Vol. 121 No.

1 January 2016

Smoking habits and clinical patterns can alter the

inflammatory infiltrate in oral lichenoid lesions
Mohammad S. Alrashdan, BDS, MSD, DCD,a Christopher Angel, BDSc, MDSc, MBBS, FRCPA,b
Nicola Cirillo, DMD, PhD,c and Michael McCullough, BDSc, MDSc, PhDc

Objective. The present immunohistochemical study aimed to investigate the possible correlation between demographic
variables and clinical presentation of oral lichenoid lesions (OLL), in addition to the potential effects of these variables and
smoking status on OLL inflammatory infiltrate.
Study Design. A total of 53 patients with OLL were assigned, according to their smoking status at the time of diagnosis, to
either a smokers group (n ¼ 27) or a nonsmokers group (n ¼ 26). Demographic and clinical data, including the site and pattern
of the OLL, symptoms, and medical history, were analyzed. Immunohistochemical expression of clusters of differentiation,
including CD3, CD4, CD8, CD68, and CD1a, was compared between the two groups.
Results. Gingival involvement in OLL was found to be significantly associated with older age. Buccal mucosa as
the sole OLL site showed a significantly higher expression of CD3þ cells compared with other sites (P < .05). OLL
presenting as a reticular type alone was significantly associated with less CD3þ expression (P < .05), whereas a
significantly higher CD1aþ expression was seen with plaque-like type OLL (P < .05). Smoking was significantly
associated with less expression of macrophages (CD68þ cells) and less clinical symptoms (P < .05 and P < .01,
respectively).
Conclusion. The inflammatory infiltrate in OLL can be affected by their clinical distribution and presentation. Smoking
reduces the expression of macrophages in OLL, and this may alter the immune surveillance and the mechanisms of malignant
transformation. (Oral Surg Oral Med Oral Pathol Oral Radiol 2016;121:49-57)

Oral lichen planus (OLP) is a chronic, inflammatory,
immune-mediated disease affecting the oral stratified
squamous mucosa and occurring in approximately 1%
to 2% of the general population, with characteristic
relapses and remissions.1-3 The most common oral sites
involved are the buccal mucosa, lateral surface of the
tongue, and the gingivae, respectively.1,2
OLP is twice as common in women as in men and
commonly affects adults over 40 years of age; however,
younger ages can also be affected.4 People from all
ethnic backgrounds seem to be susceptible to OLP.5
Four clinical types of OLP lesions are usually
encountered, individually or combined: Reticular,
plaque-like, atrophic, and erosive/ulcerative. Papular
and bullous OLP, the other two forms described in
literature, are considered uncommon.4,5 Lesions that are
clinically and histologically similar to OLP may
This work was partially funded by the Melbourne Dental School as a
postgraduate research project.
a
Assistant Professor of Oral Medicine, Faculty of Dentistry, Jordan
University of Science and Technology, Irbid, Jordan.
b
Anatomic Pathologist, Department of Pathology, Peter MacCallum
Cancer Centre, East Melbourne, Australia.
c
Associate Professor of Oral Pathology, Diagnosis and Medicine,
Melbourne Dental School, The University of Melbourne, Victoria,
Australia.
Received for publication Jun 9, 2015; returned for revision Aug 13,
2015; accepted for publication Aug 24, 2015.
Crown Copyright Ó 2016 Published by Elsevier Inc. All rights
reserved.
2212-4403/$ - see front matter
http://dx.doi.org/10.1016/j.oooo.2015.08.020
develop as a reaction to systemic medications or dental
materials, and these are called oral lichenoid reactions
(OLRs). The collective term that includes both OLP and
OLRs is oral lichenoid lesions (OLL), which will be
used here whenever reference to both is made. The rate
of malignant transformation of OLP varies according to
different studies6 and is commonly associated with
erosive/erythematous patterns.7
The clinical types and distribution of OLL within the
oral cavity are associated with different biologic and
clinical behaviors; however, the correlation among the
type of inflammatory infiltrate, the intraoral site
affected, and the clinical type of an OLL has not been
thoroughly investigated so far.
The key role of the inflammatory infiltrate in the
pathophysiology of OLP has been well documented.5,8
The majority of OLP-related inflammatory cells are
activated cytotoxic (CD8þ) T-lymphocytes, which are
thought to interact with other inflammatory cells such
as helper (CD4þ) subpopulations, Langerhans cells
(CD1aþ), and macrophages (CD68þ), as well as basal
Statement of Clinical Relevance
Oral lichenoid lesions (OLL) are common oral
mucosal condition with variable clinical presentation
and distribution and the underlying inflammatory
infiltrate is affected by some of these variables as
well as the smoking status of the patient.

