Cellular immunosuppression in oral lichen planus
Tetsuya Yamamoto, Kazunori Yoneda, Eisaku Ueta and Tokio Osaki Department ot Oral Surgery, Kochi tvledicai School, Nankoku City, Kochi, Japan
Yamamoto T, Yoneda K, Ueta E, Osaki T: Cellular immunosuppression in oral lichen planus, J Oral Pathol Med 1990; 19: 464-70. Functions of peripheral blood lymphocytes and neutrophils from 30 oral lichen planus (OLP) patients were examined using healthy persons as controls. Twocolor flow cytometry of lymphocytes revealed no proportional difference in CD3 or CD4 cells between OLP and controls. CD8CD1 lb (suppressor T) and CD3HLA-DR*^ cell populations increased significantly in OLP when compared with controls, and, CD4/CD8 cell ratio decreased in OLP. Mitogenic response of patients" CD8 and CD4Leu8" cells was similar to that in controls. However, weaker blastogenesis of CD4Leu8^ cells, the most excellent responders in T cell subsets, was observed in OLP Serum IFNP level in OLP (8.4 ±4.8 IU/ml) was significantly lower than in controls (13.7 + 5.0 IU/m!) whereas no difference between the two groups could be found in IFNa or y. As for in vitro cytokine production by IL-2-stimulated lymphocytes, there was no difference in GM-CSF generation between the two groups, but, IFNy and ILip production of patients' lymphocytes was less than that in healthy donors (57.6 + 50,7 VS 78,7 ±39,6 u/ ml, 152,3 ±93,5 VS 258,7 ±65,4 pg/ml, respectively). Moreover, superoxide generation of patients' neutrophils by PMA stimulation was significantly insufficient as compared with controls' (84,9 ±30,9 VS II0,8 ±24,1 pmol/min/lO" cells). Nevertheless, natural killer eell activities of both groups distributed in the same range. These results suggest that OLP patients' lymphocyte and neutrophil functions are impaired, and that cellular immunosuppression is a pathologic characteristic of OLP,
Lichen planus, a chronic infiammatory mucocutaneous disease of unknown etiology, often occurs in the oral mucosa. Complication of lichen planus with autoimmune diseases such as myasthenia gravis, ulcerative colitis and systemic lupus erythematosus (SLE) has recently been reported (1-3). It now seems more accurate to suppose that lichen planus is based on a disturbed immune condition, and may be a localized autoimmune disease. Recent immuno-histochemical studies on oral lichen planus (OLP) have identified each lymphocyte infiltrate subset and accessory cells (4-6). As for accumulated immunohistochemical results, OLP can be thought of as a clinical manifestation of infiltrated T cell and accessory cell response to modified autologous epithelial cells. The interesting observation that peripheral blood lymphocytes may exert nonspecific cytotoxocity toward keratinocytes was recently reported (7), However, no direct proof that autologous lymphocytes attack and lyse the basal cells or inferior spinosum layer cells has been obtained. Previous studies demonstrated T cell subsets in peripheral blood of patients
with OLP (8, 9), but investigations of functional lymphocyte subsets by twocolor analysis are scarce. There are few studies regarding cellular immunity in OLP (10-12), and no investigator has clarified abnormalities which identify OLP as a disease related to impaired cellular immunity. In the present study, we aimed to evaluate cellular immunities of OLP patients, and investigated peripheral blood lymphocyte subsets by two-color flow cytometric analysis, natural killer cell (NK) activity and blastogenesis. Furthermore, functions such as cytokine producibility of lymphocytes by interleukin 2(lL-2) stimulation, superoxide(O2~) generation of neutrophils, and interferon (IFN) activity in serum were investigated. Material and methods Subjects
Thirty patients with clinically diagnosed and histologically confirmed OLP were evaluated. The clinical characteristics of the patients are given in Table 1. No patient had been given topical or systemic drugs which could modify the im-
Key words: cylokine and superoxide generation: double color flow cytometry: lymphoblastogenesis; oral lichen planus: serum interferon. T. Yamamoto, Department of Oral Surgery, Kochi Medical School Kohasu Oko-cho, Nankoku-city, Kochi 783 Japan Accepted for publication August 3, 1990.
