Imunidade em Lichen Planus

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Assessment of the peripheral immunocompetent cells in patients
with reticular and atrophic-erosive lichen planus
Katarzyna Charazinska-Carewicz, MD, DDS, PhD,a Ewa Ganowicz, DDS,b Maria Krol, PhD,c
and Renata Gorska, DDS, PhD,d Warsaw, Poland
MEDICAL UNIVERSITY OF WARSAW
Objective. To assess the cellular and humoral immunity in patients with reticular and atrophic-erosive oral lichen
planus (OLP).
Study design. Lymphocyte phenotype analysis of 50 patients with OLP and 16 control subjects was performed by
means of flow cytometry. The results were analyzed by the Mann-Whitney U test.
Results. In the patient group the percentage of naïve helper T cells was significantly decreased, while that of memory
cells was increased, resulting in a significantly higher ratio of memory to naïve cells in the OLP group than in the
control group. In the group of patients with atrophic-erosive OLP, the percentages of cytotoxic/suppressor T cells and
cytotoxic/suppressor naïve T cells were found to be slightly decreased.
Conclusions. The results indicate that the relative ratio of immunocompetent cells in the peripheral blood is altered in
patients with lichen planus and that the exact character of these disturbances depends on OLP form. Some of the
alterations observed in these patients are typical to autoimmune diseases, thus promoting the concept of autoimmune
etiopathogenesis of lichen planus. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2008;105:202-5)
Lichen planus is a chronic disease affecting the skin
and mucous membranes. Oral lichen planus (OLP) may
present with papules, plaques, reticular pattern, ero-
sions or, rarely, blisters. In clinical practice, a simpli-
fied classification according to Bagan-Sebastian is
used.1,2 It distinguishes a reticular form (only white
papules or reticular pattern) and a more severe atrophic-
erosive form of the disease. The latter is characterized
by erosions and skin atrophy.
Even though the pathogenesis of lichen planus is
poorly understood, it is commonly believed to be of
immune, namely, autoimmune, nature. The histochem-
ical analysis of affected mucous membrane reveals
profuse inflammatory infiltrate consisting predomi-
nantly of lymphocytes.2 On the basis of earlier studies,
it has been stated that the infiltrate composition depends
on the clinical form of disease.3 In initial lesions, cor-
responding with reticular form of OLP, the predomi-
nant lymphocyte subset constitute CD4� (helper) cells,
whereas in more advanced atrophic-erosive lesions the
number of CD8� (cytotoxic/suppressor) cells in-
aLecturer, Department of Periodontology and Oral Diseases.
bPhD student, Department of Periodontology and Oral Diseases.
cResearch staff, Department of Hematology, Oncology, and Internal
Diseases.
dProfessor and Head, Department of Periodontology and Oral Diseases.
Received for publication Jul 28, 2006; returned for revision Mar 25,
2007; accepted for publication Mar 26, 2007.
1079-2104/$ - see front matter
© 2008 Mosby, Inc. All rights reserved.
doi:10.1016/j.tripleo.2007.03.031
202
creases substantially.4 Moreover most of the CD4�
cells belong to the memory lymphocyte subset
(CD4�CD45R0�).2
Some authors have also investigated the alterations
of lymphocyte subsets in peripheral blood in patients
with OLP. Even though the results obtained correspond
partially to the local infiltrate composition, they are not
unambiguous.2,3 That is why the aim of presented study
was to compare the B-cell, NK-cell, and T-cell sub-
population levels in the peripheral blood of patients
with the reticular and atrophic-erosive forms of OLP
and of a control group.
MATERIAL AND METHODS
The study group consisted of 50 consecutive lichen
planus patients presenting to the Department of Peri-
odontology and Oral Diseases at the Medical Univer-
sity of Warsaw, Poland, among them 42 women and 8
men, aged 61.5 � 13.0 years. All the patients gave
written informed consent.
The preliminary diagnosis was made on the basis of
clinical presentation. A thorough medical history and
clinical examination were performed to exclude pa-
tients likely to suffer from oral lichenoid lesions, for
example patients after bone marrow transplantation. In
questionable cases (n � 11; only patients with atrophic-
erosive form of disease) a histopathologic examination
was performed.
Depending on the clinical signs the patients were
classified according to the Bagan-Sebastian classifica-
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Volume 105, Number 2 Charazinska-Carewicz et al. 203
tion1 into 2 groups: reticular (n � 11) and atrophic-
erosive (n � 39).
The control group consisted of 16 healthy gender-
matched individuals.
Peripheral blood lymphocyte subsets assessment
Two milliliters of peripheral venous blood was sam-
pled into an EDTA-containing test tube. In all of the
patients blood was sampled in the morning on a clinical
examination day.
The complete blood count with differential was as-
sessed by means of a Cell-Dyn 1700 device (Abbott,
Chicago, IL).