49

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ORAL AND MAXILLOFACIAL PATHOLOGY
50 Alrashdan et al.
keratinocytes, leading ultimately to keratinocyte
apoptosis.8 Interestingly, it has been suggested that the
malignant transformation of OLP may be related to, or
dependent on, a series of molecular stimuli originating
in the inflammatory infiltrate, rather than external
carcinogens.9
Differences in OLL inflammatory infiltrate according
to age, gender, clinical presentation, and the possible in-
fluence of known carcinogens (e.g., smoking) on this
infiltrate have not been well addressed in the literature.
Characterization of the potential differences has salient
clinical implications because all of these variables can
affect the clinical behavior of an OLL, including response
to therapy and the likelihood of malignant transformation.
Therefore, this study was designed to clarify whether
demographic and clinical variables can alter the features
of OLL inflammatory infiltrate and, more importantly, to
investigate the possible effects of smoking on OLL in-
flammatory infiltrate by means of immunohistochemistry.
MATERIALS AND METHODS
Patients
A total of 53 patients with OLL (25 males and 28 fe-
males, mean age ¼ 59 years) were enrolled in this
retrospective study. The study was reviewed and
approved by the Health Sciences Human Ethics com-
mittee of The University of Melbourne (ID: 1136916).
All patients had an OLL diagnosis, which was based on
both clinical and histopathologic examinations of
representative intraoral biopsy specimens.
All patients were diagnosed, managed, and followed
up at the Oral Medicine Department of the Royal
Dental Hospital of Melbourne in the period be-
tween1998 and 2006, thus, a follow-up period of at
least 7 years was applied to all of our patients.
Diagnosis of OLL was based on clinical and histo-
logic features in each patient. Clinical features included
the presence of at least one of the following patterns:
white striations (reticular), white plaques (homoge-
neous, slightly elevated smooth white patches),
erythematous, erosive, or ulcerative lesions. Clinical
features suggestive of OLRs to dental materials, such as
localized lesions in close proximity to a particular
material, were taken into consideration, and such le-
sions were excluded. However, OLRs to systemic
medications are very difficult to differentiate from
classic OLP, and so both were accepted in this study as
OLL. Diagnostic histologic features included a sub-
epithelial band-like inflammatory infiltrate, liquefactive
degeneration of basal keratinocytes, and the presence of
colloid bodies. Lesions not presenting with these fea-
tures or showing signs of epithelial dysplasia or
abnormal keratinization were also excluded.
Patients were allocated to either the experimental
group (smokers) or the control group (nonsmokers)
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OOOO
January 2016
based on their smoking status at the time of the biopsy.
The two groups were matched for age and gender. A
smoker was defined as a patient who smoked tobacco
for at least 1 year before the OLL diagnosis and was
still smoking at the time of diagnosis. Nonsmokers
were defined as those patients who have never smoked.
Five patients from the smokers group and four from the
nonsmokers group were occasional alcohol drinkers.
None of the patients in either group used topical oral
medications before the OLL diagnosis.
Demographic data and clinical information regarding
site, pattern of OLL, presence or absence of symptoms,
and medical history were collected. To facilitate anal-
ysis, the clinical sites of OLL were assigned to one of
three groups: (1) buccal mucosa only; (2) tongue, with
or without other sites; and (3) gingivae, with or without
other sites. The clinical patterns were assigned to one of
four groups: (1) reticular only; (2) plaque-like, with or
without other patterns; (3) desquamative gingivitis
(defined as erythematous and/or erosive/ulcerative OLL
affecting all or parts of the gingivae), with or without
other patterns; and (4) others, for patients presenting
with patterns or combinations not fitting into any of the
first three categories.
Immunohistochemistry
Immunohistochemical reactions were assessed by using
a standard streptavidin-biotin protocol against the
following clusters of differentiation: CD1a, CD3, CD4,
CD8, and CD68. Sections of 3 mm were obtained from
routinely processed paraffin-embedded blocks and
mounted on 3-aminopropyltriethoxysilane (Sigma-
Aldrich, St. Louis, MO, USA). Pretreatment of depar-
affinized and hydrated tissue sections with heat-induced
epitope retrieval was performed using Dako Target
Retrieval Solution (code S1700 for CD1a and CD68,
code S3308 for CD3 and CD8, code K8004/8014 for
CD4) (Dako Corporation, Carpinteria, CA, USA) at
100 C for 20 minutes. Sections were then incubated
with Dako peroxidase block (code S2001) for 5 minutes
and thoroughly rinsed with distilled water and then with
phosphate buffer saline. Monoclonal mouse antihuman
antibodies were diluted in Dako Antibody Diluent
(code S0809) at the following concentrations: CD1a
(1:50) (clone 010, code M3571, Dako Cytomation),
CD3 (1:25) (clone F7.2.38, code M7254, Dako Cyto-
mation), CD4 (1:40) (clone 4B12, code M7310, Dako
Cytomation), CD8 (1:50) (clone C8/144B, code
M7103, Dako Cytomation), CD68 (1:100) (clone PG-
M1, code M0876, Dako Cytomation) (Dako Corpora-
tion, Carpinteria, CA, USA).
The sections were incubated with primary antibodies
for 30 minutes and then rinsed with phosphate buffer
saline. Universal Dako labeled streptavidin biotin
OOOO ORIGINAL ARTICLE
Volume 121, Number 1 Alrashdan et al. 51

