Polymorphonuclear neutrophil function in recurrent aphthous stomatitis
D, Wray^ and J. Charon^ 'Department ol Oral Medicine & Oral Pathology, University ot Edinburgh, Scotland and 'Periodontist, Lille, France
Wray D, Charon J; Polymorphonuclear neutrophil function in recurrent aphthous stomatitis. J Oral Pathol Med 1991; 20; 392-4. Polymorphonuclear neutrophils (PMN) are the predominant cell type in the established lesion of recurrent aphthous stomatitis (RAS). The function of these cells was studied in 15 patients with RAS and 15 age and sex matched controls. Production of oxygen radicals was assessed using cytochrome-c and nitroblue tetrazolium dye (NBT) reduction assays. Oxidative metabolism was found to be similar for experimental and control groups in both stimulated and nonstimulated assays. Adherence to glass surfaces by PMN was also measured. Adherence of PMN, both from patients and controls, was significantly reduced by serum, but to a lesser extent by serum from patients compared to controls. This effect of .serum on adherence was reversed by phorbol myristate acetate (PMA) stimulation. These studies suggest that increased adherence of stimulated PMN, due to humoral factors, may be involved in perpetuating ulcerative lesions in RAS.
Polymorphonuclear neutrophils (PMN) are the predominant cell type in recurrent aphthous stomatitis (RAS) lesions (1, 2). They may be attracted there by chemotactic split products of complement which have been activated by immune complexes deposited in the oral mucosa (3). Enhanced PMN chemotaxis has been demonstrated in Behcet's syndrome (4-6) but PMN chemotactic activity and spontaneous migration have been shown to be normal in RAS (7. 8). PMN are important for phagocytosing and eliminating antigenic material or products of tissue damage; production of oxygen radicals is an integral part of this function (9). We examined the function of peripheral blood neutrophils in recurrent aphthous stomatitis, using cytochrome-r and nitroblue tetrazolium reduction as parameters of oxidative metabolism, and adherence to glass surfaces. Material and methods Patients
Fifteen patients with active minor recurrent aphthous stomatitis, diagnosed by the criteria of LEHNER (10) were used in this study. The ages of the patients ranged from 25 to 65 yr (mean age 43.4 yr); six were men (mean age 42.3 yr) and nine were women (mean age 44,1 yr). Fifteen age and sex matched volun-
teers without recurrent aphthous stomatitis were used as controls.
Key words: mouth, diseases; polymorphonuclear neutrophils: recurrent aphthous stomatitis, D, Wray, Department of Oral Medicine & Oral Pathology, University ot Edinburgh, High School Yards, Edinburgh, EH1 1NR, UK, Accepted for publication April 7, 1991
using a microtitre plate assay (12). Briefiy, 100,000 viable neutrophils in Hank's BSS were plated into each well of a flat bottomed 96-well microtitre Methods plate (Flow Laboratories) and incubatPreparation of peripheral blood potymor- ed in the absence of serum for one hour phortuetectr tu'titrophits (PB-PMN) ~ Pe-at 37' C in a humidified, 5% CO, atmoripheral blood polymorphonuclear neu- sphere. The cells were then washed twice trophils (PB-PMN) were separated with warm Hank's BSS leaving a monofrom heparinized (20 U/ml) whole layer of adherent PMN with more than blood as described previously by BOYUM 98% viability as assessed by trypan blue (II). Briefiy , the heparinized blood, di- exclusion. Each vertical row of eight luted 1:1 in phosphate buffered saline wells immediately received the NBT so(PBS, 1.15 M, pH 7,5) was layered onto lution at a final dilution of 1 mg/ml, a Ficoll-hypaque solution (density 1,077 with or without phorbol myristate aceSG) and centrifuged at 400 x g for 30 tate (PMA, Sigma) which constituted min at 2O'C. The pellet containing red the positive control (13). Initial dose reblood cells and neutrophils was diluted sponse studies indicated that the optiin an equal volume of autologous plas- mal concentration for PMA was 10 ng/ ma and the resulting solution was ml. This concentration was used in subfurther diluted 1; 1 in three per cent Dex- sequent experiments. Thus, each experitran (Pharmacia). After 20 min sedi- ment was conducted on eight replicate mentation at room temperature, the samples. The plates were then incubated PMN rich top layer was washed twice for 1 h at 37 C in a 5% CO, containing in Hank's balanced salt solution (BSS). atmosphere. The optical density (O.D.) Residual erythrocytes were lysed by two of each well was then read at 550 nm cycles of hypotonic shock and the PMN using a Titertek Multi.scan (Flow Laboresuspended in Hank's BSS. This proce- ratories) with a blank consisting of dure yielded a population of more than PMN incubated with 10 mM of idioace95 per cent PMN containing more than tamide (Sigma). 