mune condition, and blood samples were obtained from untreated OLP patients. The samples from patients and healthy controls were synchronously examined. Healthy volunteers were chosen with the aim of matching their sex and age with the patients. Cell preparation and culture
Lymphocytes and polymorphonuclear leukocytes (PMNL) were obtained by the Ficoll-Paque gradient method, HepTable I, Characteristics of OLP patients t Sex (m/f) t Age (yrs) Clinical type* reticular plaque type atrophie ulcerative Duration of symptoms -- 1 mo. -6 mo. -1 yr. 1 yr. ~
10/20 59.4±13.4 (25-83) 16 5 3 6 5
10 5 10
t Healthy controls: lO/tO, 49.9±l2.l (28-80) * Clinical types were determined according to ANDREASEN'S classit'ieation (15).
Cellular imtnunitv in oral liehen planus 465 arinized peripheral blood was centrifuged on Ficoll-Paque gradients at 400 X g for 30 min. Mononuclear leukocytes were drawn from the upper layer of the gradients and PMNL were collected at the interface just above the red blood cell layer. For removing contaminated red blood cells, cold distilled water was added to PMNL pellets. After 10 s, an equal volume of 2 x PBS was added to the PMNL hypotonic solution and restored isotonicity. Isolated mononuclear leukocytes and PMNL were washed appropriately, and suspended in RPMl 1640 (Nissui Pharma,, Co,, Ltd, Tokyo Japan) supplemented with fetal calf serum and antibodies, and in Hank's balanced salt solution (HBSS), respectively. Microscopic examination of Giemsa-stained cells revealed that PMNL preparations were composed of over 98% PMNL, Mononuclear leukocytes were incubated in plastic petri dishes in a COi-ineubator at 37''C for 120 min. The dishes were then gently swabed and lymphocytes were isolated, T cells were separated from non-T cells by a single step rosetting method (13) with 2-amino-ethylisothiouronium bromide hydrobromide (Sigma Chemical Co,, St, Louis, MO)-treated sheep red blood cells (AET-SRBC), To prepare T cell subsets, a panning method (14) was used, Rosetted T cells were incubated with 25 |i g/ml anti-CD8 monoclonal antibody (mAb, Becton Dickinson, California, USA) for 30 min at 4 ^ , and then cultured in petri dishes that had been coated with 2 |ig/ml goat antimouse IgG antiserum (Kirkegaard & Perry Laboratories Inc, Gaithersburg, Md.). After 70 min incubation, adherent cells (CD8* cells) were recovered by scraping with a rubber policeman and removal of non-adherent cells (CD8" cells). CD8^ T cells (over 95% of which were CD4 ') were further fractionated into Leu8^ and Leu8" subpopulations by a second panning set with anti-Leu8 mAb (10 |ig/ml, Becton Dickinson), The composition of isolated CD8^, CD4 ' Leu8+ and CD4' Leu8- cells was ascertained by flow cytometry, and more than 90% purity was proven in all subsets. With cell preparation, serum samples were collected for IFN assay, IVvo-color flow cytometry
For double color analysis of lymphocytes, both monoclonal FITC-conjugated (anti-CD8, anti-CD4, anti-CD3, anti-CD57) and PE-conjugated (antiLeu8, anti-CD16, anti-CDIlb, anti-