Lymphocyte phenotype analysis was performed by
means of 2-color, 4-parameter flow cytometry using
FACS Calibur (BDIS, Mountain View, CA). Five mi-
croliters of an appropriate monoclonal antibody was
added to 50 �L peripheral blood and incubated for 20
minutes in the dark at room temperature. The blood
cells were then incubated for 10 minutes with a lysing
agent (FACS Lysing Solution; BDIS) to remove eryth-
rocytes and washed twice with phosphate-buffered sa-
line. The washed cells were fixed in 1% paraformalde-
hyde. Lymphocyte phenotype analysis was performed
using monoclonal antibodies to distinguish cells ex-
pressing CD19, CD3, CD16/56, CD4, CD8, CD45RA,
and CD45R0 antigens (CD3 FITC/CD4 RPE, CD3
FITC/CD8 RPE, CD19 RPE, CD45RO FITC/CD4
RPE, CD45RO FITC/CD8 RPE, CD45RA RPE/CD4
FITC, and CD45RA RPE/CD8 FITC [Dako, Glostrup,
Denmark] and CD3 FITC/CD16�56 PE [BDIS]).
When leukocytes are added to the reagent, the fluoro-
Table I. Comparison of B-cell, NK-cell, and T-cell sub
planus (reticular and atrophic-erosive form) and contro
Cell population/subpopulation [surface antigens] Reticu
B cells [CD19�] 9.83
NK cells [CD3�(CD16�CD56)�] 12.08
T cells [CD3�] 74.83
T helper cells [CD3�CD4�] 42.92
T cytotoxic/suppressor cells [CD3�CD8�] 31.00
T helper naïve cells [CD4�CD45RA�] 16.17
T helper memory cells [CD4�CD45R0�] 29.08
CD4�CD45R0�:CD4�CD45RA� 4.01
T cytotoxic/suppressor naïve cells [CD8�CD45RA�] 23.83
T cytotoxic/suppressor memory cells
[CD8�CD45R0�]
14.00
CD8�CD45R0�:CD8�CD45RA� 0.60
Data are presented as mean percentage � SD.
*P � .05 compared with control.
†P � .001 compared with control.
‡P � .1 compared with reticular form.
chrome-labeled antibodies bind specifically to leuko-
cyte surface antigens. During acquisition, the cells
travel past the laser beam and scatter the laser light. The
stained cells fluoresce. These scatter and fluorescence
signals, detected by the cytometer, provide information
about the cell’s size, internal complexity, and relative
fluorescence intensity.
Cells were considered to be positive for tested anti-
gens if they revealed higher fluorescence intensity than
cells stained with the isotype-matched control antibody.
The results were presented as the percentage of cells
gated in FSC/SSC lymphocyte-gate expressing the as-
sessed antigens.
Statistical analysis
The obtained results were analysed by means of the
nonparametric Mann-Whitney U test. The level of sig-
nificance was set to .05.
RESULTS
Table I presents the percentages of lymphocyte pop-
ulations and subpopulations in all of the study groups.
The B-cell level (CD19�) did not differ significantly
among the groups. The NK-cell level (CD3�(CD16�
CD56)�) did not differ between the patient and control
groups but was slightly elevated in the patients with
atrophic-erosive OLP compared with the reticular OLP
group.
In the group of patients suffering from atrophic-
erosive lichen planus, the percentage of T cells (CD3�)
was found to be slightly decreased, even though no
differences were found betweenthe patient and control
groups. The percentage of naïve helper T cells was
significantly decreased in the OLP group, whereas the
ations (mean percentage � SD) in patients with lichen
p
Lichen planus [%]
Atrophic-erosive
form Total
Control group
[%]
10.08 � 4.06 10.02 � 4.03 9.75 � 3.75
16.59 � 7.66‡ 15.51 � 7.51 15.62 � 7.85
68.74 � 8.92‡ 70.23 � 9.15 70.29 � 7.12
42.28 � 9.30 42.44 � 9.36 42.69 � 6.38
26.58 � 7.85‡ 27.64 � 7.93 26.38 � 6.67
13.11 � 7.66 13.84 � 9.56 18.56 � 6.93*
31.70 � 7.99 31.07 � 7.60 25.12 � 4.63*
3.56 � 2.59 3.66 � 2.95 1.69 � 1.25†
21.58 � 6.77‡ 22.12 � 6.20 21.06 � 5.65
14.92 � 6.13 14.70 � 6.10 12.81 � 6.26
0.75 � 0.35 0.71 � 0.34 0.64 � 0.37
popul
l grou
lar form
� 4.11
� 6.10
� 8.63
� 9.95
� 7.51
� 14.19
� 6.11
� 3.98
� 3.59
� 6.24
� 0.31
memory cell level was increased, resulting in a signi-
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204 Charazinska-Carewicz et al. February 2008
ficantly increased CD4�CD45R0�:CD4�CD45RA�
ratio in the OLP group compared with the control
group. No statistically significant differences were ob-
served between the 2 disease forms regarding helper T
cells (CD3�CD4�), naïve helper T cells (CD4�
CD45RA�), and memory helper T cells.