97% viable cells as determined by tryCytoehrotne-e redttetioti as.uty - The pan blue exclusion. cytochrome-c- reduction assay was perNitrohlue tetrazotititn reduetion assay. formed using a microassay (14). Briefiy, ~ The capacity to reduce the nitroblue 50 |il of neutrophils (0.4xl0'Vml in tetrazolium dye by PB-PMN was tested Hank's BSS) were added to 100 ^tl of a
Netttroptn't ftinction in reetirrent aphthae
160 uM cytochrome-f solution (ferrocytochrome-c, type III, Sigma) in Hank's BSS in each well of a microtitre plate. Neutrophils were stimulated with PMA either in the presence or absence of superoxide dismutase (SOD) (from bovine blood type I, Sigma) at a final concentration of 400 U/ml. After 90 min incubation in a 5% COj containing atmosphere, the amount of reduced cytochrome-c was measured spectrophotometrically as absorbance at 550 nm (Titertek Multiskan, Flow Laboratories) with a blank consisting of cells
with SOD but without PMA. The O.D. values were multiplied by a factor of 15.873, giving the amount of reduced cytochrome-c in nanomoles per well (14). The superoxide-dependent reduction of cytochrome-f was calculated by subtracting the amount of cytochromee in the presence of SOD from that in its absence. All assays were conducted on eight replicate samples. Initial dose response studies indicated that the optimal concentration for PMA was 10 ng/ ml. This concentration was used in subsequent experiments.
Table I. Reduction of cytochromc-f by peripheral blood polymorphonuclear neutrophils Number Recurrent aphthae Patients
10
Controls
10
PMA (10 ng/ml)* 3,94 + 0,24** NS*** 4,37 + 0.37
Stimulus
None
P
D, Wray^ and J. Charon^ 'Department ol Oral Medicine & Oral Pathology, University ot Edinburgh, Scotland and 'Periodontist, Lille, France
Wray D, Charon J; Polymorphonuclear neutrophil function in recurrent aphthous stomatitis. J Oral Pathol Med 1991; 20; 392-4. Polymorphonuclear neutrophils (PMN) are the predominant cell type in the established lesion of recurrent aphthous stomatitis (RAS). The function of these cells was studied in 15 patients with RAS and 15 age and sex matched controls. Production of oxygen radicals was assessed using cytochrome-c and nitroblue tetrazolium dye (NBT) reduction assays. Oxidative metabolism was found to be similar for experimental and control groups in both stimulated and nonstimulated assays. Adherence to glass surfaces by PMN was also measured. Adherence of PMN, both from patients and controls, was significantly reduced by serum, but to a lesser extent by serum from patients compared to controls. This effect of .serum on adherence was reversed by phorbol myristate acetate (PMA) stimulation. These studies suggest that increased adherence of stimulated PMN, due to humoral factors, may be involved in perpetuating ulcerative lesions in RAS.
Polymorphonuclear neutrophils (PMN) are the predominant cell type in recurrent aphthous stomatitis (RAS) lesions (1, 2). They may be attracted there by chemotactic split products of complement which have been activated by immune complexes deposited in the oral mucosa (3). Enhanced PMN chemotaxis has been demonstrated in Behcet's syndrome (4-6) but PMN chemotactic activity and spontaneous migration have been shown to be normal in RAS (7. 8). PMN are important for phagocytosing and eliminating antigenic material or products of tissue damage; production of oxygen radicals is an integral part of this function (9). We examined the function of peripheral blood neutrophils in recurrent aphthous stomatitis, using cytochrome-r and nitroblue tetrazolium reduction as parameters of oxidative metabolism, and adherence to glass surfaces. Material and methods Patients
Fifteen patients with active minor recurrent aphthous stomatitis, diagnosed by the criteria of LEHNER (10) were used in this study. The ages of the patients ranged from 25 to 65 yr (mean age 43.4 yr); six were men (mean age 42.3 yr) and nine were women (mean age 44,1 yr). Fifteen age and sex matched volun-
teers without recurrent aphthous stomatitis were used as controls.
