Suppressor Cell Function in Oral Lichen Planus P.B. SUGERMAN, P.A. ROLLASON, N.W. SAVAGE, and G.J. SEYMOUR Immunopathology Unit, Division of Oral Biology and Pathology, Department ofDentistry, The University of Queensland, St. Lucia, Brisbane, 4072, Australia
Oral lichen planus (OLP) is a common inflammatory condition ofthe oral mucous membranes which affects between one and two percent of the general population. In accordance with the protracted clinical course of OLP and its association with known auto-immune diseases, the level of self-tolerance is questionable and possibly diminished in patients with this disorder. Normal suppressor T lymphocyte function is reputedly an essential element in the maintenance of self-tolerance, and deficient cell-mediated suppressor activity is implicated in the pathogenesis of auto-immune diseases. For assessment of in vitro cell-mediated suppressor activity in OLP, peripheral blood mononuclear cells (PBMC) from ten patients with OLP and from 11 control subjects were activated with the plant mitogen concanavalin A (Con A), followed by co-culture with autologous responder cells. The ability of irradiated Con A-activated cells to suppress the proliferation of Con A-stimulated responder cells was determined. ConA-induced suppressor activity of PBMC in the OLP patients was significantly less than that in control subjects (p = 0.001). Results of the present investigation complement previous in vitro findings which provided indirect evidence of deficient cellmediated suppressor activity in OLP, particularly a decreased proportion of circulating CD4+CD45RA+ lymphocytes and reduced Con A-stimulated PBMC proliferation. The depressed Con Ainduced suppressor activity ofPBMC in the OLP patients provides direct evidence of deficient in vitro cell-mediated suppressor function in OLP, and suggests that defective cell-mediated suppressor circuits and reduced self-tolerance maybe involved in the pathogenesis of this disorder.
result from defective T cell-mediated suppressor circuits in OLP patients. Two previous experimental findings provided indirect evidence for defective suppression in OLP. First, the proportion of CD4+CD45RA+ naive suppressor-inducer T lymphocytes was found to be decreased significantly in the peripheral blood ofOLP patients (Sugerman et al., in press). Second, Con A-stimulated peripheral blood lymphocyte proliferation in OLP patients was decreased significantly (Yamamoto et al., 1990; Sugerman et al., in press). The aim of the present study was to assess directly in vitro cell-mediated suppression in a group of OLP patients by determination of the suppressor activity of PBMC, following stimulation with the plant mitogen Con A, in an autologous assay system. Depressed Con Ainduced suppressor activity has been reported previously in mucosal (Davidsen and Kristensen, 1986) and auto-immune (Silverman et al., 1990) diseases, although not previously in lichen planus.
Materials and methods.
Ten patients with histologically confirmed OLP were selected at random from the Oral Medicine Clinic of the Dental Faculty, University of Queensland. World Health Organization (WHO) criteria for the histological diagnosis of OLP were applied to hematoxylin-and-eosin-stained biopsy sections (WHO, 1978). This group was composed of three males and seven females with an age range of 44 to 72 years (56.1 ± 8.4 years, mean ± SD). Three patients had reticular lesions, five had atrophic, and two had erosive lesions. The duration oflesions varied from 1.0 to 12 years (6.6 ±4.6 years). Nine patients had lesions involving the buccal mucosa and usually one J Dent Ries 71(12):1916-1919, December, 1992 other site, while only one patient had lesions confined to the gingivae. Lesions were distributed bilaterally in all cases. Eight patients infrequently used topical steroid preparations for the Introduction. control of oral lesions, while for two patients, active treatment was not required. Six patients were taking antihypertensive medicaLichen planus (LP) is a chronic mucocutaneous disease which was tions, and one patient was taking non-steroidal anti-inflammatory first described by Erasmus Wilson (1869). The features