HLA-DR) antibodies were used. For dual analysis, combinations of antiCD8 with anti-CD lib, anti-CD4 with anti-Leu8, anti-CD 16 with anti-CD57, and anti-CD3 with anti HLA-DR were used. Isolated lymphocytes of 5x10' cells were resuspended in 100 nl of PBS containing 0,1% azide (volume %) and packed into culture tubes. After saturating with both FITC- and PE-conjugated mAb solutions, tubes were mildly agitated and incubated for 30 min at 4°C in a dark place. Cells were washed twice, resuspenced in a 0,1% azide solution, and analyzed with an EPICS V flow cytometer (Coulter Electronics, Inc, USA), Monocolor analysis was also applied to CD3 ', CD25 ' and HLA-DR + cells. Lymphocyte blastogenesis
Lymphocyte blastogenesis was also routinely examined. Briefly, lymphocytes of 1x10-' cells or each T cell subset of 5x lO'' cells in 0,1 ml of RPMI were seeded into each round-bottomed microwell, and an equal volume (100 |il) of phytohemagglutinin-P (PHA, Difco, l|ig/ml) or concanavalin A (Con A, Difco, 10 |ig/ml) solution was added to the well. Cells were incubated in a COiincubator at 37''C for 72 h, and during the final 6 h 1 nCi of pH]-thymidine
50T
Table 2. Lymphocyte subset proportions in OLP patients and healthy persons Lymphoeyte subsets
OLP
patients
Controls (» = 20)
61.4+11.8 61,6 + 9,6 35.3 + 8.9 35.2+10.9 11.4+3.7 10.3 + 4.5 23.9 + 8.7 24.9 + 9.9 14.4 + 5.9 11.5 + 5.7 13.1+5.7 10.9 + 5.7 1.3 + 1.2* 0.6 + 0.5 2.7+1.2 3.2 + 2.2 5.0 + 2.8 4.5+1.5 CD16'CD57CD16+CD57+ 5.0 + 4.0 5.4 + 4.5 CD16-CD57" 13.6 + 7.0 12.1+7.0 HLA-DR14.8 + 6.5 11.4 + 3.7 CD3 + HLA-DR* 3.2+1.5* 1.3 + 0.6 CD25^ 1,1+0,7 1,2 + 0,3 CD3 + CD4* CD4+Leu8CD4+Leu8 + CD8 + CD8^CDllbCD8*CDllb+ CD4+/CD8*
All antibodies used are products of Becton Dickinson (Cahfornia, USA) Each value represents mean±SD (%) * Statistical significance of t-test in OLP patients VS controls - CD8*CDlIb-: P = 0,02, CD3*HLA-DR^ P
Tetsuya Yamamoto, Kazunori Yoneda, Eisaku Ueta and Tokio Osaki Department ot Oral Surgery, Kochi tvledicai School, Nankoku City, Kochi, Japan
Yamamoto T, Yoneda K, Ueta E, Osaki T: Cellular immunosuppression in oral lichen planus, J Oral Pathol Med 1990; 19: 464-70. Functions of peripheral blood lymphocytes and neutrophils from 30 oral lichen planus (OLP) patients were examined using healthy persons as controls. Twocolor flow cytometry of lymphocytes revealed no proportional difference in CD3 or CD4 cells between OLP and controls. CD8CD1 lb (suppressor T) and CD3HLA-DR*^ cell populations increased significantly in OLP when compared with controls, and, CD4/CD8 cell ratio decreased in OLP. Mitogenic response of patients" CD8 and CD4Leu8" cells was similar to that in controls. However, weaker blastogenesis of CD4Leu8^ cells, the most excellent responders in T cell subsets, was observed in OLP Serum IFNP level in OLP (8.4 ±4.8 IU/ml) was significantly lower than in controls (13.7 + 5.0 IU/m!) whereas no difference between the two groups could be found in IFNa or y. As for in vitro cytokine production by IL-2-stimulated lymphocytes, there was no difference in GM-CSF generation between the two groups, but, IFNy and ILip production of patients' lymphocytes was less than that in healthy donors (57.6 + 50,7 VS 78,7 ±39,6 u/ ml, 152,3 ±93,5 VS 258,7 ±65,4 pg/ml, respectively). Moreover, superoxide generation of patients' neutrophils by PMA stimulation was significantly insufficient as compared with controls' (84,9 ±30,9 VS II0,8 ±24,1 pmol/min/lO" cells). Nevertheless, natural killer eell activities of both groups distributed in the same range. These results suggest that OLP patients' lymphocyte and neutrophil functions are impaired, and that cellular immunosuppression is a pathologic characteristic of OLP,