In the group of patients suffering from atrophic-
erosive lichen planus, the percentages of cytotoxic/
suppressor T cells (CD3�CD8�) and cytotoxic/
suppressor naïve T cells (CD8�CD45RA�) were
found to be slightly decreased, even though no diffe-
rences were found between the patient and control
groups. No statistically significant differences were ob-
served between all of the groups regarding memory
cytotoxic/suppressor T cells (CD8�CD45R0�).
DISCUSSION
Even though it is widely accepted that immune
mechanisms are important in the pathogenesis of OLP,
the exact etiology of this disease is still unexplained.2,3
Histopathologic and histochemical studies performed at
different stages of lesions have revealed a diffuse lym-
phocyte infiltrate, comprising principally T cells, with a
gradual accumulation of CD8� cells.2,5 The composi-
tion of the accumulated cells differs depending on the
clinical form of lichen planus. It seems that some
differences can also be observed on the periphery.2
Still, whether the primary reason for this picture is a
change in keratinocytes that triggers an immunologic
reaction and attraction of lymphocytes or a primary
general immunologic disturbance leading to the change
and degeneration of keratinocytes remains unknown.2,6
In the present study, we assessed the percentage of
different immunocompetent cells and their subsets to
determine whether any disorders could be identified in
patients with lichen planus, also taking into consider-
ation 2 distinct forms of the disease.
It is usually assumed that the cellular immune re-
sponse is crucial in the etiopathogenesis of lichen pla-
nus.2,7,8 In accordance with this, no statistically signi-
ficant difference between study groups was found in
B-cell levels (CD19�). There are only a few reports
about slight aberrations in serum antibody levels in
patients with lichen planus.9 These changes seem to be
secondary to the impairment of keratinocytes and thus
unrelated to lichen planus etiopathogenesis.2
An increase in the level of NK cells (CD3�
(CD16�CD56)�) was found in the atrophic-erosive
OLP group compared with the reticular form of disease,
even though it was not statistically significant. We
found only 2 reports in which the percentage of NK
cells in the peripheral blood of lichen planus patients
was analyzed. Biocina-Lukenda et al. found the NK cell
level to be decreased in lichen planus patients,10,11
especially in the atrophic-erosive form of the disease.
The present results, therefore, disagree with those in the
available reports. It can be assumed that NK cells could
take part in the destruction of basal layer keratinocytes,
a process observed in the histopathology of erosions.
In the present results, no differences were found in
the levels of T cells (CD3�) or T helper cells (CD4�)
between the OLP and control groups.
The percentage of naïve helper T cells was signifi-
cantly decreased in the OLP group, whereas that of
memory cells was increased, resulting in a significantly
increased CD4�CD45R0�:CD4�CD45RA� ratio in
the OLP group compared with the control group. The
standard deviation of the CD4�CD45R0�:CD4�
CD45RA� ratio is relatively high (Table I), yet the
statistical analysis by means of Mann-Whitney test,
designed to compare data when their distribution is not
normal, revealed the statistically significant difference
between the OLP and control groups (P � .001). Other
authors have also observed an increase in memory
helper T-cell levels with a concomitant decrease in
naïve helper T cells.2,12 According to Sugerman et al.,
the causative factors of lichen planus stimulate naïve
helper T-cell conversion into memory cells.13 This
leads to a decrease in the percentage of naïve cells
responsible for suppression and accumulation of acti-
vated cells exhibiting helper activity.2,12,14 Current evi-
dence suggests that the imbalance between lymphocyte
subpopulations could lead to the impairment of sup-
pressor functions, although data concerning that prob-
lem are limited.5 A similar phenomenon is observed in
some autoimmune diseases.8,15
No differences were found in the level of helper T
cells (CD4�) or their subpopulations between the re-
ticular and atrophic-erosive forms of OLP. In contrast,
in lichen planus patients with atrophic-erosive lesions
Rodriguez-Nunez et al. reported an increased percen-
tage of naïve and memory helper T cells (CD4�
CD45R0� and CD4�CD45DR�), but no change in
their ratio.2 Thus, it seems that the shift of helper T
cells toward memory helper cells may only be observed
at early stages of disease and not when the lesions
differentiate from reticular toward atrophic-erosive
form. It is also possible that this process only takes
place in situ and no such phenomenon can be observed
in the peripheral blood.