which drugs for chronic lower back pain. Both drug categories are implidistinguish lichen planus and associated lichenoid lesions from cated in the pathogenesis of oral lichenoid lesions (Potts et al., 1987; other chronic dermatoses are epithelial basal cell destruction and a Firth and Reade, 1989). Control subjects consisted of 11 volunteers band-like inflammatory cell infiltrate in the papillary dermis/ (five males and six females) with ages ranging from 42 to 75 years superficial lamina propria (Ellis, 1967; Krameret al., 1970; Hedberg (51.3 ± 9.3 years). There was no significant difference in mean ages et al., 1986). Although the etiology of OLP is unknown, previous between the patient and control groups. Informed consent was investigations indicate that immunological mechanisms are in- obtained from all subjects participating in this study, for which volved in the initiation and/or progression of OLP lesions, and, by Institutional Ethics Committee approval was granted. A thorough association, the cell-mediated arm of the immune system is impli- medical history was undertaken, and subjects with conditions or cated almost exclusively (Scully and El-Kom, 1985; Ishii, 1987; taking drugs that may have influenced results were excluded. For Malmstrom et al., 1988; Jungell et al., 1989; Rich and Reade, 1989; example, progressive multiple sclerosis (Oger et al., 1986) and Walsh et al., 1990). systemic lupus erythematosus (Sakane et al., 1986) are associated An immune response to a non-self antigen usually adheres to a with depressed Con A-induced suppressor activity, while chronic defined time course, and, following elimination of the antigen, this periodontal disease (Evans et al., 1989) and corticosteroids, both type of response disappears. However, the clinical course of OLP is endogenous (Indiveri et al., 1985) and systemically-administered protracted, with lesions persisting for more than 20 years. The (Hahn et al., 1980), are known to influence results in autologous in possibility therefore arises of an auto-immune mechanism acting as vitro immunological investigations. No participating subject had at least a contributing factor in the pathogenesis of OLP, particu- taken systemic steroids for at least six months prior to the current larly in the development of lesion chronicity. This scenario may investigation, and blood was collected at the same time (eight a.m.) on each day of experimentation, to control for circadian variations in endogenous cortisol levels. Received for publication April 3, 1992 Media for all phases of this study consisted of RPMI-1640 Accepted for publication July 13, 1992 This investigation was supported by The National Health and Medical [Commonwealth Serum Laboratories (CSL), Melbourne, AustraResearch Council of Australia and The Australian Dental Research Fund, lia], supplemented with 10% pooled, heat-inactivated human AB Incorporated. serum (Red Cross Blood Bank, Brisbane, Australia), 50 IU/mL 1916
Downloaded from jdr.sagepub.com at FUDAN UNIV LIB on May 2, 2015 For personal use only. No other uses without permission.
Vol. 71 No. 12
SUPPRESSOR ACTIVITY IN ORAL LICHEN PLANUS
penicillin G, 50 pg/mL streptomycin sulphate (CSL, Melbourne, Australia), and 2 mmolJL glutamine (Sigma, St. Louis, MO). The same batch of pooled human serum was used throughout this study. PBMC were isolated from 84 mL of heparinized whole blood by density gradient centrifugation with Ficoll-Paque (Pharmacia, Uppsala, Sweden). Following three washes with RPMI-1640, viability was assessed with ethidium bromide/acridine orange and found to be always greater than 95%. The PBMC were adjusted to 107 cells/mL medium. Extensive preliminary kinetic investigations with healthy control subjects were undertaken to establish the conditions required for optimal in vitro Con A-induced cell-mediated suppressor activity. Maximum suppression was obtained with stored responder cells, at a Con A concentration of 10 pg/mL during induction and with 20 Gy of y radiation to inhibit proliferation of the effector cells. The same batch of Con A was used throughout this experiment. The PBMC suspension was separated into three components duringthe