Fig. 1. Image analysis using ImageJ and IHC Profiler. A, original (red-green-blue [RGB]) image showing immunohistochemical
reaction against CD3. The image is split by IHC Profiler into DAB-stained B, and hematoxylin-stained C, images. The positive
cells in each image are selected with the use of an adjustable threshold function; positive DAB-stained D, and positive
hematoxylin-stained E, cells. The percentage of CD3þ cells ¼ (positive cells in image D/positive cells in image E)  100%.

(LSABþ) with peroxidase (LSABþ kit, HRP) (code
K0679) (Dako Corporation, Carpinteria, CA, USA), was
used for the application of the biotinylated link antibody
and peroxidase-labeled streptavidin. 3,30 dia-
minobenzidine (DABþ, code K3468) (Dako Corpora-
tion, Carpinteria, CA, USA) was then used as a
chromogen solution. Finally, the sections were coun-
terstained with Harris’s hematoxylin, thoroughly washed
in water, dehydrated, and mounted with cover slips and
DPX mountant (Sigma-Aldrich, St. Louis, MO, USA).
A negative control for each CD was obtained by omit-
ting the primary antibody, and sections of tonsillar and
gingival tissues were used as positive controls.
Final images were captured by using a digital SLR
camera (Nikon D5200, Nikon Corp, Tokyo, Japan)
attached to a light microscope (Laborlux S, Leica Mik-
roskope und Systeme GmbH, Wetzlar, Germany). At least
three images (range 3-5) (100) of representative areas of
each section were captured for analysis. The selection of
the representative areas took into account that they
showed the basic histopathologic features of OLL (i.e., the
band-like subepithelial inflammatory infiltrate, degener-
ation of basal keratinocytes, and colloid bodies) and that
they were devoid of processing artefacts.
Image analysis
Image analysis was conducted by using the NIH ImageJ
1.45q software (National Institutes of Health, Bethesda,
MD, USA) and a compatible specialized plugindthe
IHC Profiler. IHC Profiler (National Institutes of
Health, Bethesda, MD, USA) is a software module
designed for the quantitative evaluation and automated
scoring of digital immunohistochemistry (IHC) images
in imageJ.10 It is basically a color deconvolution
software that allows splitting of the red, green, and

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ORAL AND MAXILLOFACIAL PATHOLOGY OOOO
52 Alrashdan et al. January 2016

blue channel image (original image) opened in ImageJ

*The OLP site was divided into buccal mucosa alone; tongue, with or without other sites; and gingivae, with or without other sites. The numbers do not add up because some patients had OLP involving
medical history

The clinical pattern of OLP was divided into reticular pattern only; plaque-like pattern, with or without other patterns; desquamative gingivitis, with or without other patterns; and others, for patients who did
Patients with
into a hematoxylin-stained and a DAB-stained sepa-

3
rate images, selection of stained cells (blue in hema-
toxylin and brown in DAB) in each of the resultant
images via an adjustable threshold function, and finally
calculation of the number of stained particles and

Patients with
symptoms
staining intensity by using the incorporated IHC Pro-
filer macro. The percentage of positive cells in each

14

3
image was calculated as follows: (mean positive DAB
stained cells/positive hematoxylin stained cells) 
100%. The image analysis procedure is illustrated in

Others
Figure 1.