Neither the cytotoxic/suppressor T-cell population
(CD3�CD8�) nor its subpopulations differed between
patients with OLP and the control subjects. The cyto-
toxic/suppressor T-cell population level was found to
be slightly decreased in patients with the atrophic-
erosive form of lichen planus compared with the reti-
cular form. According to Eversole, the decreased lym-
phocyte count in the peripheral blood is related mainly
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Volume 105, Number 2 Charazinska-Carewicz et al. 205
to the migration of these cells from the blood vessels
and infiltration of the basal skin/mucous membrane
layer.7 This hypothesis is supported by the finding that
proliferating CD8� lymphocytes are scarce in the li-
chen planus infiltration.16 This would suggest that most
of them originate from the peripheral blood. Moreover,
as the disease progresses and the clinical changes in-
tensify, evolving into erosions, cytotoxic/suppressor
cells predominate in the infiltration.2 The very same
subpopulation is decreased in the peripheral blood
when the atrophic-erosive lesions are present.17 This
can contribute to the changes observed in the peripheral
blood of lichen planus patients, depending on the clinical
picture of OLP. According to this theory, the aberrations
observed in the peripheral blood are secondary to the local
process (infiltration of mononuclear cells).6,7
The differences observed between groups could
also suggest aberrations in the suppressor function in
the atrophic-erosive group.2 According to Biocina-Lu-
kenda and some other authors, the suppressor activity is
lower in patients with OLP.2,7,11 Suppressor-cell defi-
ciency may lead to uncontrolled lymphocyte activation,
including autoreactive immunocompetent cells.4 Ac-
cording to this theory, the general disturbances in im-
mune system function lead to the onset of the disease.3
In the atrophic-erosive form of the disease, the level
of naïve cytotoxic/suppressor cells (CD8�CD45RA�)
was also found to be slightly decreased. No difference
was found in the percentage of memory cytotoxic/suppressor cells. A decrease in the percentage of naïve
lymphocytes is also observed in other autoimmune
disorders and can be due to excessive antigen stimula-
tion or disturbances in suppressor function.12
Nevertheless, care must be taken in interpreting the
data achieved, because not all of the patients had a
diagnosis of OLP confirmed by biopsy. Out of 50
patients, only 11 agreed to the biopsy and none of them
with the reticular form of disease.
CONCLUSIONS
The present results show that the percentages of
immunocompetent cells in lichen planus differ from
normal values and that these differences also depend on
the clinical form of OLP.
In patients with OLP (compared with the control
group) the main alteration was a decrease in naïve
helper T cells (CD4�CD45RA�) accompanied by an
increase in memory helper T cells (CD4�CD45R0�).
It may reflect a shift toward memory helper T cells in
the initial steps of OLP etiopathogenesis.
In the atrophic-erosive group, the observed distur-
bances mainly pertained to cytotoxic/suppressor cells
compared with the reticular group. The observed de-
crease in cytotoxic/suppressor T cells, especially their
naïve subpopulation, is typical of autoimmune diseases,
thus promoting the concept of autoimmune etiopatho-
genesis of lichen planus.
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Peripheral T-cell subsets in patients with reticular and atrophic-
erosive oral lichen planus. Oral Surg Oral Med Oral Pathol Oral
Radiol Endod 2001;91:180-8.
3 . Al-Fouzan AS, Habib MA, Sallam TH, El-Samahy MH, Rostom
A I . Detection of T lymphocytes and T lymphocyte subsets in lichen
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1 3 . Sugerman PB, Rollason PA, Savage NW, Seymour GJ. Suppressor
cell function in oral lichen planus. J Dent Res 1992;71:1916-9.
14. Clement LT, Yamashita N, Martin AM. The functionally distinct
subpopulations of human CD4� helper/inducer T lymphocytes
defined by antiCD45R antibodies derive sequentially from a
differentiation pathway that is regulated by activation-dependent
post-thymic differentiation. J Immunol 1988;141:1464-70.
15. Silverman ED, Somma C, Khan M, Melmon KL, Engleman EG.
Abnormal T suppressor cell function in juvenile rheumatoid
arthritis. Arthritis Rheum 1990;33:205-11.
16. Deguchi M, Ohtani H, Sato E, Naito Y, Nagura H, Aiba S, et al.
Proliferative activity of CD8(�) T cells as an important clue to
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17. Gadenne AS, Strucke R, Dunn D, Wagner M, Bleicher P, Bigby
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Reprint requests:
Dr. K. Charazinska-Carewicz
Department of Periodontology and Oral Diseases
Medical University of Warsaw
18, Miodowa St.
00-246 Warsaw
Poland
sluzowki@am.edu.pl
mailto:sluzowki@am.edu.pl
	Assessment of the peripheral immunocompetent cells in patients with reticular and atrophic-erosive lichen planus
	MATERIAL AND METHODS
	Peripheral blood lymphocyte subsets assessment
	Statistical analysis
	RESULTS
	DISCUSSION
	CONCLUSIONS
	REFERENCES