induction phase: (1) activated effectors (AE), (2) non-activated effectors (NE), and (3) stored responders (SR). To obtain the AE, aliquots of 2 mL of the PBMC suspension (2 x 107 cells) were placed into two wells of a 6-well plate (Nunclon, Denmark), followed by the addition of 2 mL of Con A (Pharmacia, Uppsala, Sweden) in medium to give a final concentration of 5 x 106 PBMC/mL. To one well was added Con A at 20 pg/mL, and to the other was added Con A at 5 pg/mL, resulting in final Con A concentrations of 10 and 2.5 /mL. The resultant cell populations were designated AE 1 and AE25, respectively. NE were obtained by the addition of 2 mL of medium alone to 2 mL of the PBMC suspension in a third well of the 6-well tray. The AE25, AE10, and NE were incubated at 37°C for 48 h in a humidified atmosphere of 5% CO2 and air. The SR were adjusted to 106 PBMC/mL in medium and stored at 40C for 48 h. The responding phase of the suppressor assay was commenced on day 2. The SR were washed with RPMI-1640 and adjusted to 2 x 106 cells/mL. The AE25, AE10, and the NE were harvested and washed three times with 20 mL of 50 mmolbcx-methyl-D-mannoside in RPMI- 1640 for removal of surface-bound Con A (Powell and Leon, 1970). These cells were then washed once with RPMI and adjusted to 2 x 106 cells/mL. Both the AE and the NE were irradiated with 20 Gy of y radiation from a 6Co source to inhibit their proliferation in subsequent culture. Six autologous assays were set up for each subject (each with 5 replicates) in 200 pL of medium in roundbottomed wells of 96-well plates (Nunclon, Denmark), as follows:
follows: % Suppression = [1 - (A -
Results. Results of the Con A-induced suppressor assays are presented in Fig. 1. Con A-induced suppressor activity of PBMC in the OLP patients was significantly less than that in control subjects, but only at a concentration of 4 pg Con A per mL in the responder system. Following induction at 2.5 pg Con A per mL (Fig. 1A), percent suppression was 44 (38-49) vs. 29 (14-32) (median and 95% confidence intervals of control and OLP groups, respectively), with a significance level of p = 0.001. This difference was less remarkable following induction at 10 p/mL (Fig. 1B). At this concentration, control and OLP percent suppression were 46 (41-53) vs. 34 (31-45), respectively, with a significance level of p < 0.05.
A
* OLP PATIENTS
100
(NS)
o CONTROLS
(NS)
80
(NS)
0-
z 0
Couo
Cn
00
(p=0,00 1)
60
0
40
I
0
. .0
coo
0
I
0.
0. CL0n
0
i00
I..C-
0:
J.0I o.I
Off.
20-
n
p/mL;
A further five replicate cultures containing 105 SR alone stimulated with Con A at 4 pg/mL were established as part of an exclusion criterion to confirm that the SR were actually capable of proliferating following storage at 40C for two days. Cultures were incubated at 370C for four days in a humidified atmosphere of 5% CO2 and air. For the final 24 h of culture, 0.5 pCi tritiated thymidine (999 GBq/ mmol, Amersham, UK) in 50 pL of medium was addedperwell. Well contents were harvested (Titertek Cell Harvester 530, Flow Laboratories, Australia) onto glass-fiber discs for liquid scintillation spectroscopy in a B3 emission counter (Beckman Instruments, Fullerton, CA). Each datum point was the geometric mean of the five replicates. The percent suppressor activity at each concentration of Con A during the induction and responder phases was calculated as
E)] X 100
The geometric mean of replicate cultures was used as the value for each subject at each mitogen concentration, since exponential growth of cultured lymphocytes results in non-normal distribution of data (Oppenheim et al., 1975). The Mann-Whitney test was used for comparison ofthe median percents of suppression of patient and control groups at each mitogen concentration, with significance indicated by a "p" value of less than 0.05.
1r
(A) 105 SR + 105 AE2.5J10 stimulated with Con A at 1, 2, 3, and 4 (B) 105 SR + 105 AE2.5/10 without addition of Con A; (C) 105 SR + 101 NE stimulated with Con A at 1, 2, 3, and 4 pg/ mL; (D) 105 SR + 105 NE without addition of Con A; (E) 105 AE2.5J10 alone stimulated with Con A at 1, 2, 3, and 4 pg/ mL; and (F) 105 NE alone stimulated with Con A at 1, 2, 3, and 4 pg/mL.