Lichen planus, a chronic infiammatory mucocutaneous disease of unknown etiology, often occurs in the oral mucosa. Complication of lichen planus with autoimmune diseases such as myasthenia gravis, ulcerative colitis and systemic lupus erythematosus (SLE) has recently been reported (1-3). It now seems more accurate to suppose that lichen planus is based on a disturbed immune condition, and may be a localized autoimmune disease. Recent immuno-histochemical studies on oral lichen planus (OLP) have identified each lymphocyte infiltrate subset and accessory cells (4-6). As for accumulated immunohistochemical results, OLP can be thought of as a clinical manifestation of infiltrated T cell and accessory cell response to modified autologous epithelial cells. The interesting observation that peripheral blood lymphocytes may exert nonspecific cytotoxocity toward keratinocytes was recently reported (7), However, no direct proof that autologous lymphocytes attack and lyse the basal cells or inferior spinosum layer cells has been obtained. Previous studies demonstrated T cell subsets in peripheral blood of patients
with OLP (8, 9), but investigations of functional lymphocyte subsets by twocolor analysis are scarce. There are few studies regarding cellular immunity in OLP (10-12), and no investigator has clarified abnormalities which identify OLP as a disease related to impaired cellular immunity. In the present study, we aimed to evaluate cellular immunities of OLP patients, and investigated peripheral blood lymphocyte subsets by two-color flow cytometric analysis, natural killer cell (NK) activity and blastogenesis. Furthermore, functions such as cytokine producibility of lymphocytes by interleukin 2(lL-2) stimulation, superoxide(O2~) generation of neutrophils, and interferon (IFN) activity in serum were investigated. Material and methods Subjects
Thirty patients with clinically diagnosed and histologically confirmed OLP were evaluated. The clinical characteristics of the patients are given in Table 1. No patient had been given topical or systemic drugs which could modify the im-
Key words: cylokine and superoxide generation: double color flow cytometry: lymphoblastogenesis; oral lichen planus: serum interferon. T. Yamamoto, Department of Oral Surgery, Kochi Medical School Kohasu Oko-cho, Nankoku-city, Kochi 783 Japan Accepted for publication August 3, 1990.
mune condition, and blood samples were obtained from untreated OLP patients. The samples from patients and healthy controls were synchronously examined. Healthy volunteers were chosen with the aim of matching their sex and age with the patients. Cell preparation and culture
Lymphocytes and polymorphonuclear leukocytes (PMNL) were obtained by the Ficoll-Paque gradient method, HepTable I, Characteristics of OLP patients t Sex (m/f) t Age (yrs) Clinical type* reticular plaque type atrophie ulcerative Duration of symptoms -- 1 mo. -6 mo. -1 yr. 1 yr. ~
10/20 59.4±13.4 (25-83) 16 5 3 6 5
10 5 10
t Healthy controls: lO/tO, 49.9±l2.l (28-80) * Clinical types were determined according to ANDREASEN'S classit'ieation (15).