3
Desquamative
Semiquantitative analysis of immunostaining

gingivitis
A semiquantitative scoring system was applied to ex-

3
Clinical patterny
press both staining extent and intensity, based on the
results obtained from IHC Profiler (National Institutes
of Health, Bethesda, MD, USA). The extent of staining

Plaque-like
was defined as the percentage of positive (DAB-

14
7
stained) cells in relation to the overall (hematoxylin-
stained) cells in each section. Because of the wide range
and variability of the resultant scores between different

Reticular
antibodies, a universal semiquantitative scoring system

only
7

7
could not be applied; instead, three scoring systems,
each on a scale of 0 to 2, were adopted as follows: for
CD1a and CD68; 0 ¼ <2% positive cells, 1 ¼ 2%-5% Gingivae
involved
positive cells, 2 ¼ >5% positive cells, for CD3 and
7

2
CD8; 0 ¼ <20% positive cells, 1 ¼ 20%-50% positive
cells, 2 ¼ >50% positive cells and for CD4; 0 ¼ <5%
positive cells, 1 ¼ 5%-30% positive cells and 2 ¼
involved
Tongue

>30% positive cells.

10

14
Site*

The staining intensity was scored on a scale of 0 to 3,

Table I. Demographic and clinical data of the study population

with each score corresponding to a percentage

Buccal mucosa

expressed by the IHC Profiler (National Institutes of

Health, Bethesda, MD, USA) (100% being the stron-
only
11

gest intensity). As cells within the same DAB image are

likely to show different staining intensities, the IHC
Profiler (National Institutes of Health, Bethesda, MD,
USA) expresses the average intensity, and scores were
12

13
14

14
Gender

defined as follows: <25% ¼ 0 (negative), 25%-50% ¼

¼

¼
¼

1 (weak/low positive), 50%-75% ¼ 2 (moderate/posi-

M

M
F

tive) and >75% ¼ 3 (strong/high positive). It is

30-78 (60  12)

33-82 (58  13)

important to note that an intensity score of 0 (negative)

(mean  SD)
Age range

does not mean a total absence of positive cells but

rather that the average intensity is less than 25% of the
maximum.
not fit into any other category.

The percentages (calculated from means of original

counts) of positive cells and means of staining in-
Nonsmokers (n ¼ 26)

tensities for each specimen were assigned to the cor-

Smokers (n ¼ 27)

responding semiquantitative score and used for

Group

multiple sites.

comparison between the two groups.

In addition, the presence or absence of positive cells
within the epithelium was scored as 0 ¼ absent or 1 ¼
present and compared between the two groups.
y

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OOOO ORIGINAL ARTICLE
Volume 121, Number 1 Alrashdan et al. 53

Table II. Summary of immunohistochemistry results showing the percentage of positive cells reacting to each CD in
each group in addition to the number of specimens corresponding to each staining intensity score
Nonsmokers Smokers

Cluster of Percentage of positive Staining intensity Percentage of positive Staining intensity

differentiation cells % (SD) 0 1 2 3 cells % (SD) 0 1 2 3
CD1a 4.3 (1.2) 19 7 0 0 4.7 (1.3) 19 8 0 0
CD3 38.8 (4.1) 9 13 3 1 42.6 (3.6) 9 14 3 1
CD4 17.2 (3.2) 9 13 3 1 21.9 (2.7) 9 13 4 1
CD8 38.1 (4.5) 8 11 7 0 40.9 (4.8) 8 14 4 1
CD68 4.2 (1.6) 25 1 0 0 3.3* (1.8) 27 0 0 0
0 ¼ negative; 1 ¼ weak, 2 ¼ moderate, 3 ¼ strong.
*Significantly less CD68þ cells were expressed in the smokers group (Chi-square ¼ 8.046, P ¼ .018). Otherwise, the two groups did not show any
significant difference in the percentage of positive cells or staining intensity of any of the CDs studied.