1917
B
100-
80-] 2 0 Co
a.
60 80 -
3
2
4
(NS) (NS)
0.
(NS)
(p
Oral lichen planus (OLP) is a common inflammatory condition ofthe oral mucous membranes which affects between one and two percent of the general population. In accordance with the protracted clinical course of OLP and its association with known auto-immune diseases, the level of self-tolerance is questionable and possibly diminished in patients with this disorder. Normal suppressor T lymphocyte function is reputedly an essential element in the maintenance of self-tolerance, and deficient cell-mediated suppressor activity is implicated in the pathogenesis of auto-immune diseases. For assessment of in vitro cell-mediated suppressor activity in OLP, peripheral blood mononuclear cells (PBMC) from ten patients with OLP and from 11 control subjects were activated with the plant mitogen concanavalin A (Con A), followed by co-culture with autologous responder cells. The ability of irradiated Con A-activated cells to suppress the proliferation of Con A-stimulated responder cells was determined. ConA-induced suppressor activity of PBMC in the OLP patients was significantly less than that in control subjects (p = 0.001). Results of the present investigation complement previous in vitro findings which provided indirect evidence of deficient cellmediated suppressor activity in OLP, particularly a decreased proportion of circulating CD4+CD45RA+ lymphocytes and reduced Con A-stimulated PBMC proliferation. The depressed Con Ainduced suppressor activity ofPBMC in the OLP patients provides direct evidence of deficient in vitro cell-mediated suppressor function in OLP, and suggests that defective cell-mediated suppressor circuits and reduced self-tolerance maybe involved in the pathogenesis of this disorder.
result from defective T cell-mediated suppressor circuits in OLP patients. Two previous experimental findings provided indirect evidence for defective suppression in OLP. First, the proportion of CD4+CD45RA+ naive suppressor-inducer T lymphocytes was found to be decreased significantly in the peripheral blood ofOLP patients (Sugerman et al., in press). Second, Con A-stimulated peripheral blood lymphocyte proliferation in OLP patients was decreased significantly (Yamamoto et al., 1990; Sugerman et al., in press). The aim of the present study was to assess directly in vitro cell-mediated suppression in a group of OLP patients by determination of the suppressor activity of PBMC, following stimulation with the plant mitogen Con A, in an autologous assay system. Depressed Con Ainduced suppressor activity has been reported previously in mucosal (Davidsen and Kristensen, 1986) and auto-immune (Silverman et al., 1990) diseases, although not previously in lichen planus.
Materials and methods.
Ten patients with histologically confirmed OLP were selected at random from the Oral Medicine Clinic of the Dental Faculty, University of Queensland. World Health Organization (WHO) criteria for the histological diagnosis of OLP were applied to hematoxylin-and-eosin-stained biopsy sections (WHO, 1978). This group was composed of three males and seven females with an age range of 44 to 72 years (56.1 ± 8.4 years, mean ± SD). Three patients had reticular lesions, five had atrophic, and two had erosive lesions. The duration oflesions varied from 1.0 to 12 years (6.6 ±4.6 years). Nine patients had lesions involving the buccal mucosa and usually one J Dent Ries 71(12):1916-1919, December, 1992 other site, while only one patient had lesions confined to the gingivae. Lesions were distributed bilaterally in all cases. Eight patients infrequently used topical steroid preparations for the Introduction. control of oral lesions, while for two patients, active treatment was not required. Six patients were taking antihypertensive medicaLichen planus (LP) is a chronic mucocutaneous disease which was tions, and one patient was taking non-steroidal anti-inflammatory first described by Erasmus Wilson (1869). The features which drugs for chronic lower back pain. Both drug categories are implidistinguish lichen planus and associated lichenoid lesions from cated in the