Cellular imtnunitv in oral liehen planus 465 arinized peripheral blood was centrifuged on Ficoll-Paque gradients at 400 X g for 30 min. Mononuclear leukocytes were drawn from the upper layer of the gradients and PMNL were collected at the interface just above the red blood cell layer. For removing contaminated red blood cells, cold distilled water was added to PMNL pellets. After 10 s, an equal volume of 2 x PBS was added to the PMNL hypotonic solution and restored isotonicity. Isolated mononuclear leukocytes and PMNL were washed appropriately, and suspended in RPMl 1640 (Nissui Pharma,, Co,, Ltd, Tokyo Japan) supplemented with fetal calf serum and antibodies, and in Hank's balanced salt solution (HBSS), respectively. Microscopic examination of Giemsa-stained cells revealed that PMNL preparations were composed of over 98% PMNL, Mononuclear leukocytes were incubated in plastic petri dishes in a COi-ineubator at 37''C for 120 min. The dishes were then gently swabed and lymphocytes were isolated, T cells were separated from non-T cells by a single step rosetting method (13) with 2-amino-ethylisothiouronium bromide hydrobromide (Sigma Chemical Co,, St, Louis, MO)-treated sheep red blood cells (AET-SRBC), To prepare T cell subsets, a panning method (14) was used, Rosetted T cells were incubated with 25 |i g/ml anti-CD8 monoclonal antibody (mAb, Becton Dickinson, California, USA) for 30 min at 4 ^ , and then cultured in petri dishes that had been coated with 2 |ig/ml goat antimouse IgG antiserum (Kirkegaard & Perry Laboratories Inc, Gaithersburg, Md.). After 70 min incubation, adherent cells (CD8* cells) were recovered by scraping with a rubber policeman and removal of non-adherent cells (CD8" cells). CD8^ T cells (over 95% of which were CD4 ') were further fractionated into Leu8^ and Leu8" subpopulations by a second panning set with anti-Leu8 mAb (10 |ig/ml, Becton Dickinson), The composition of isolated CD8^, CD4 ' Leu8+ and CD4' Leu8- cells was ascertained by flow cytometry, and more than 90% purity was proven in all subsets. With cell preparation, serum samples were collected for IFN assay, IVvo-color flow cytometry
For double color analysis of lymphocytes, both monoclonal FITC-conjugated (anti-CD8, anti-CD4, anti-CD3, anti-CD57) and PE-conjugated (antiLeu8, anti-CD16, anti-CDIlb, anti-
HLA-DR) antibodies were used. For dual analysis, combinations of antiCD8 with anti-CD lib, anti-CD4 with anti-Leu8, anti-CD 16 with anti-CD57, and anti-CD3 with anti HLA-DR were used. Isolated lymphocytes of 5x10' cells were resuspended in 100 nl of PBS containing 0,1% azide (volume %) and packed into culture tubes. After saturating with both FITC- and PE-conjugated mAb solutions, tubes were mildly agitated and incubated for 30 min at 4°C in a dark place. Cells were washed twice, resuspenced in a 0,1% azide solution, and analyzed with an EPICS V flow cytometer (Coulter Electronics, Inc, USA), Monocolor analysis was also applied to CD3 ', CD25 ' and HLA-DR + cells. Lymphocyte blastogenesis
Lymphocyte blastogenesis was also routinely examined. Briefly, lymphocytes of 1x10-' cells or each T cell subset of 5x lO'' cells in 0,1 ml of RPMI were seeded into each round-bottomed microwell, and an equal volume (100 |il) of phytohemagglutinin-P (PHA, Difco, l|ig/ml) or concanavalin A (Con A, Difco, 10 |ig/ml) solution was added to the well. Cells were incubated in a COiincubator at 37''C for 72 h, and during the final 6 h 1 nCi of pH]-thymidine
50T
Table 2. Lymphocyte subset proportions in OLP patients and healthy persons Lymphoeyte subsets
OLP
patients
Controls (» = 20)
61.4+11.8 61,6 + 9,6 35.3 + 8.9 35.2+10.9 11.4+3.7 10.3 + 4.5 23.9 + 8.7 24.9 + 9.9 14.4 + 5.9 11.5 + 5.7 13.1+5.7 10.9 + 5.7 1.3 + 1.2* 0.6 + 0.5 2.7+1.2 3.2 + 2.2 5.0 + 2.8 4.5+1.5 CD16'CD57CD16+CD57+ 5.0 + 4.0 5.4 + 4.5 CD16-CD57" 13.6 + 7.0 12.1+7.0 HLA-DR14.8 + 6.5 11.4 + 3.7 CD3 + HLA-DR* 3.2+1.5* 1.3 + 0.6 CD25^ 1,1+0,7 1,2 + 0,3 CD3 + CD4* CD4+Leu8CD4+Leu8 + CD8 + CD8^CDllbCD8*CDllb+ CD4+/CD8*
All antibodies used are products of Becton Dickinson (Cahfornia, USA) Each value represents mean±SD (%) * Statistical significance of t-test in OLP patients VS controls - CD8*CDlIb-: P = 0,02, CD3*HLA-DR^ P
