediate probing depths. J. Periodont. 58, 614-621. Metcalf J. A. (1986) Laborarory Manual of Neutrophil Funcfion, pp. 147-148. Raven Press, New York. Miller D. R., Lamster I. B. and Chasens A. I. (1984) Role of the polymorphonuclear leukocyte in periodontal health and disease. J. clin. Periodont. 11, I-15. Murphy P. (1976) The Neutrophil, p. 40. Plenum Medical Book Co., New York. Page R. C. and Schroeder H. E. (1981) Current status of the host response in chronic marginal periodontitis. J. Periodont. 52, 47749 1. Page R. C. and Schroeder H. E. (1982) Periodontiris in Man and Other Animals, p. 227. Karger, New York. Saglie R., Newman M. G., Carranza F. A. and Pattison G. C. (1982) Bacterial invasion of gingiva in advanced periodontitis in humans. J. Periodont. 53, 217-222. Saglie F. R., Pertuiset J., Rezende M. T., Nestor M., Marfany A. and Cheng J. (1988) In situ correlative
328
D. J.
immuno-identification of mononuclear infiltrates and invasive bacteria in diseased gingiva. J. Periodont. 59, 688496.
Smith R. J., Wierenga W. and Iden S. S. (1980) Characteristics of N-formyl-methionyl-leucyl-phenylalanine as an inducer of lysosomal enzyme release from human neutrophils. Inflammation 4, 73-88. Taichman N. S., Tsai C.-C., Baehni P. C., Stoller N. and McArthur W. P. (1977) Interaction of inflammatory cells and oral microorganisms. IV. In vitro release of lysosomal constituents from polymorphonuclear leukocytes exposed
PIPPIN
to supragingival and subgingival bacterial plaque. Infect. Immun. 16, 1013-1023.
Williams W. J., Beutler E., Erslev A. J. and Lichtman M. A. (1983) Hematology, 3rd edn, pp. 759-763, 794799. McGraw-Hill, New York. Zucker-Franklin D. (1975) Physiological and pathological variations in the ultrastructure of neutrophils and monocytes. In: Clinics in Haematology. Vol. 4~3. Granulocyre and Monocyte Abnormalities (Edited by Lichtman M. A.) Chap. I, p. 493. Saunders, Philadelphia, PA.