pathogenesis of oral lichenoid lesions (Potts et al., 1987; other chronic dermatoses are epithelial basal cell destruction and a Firth and Reade, 1989). Control subjects consisted of 11 volunteers band-like inflammatory cell infiltrate in the papillary dermis/ (five males and six females) with ages ranging from 42 to 75 years superficial lamina propria (Ellis, 1967; Krameret al., 1970; Hedberg (51.3 ± 9.3 years). There was no significant difference in mean ages et al., 1986). Although the etiology of OLP is unknown, previous between the patient and control groups. Informed consent was investigations indicate that immunological mechanisms are in- obtained from all subjects participating in this study, for which volved in the initiation and/or progression of OLP lesions, and, by Institutional Ethics Committee approval was granted. A thorough association, the cell-mediated arm of the immune system is impli- medical history was undertaken, and subjects with conditions or cated almost exclusively (Scully and El-Kom, 1985; Ishii, 1987; taking drugs that may have influenced results were excluded. For Malmstrom et al., 1988; Jungell et al., 1989; Rich and Reade, 1989; example, progressive multiple sclerosis (Oger et al., 1986) and Walsh et al., 1990). systemic lupus erythematosus (Sakane et al., 1986) are associated An immune response to a non-self antigen usually adheres to a with depressed Con A-induced suppressor activity, while chronic defined time course, and, following elimination of the antigen, this periodontal disease (Evans et al., 1989) and corticosteroids, both type of response disappears. However, the clinical course of OLP is endogenous (Indiveri et al., 1985) and systemically-administered protracted, with lesions persisting for more than 20 years. The (Hahn et al., 1980), are known to influence results in autologous in possibility therefore arises of an auto-immune mechanism acting as vitro immunological investigations. No participating subject had at least a contributing factor in the pathogenesis of OLP, particu- taken systemic steroids for at least six months prior to the current larly in the development of lesion chronicity. This scenario may investigation, and blood was collected at the same time (eight a.m.) on each day of experimentation, to control for circadian variations in endogenous cortisol levels. Received for publication April 3, 1992 Media for all phases of this study consisted of RPMI-1640 Accepted for publication July 13, 1992 This investigation was supported by The National Health and Medical [Commonwealth Serum Laboratories (CSL), Melbourne, AustraResearch Council of Australia and The Australian Dental Research Fund, lia], supplemented with 10% pooled, heat-inactivated human AB Incorporated. serum (Red Cross Blood Bank, Brisbane, Australia), 50 IU/mL 1916
Downloaded from jdr.sagepub.com at FUDAN UNIV LIB on May 2, 2015 For personal use only. No other uses without permission.
Vol. 71 No. 12
SUPPRESSOR ACTIVITY IN ORAL LICHEN PLANUS
penicillin G, 50 pg/mL streptomycin sulphate (CSL, Melbourne, Australia), and 2 mmolJL glutamine (Sigma, St. Louis, MO). The same batch of pooled human serum was used throughout this study. PBMC were isolated from 84 mL of heparinized whole blood by density gradient centrifugation with Ficoll-Paque (Pharmacia, Uppsala, Sweden). Following three washes with RPMI-1640, viability was assessed with ethidium bromide/acridine orange and found to be always greater than 95%. The PBMC were adjusted to 107 cells/mL medium. Extensive preliminary kinetic investigations with healthy control subjects were undertaken to establish the conditions required for optimal in vitro Con A-induced cell-mediated suppressor activity. Maximum suppression was obtained with stored responder cells, at a Con A concentration of 10 pg/mL during induction and with 20 Gy of y radiation to inhibit proliferation of the effector cells. The same batch of Con A was used throughout this experiment. The PBMC suspension was separated into three components duringthe induction phase: (1) activated effectors (AE), (2) non-activated effectors (NE), and (3) stored responders (SR). To obtain the AE, aliquots of 2 mL of the PBMC suspension (2 x 107 cells) were placed into two wells of a 6-well plate (Nunclon, Denmark), followed by the addition of 2 mL of Con A (Pharmacia, Uppsala, Sweden) in medium to give a final concentration of 5 x 106 PBMC/mL. To one well was added Con A at 20 pg/mL, and to the other was added Con A at 5 pg/mL, resulting in final Con A concentrations of 10 and 2.5 /mL. The resultant cell populations were designated AE 1 and AE25, respectively. NE were obtained by the addition of 2 mL of medium alone to 2 mL of the PBMC suspension in a third well of the 6-well tray. The AE25, AE10, and NE were incubated at 37°C for 48 h in a humidified atmosphere of 5% CO2 and air. The SR were adjusted to 106 PBMC/mL in medium and stored at 40C for 48 h. The responding phase of the suppressor assay was commenced on day 2. The SR were washed with RPMI-1640 and adjusted to 2 x 106 cells/mL. The AE25, AE10, and the NE were harvested and washed three times with 20 mL of 50 mmolbcx-methyl-D-mannoside in RPMI- 1640 for removal of surface-bound Con A (Powell and Leon, 1970). These cells were then washed once with RPMI and adjusted to 2 x 106 cells/mL. Both the AE and the NE were irradiated with 20 Gy of y radiation from a 6Co source to inhibit their proliferation in subsequent culture. Six autologous assays were set up for each subject (each with 5 replicates) in 200 pL of medium in roundbottomed wells of 96-well plates (Nunclon, Denmark), as follows:
follows: % Suppression = [1 - (A -
Results. Results of the Con A-induced suppressor assays are presented in Fig. 1. Con A-induced suppressor activity of PBMC in the OLP patients was significantly less than that in control subjects, but only at a concentration of 4 pg Con A per mL in the responder system. Following induction at 2.5 pg Con A per mL (Fig. 1A), percent suppression was 44 (38-49) vs. 29 (14-32) (median and 95% confidence intervals of control and OLP groups, respectively), with a significance level of p = 0.001. This difference was less remarkable following induction at 10 p/mL (Fig. 1B). At this concentration, control and OLP percent suppression were 46 (41-53) vs. 34 (31-45), respectively, with a significance level of p < 0.05.
A
* OLP PATIENTS
100
(NS)
o CONTROLS
(NS)
80
(NS)
0-
z 0
Couo
Cn
00
(p=0,00 1)
60
0
40
I
0
. .0
coo
0
I
0.
0. CL0n
0
i00
I..C-
0:
J.0I o.I
Off.
20-
n
p/mL;
A further five replicate cultures containing 105 SR alone stimulated with Con A at 4 pg/mL were established as part of an exclusion criterion to confirm that the SR were actually capable of proliferating following storage at 40C for two days. Cultures were incubated at 370C for four days in a humidified atmosphere of 5% CO2 and air. For the final 24 h of culture, 0.5 pCi tritiated thymidine (999 GBq/ mmol, Amersham, UK) in 50 pL of medium was addedperwell. Well contents were harvested (Titertek Cell Harvester 530, Flow Laboratories, Australia) onto glass-fiber discs for liquid scintillation spectroscopy in a B3 emission counter (Beckman Instruments, Fullerton, CA). Each datum point was the geometric mean of the five replicates. The percent suppressor activity at each concentration of Con A during the induction and responder phases was calculated as
E)] X 100
The geometric mean of replicate cultures was used as the value for each subject at each mitogen concentration, since exponential growth of cultured lymphocytes results in non-normal distribution of data (Oppenheim et al., 1975). The Mann-Whitney test was used for comparison ofthe median percents of suppression of patient and control groups at each mitogen concentration, with significance indicated by a "p" value of less than 0.05.
1r
(A) 105 SR + 105 AE2.5J10 stimulated with Con A at 1, 2, 3, and 4 (B) 105 SR + 105 AE2.5/10 without addition of Con A; (C) 105 SR + 101 NE stimulated with Con A at 1, 2, 3, and 4 pg/ mL; (D) 105 SR + 105 NE without addition of Con A; (E) 105 AE2.5J10 alone stimulated with Con A at 1, 2, 3, and 4 pg/ mL; and (F) 105 NE alone stimulated with Con A at 1, 2, 3, and 4 pg/mL.
1917
B
100-
80-] 2 0 Co
a.
60 80 -
3
2
4
(NS) (NS)
0.
(NS)
(p