Key words: mouth, diseases; polymorphonuclear neutrophils: recurrent aphthous stomatitis, D, Wray, Department of Oral Medicine & Oral Pathology, University ot Edinburgh, High School Yards, Edinburgh, EH1 1NR, UK, Accepted for publication April 7, 1991
using a microtitre plate assay (12). Briefiy, 100,000 viable neutrophils in Hank's BSS were plated into each well of a flat bottomed 96-well microtitre Methods plate (Flow Laboratories) and incubatPreparation of peripheral blood potymor- ed in the absence of serum for one hour phortuetectr tu'titrophits (PB-PMN) ~ Pe-at 37' C in a humidified, 5% CO, atmoripheral blood polymorphonuclear neu- sphere. The cells were then washed twice trophils (PB-PMN) were separated with warm Hank's BSS leaving a monofrom heparinized (20 U/ml) whole layer of adherent PMN with more than blood as described previously by BOYUM 98% viability as assessed by trypan blue (II). Briefiy , the heparinized blood, di- exclusion. Each vertical row of eight luted 1:1 in phosphate buffered saline wells immediately received the NBT so(PBS, 1.15 M, pH 7,5) was layered onto lution at a final dilution of 1 mg/ml, a Ficoll-hypaque solution (density 1,077 with or without phorbol myristate aceSG) and centrifuged at 400 x g for 30 tate (PMA, Sigma) which constituted min at 2O'C. The pellet containing red the positive control (13). Initial dose reblood cells and neutrophils was diluted sponse studies indicated that the optiin an equal volume of autologous plas- mal concentration for PMA was 10 ng/ ma and the resulting solution was ml. This concentration was used in subfurther diluted 1; 1 in three per cent Dex- sequent experiments. Thus, each experitran (Pharmacia). After 20 min sedi- ment was conducted on eight replicate mentation at room temperature, the samples. The plates were then incubated PMN rich top layer was washed twice for 1 h at 37 C in a 5% CO, containing in Hank's balanced salt solution (BSS). atmosphere. The optical density (O.D.) Residual erythrocytes were lysed by two of each well was then read at 550 nm cycles of hypotonic shock and the PMN using a Titertek Multi.scan (Flow Laboresuspended in Hank's BSS. This proce- ratories) with a blank consisting of dure yielded a population of more than PMN incubated with 10 mM of idioace95 per cent PMN containing more than tamide (Sigma). 97% viable cells as determined by tryCytoehrotne-e redttetioti as.uty - The pan blue exclusion. cytochrome-c- reduction assay was perNitrohlue tetrazotititn reduetion assay. formed using a microassay (14). Briefiy, ~ The capacity to reduce the nitroblue 50 |il of neutrophils (0.4xl0'Vml in tetrazolium dye by PB-PMN was tested Hank's BSS) were added to 100 ^tl of a
Netttroptn't ftinction in reetirrent aphthae
160 uM cytochrome-f solution (ferrocytochrome-c, type III, Sigma) in Hank's BSS in each well of a microtitre plate. Neutrophils were stimulated with PMA either in the presence or absence of superoxide dismutase (SOD) (from bovine blood type I, Sigma) at a final concentration of 400 U/ml. After 90 min incubation in a 5% COj containing atmosphere, the amount of reduced cytochrome-c was measured spectrophotometrically as absorbance at 550 nm (Titertek Multiskan, Flow Laboratories) with a blank consisting of cells
with SOD but without PMA. The O.D. values were multiplied by a factor of 15.873, giving the amount of reduced cytochrome-c in nanomoles per well (14). The superoxide-dependent reduction of cytochrome-f was calculated by subtracting the amount of cytochromee in the presence of SOD from that in its absence. All assays were conducted on eight replicate samples. Initial dose response studies indicated that the optimal concentration for PMA was 10 ng/ ml. This concentration was used in subsequent experiments.
Table I. Reduction of cytochromc-f by peripheral blood polymorphonuclear neutrophils Number Recurrent aphthae Patients
10
Controls
10
PMA (10 ng/ml)* 3,94 + 0,24** NS*** 4,37 + 0.37
Stimulus
None
P