Statistical analysis
The Minitab 17 statistical software package (Minitab
Inc., State College, PA, USA) was used for statistical
analysis. Both parametric and nonparametric statistical
tests were applied. Age correlation to other clinical
variables and to immunostaining results was analyzed
by using two-sample t test and analysis of variance test,
respectively. All other demographic and clinical data
(categorical data) and their correlation to immuno-
staining results were analyzed using the Chi-square test.
Statistical significance was set at a value of P < .05.
RESULTS
The study population comprised a total of 53 patients
with OLL: 27 smokers and 26 nonsmokers (Table I).
Gingival involvement in OLP was significantly
associated with older age. The mean age of patients
with gingival involvement was 67  8 years,
compared with a mean age (57  12 years; P ¼ .008)
of patients with OLL but without gingival involvement.
Of particular note was the presence of twice as many
patients with a plaque-like pattern in the smokers group
as in the nonsmokers group (14 vs. 7); however, this
finding did not reach statistical significance (P ¼ .064).
Interestingly, an equal number of patients (n ¼ 7) in
both groups presented with a reticular pattern of OLL as
the only clinical pattern observed (see Table I).
There was significantly less association with clinical
symptoms for: positive smoking status (Chi-square ¼
11.103; P ¼ .001); reticular pattern alone (Chi-
square ¼ 5.428; P ¼ .02); and plaque-like type OLP,
whether alone or in combination with other patterns
(Chi-square ¼ 5.052; P ¼ .025). Gingival involvement
and a desquamative gingivitis pattern were both
significantly associated with more clinical symptoms
(Chi-square ¼ 22.957 and 18.982, and P ¼ .001 and
.002, respectively).
With regard to the medical history of the study
population, the most commonly reported medical con-
ditions were hypertension (five in the nonsmokers
group, two in the smokers group) and diabetes (four in
the nonsmokers group, one in the smokers group)
without significant differences between the two groups.
Analysis of the correlation between clinical variables
and IHC results showed that when OLL are confined to
the buccal mucosa, they are significantly associated
with higher expression (Chi-square ¼ 6.859; P ¼ .032)
and higher epithelial infiltrate (Chi-square ¼ 4.154; P ¼
.042) of CD3þ cells. An OLL reticular pattern alone
was significantly associated with a lower expression of
CD3þ cells (Chi-square ¼ 6.510; P ¼ .039). Compared
with other patterns, the presence of plaque-like OLL
were associated with a significantly higher CD1aþ cells
expression (Chi-square ¼ 7.045; P ¼ .03).
A positive smoking status was significantly associ-
ated with less expression of CD68þ cells (Chi-
square ¼ 8.046; P ¼ .018); otherwise, no significant
differences in staining extent, intensity, or intra-
epithelial infiltrate of the studied cells were found be-
tween smokers and nonsmokers. A summary of the IHC
results, presented in Table II, demonstrates the lack of
variability between the two groups except for the
reduced expression of CD68þ cells in the smokers
group. Representative images of the
immunohistochemistry reaction to the different CDs
investigated are presented in Figure 2.
DISCUSSION
In our study, we found the first experimental evidence
that smoking can influence the inflammatory infiltrate
in OLL, specifically by diminished expression of
macrophages (CD68þ cells). Additionally, the data
showed that the clinical presentation and location of
OLL are not only associated with different clinical
characteristics but also with differences in the IHC
features. For example, reticular pattern and plaque-like
OLL were associated with milder symptoms as well as
with a lower expression of CD3þ cells (reticular
pattern) and a significantly higher CD1aþ cells
expression (plaque-like).

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ORAL AND MAXILLOFACIAL PATHOLOGY OOOO
54 Alrashdan et al. January 2016

Fig. 2. Images showing immunohistochemical staining against CD1a, CD3, CD4, CD8, and CD68 in both nonsmokers (NS) and
smokers (S) groups (original magnification 100). Few positive cells (<5%) reacting to CD1a and CD68 are noted in both groups,
with significantly less CD68þ cells in the smokers group (P ¼ .018). Arrows are pointing at few scattered positive cells in the
CD68 NS image. Obviously more positive cells are seen reacting to CD3, CD4, and CD8 (T lymphocytes, helper, and cytotoxic
subpopulations) without significant differences between the two groups in terms of staining extent (percentage of positive cells),
staining intensity, or intraepithelial inflammatory infiltrate.

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OOOO
Volume 121, Number 1
Macrophages are cells of the innate immune system
involved in critical regulatory activities in both health
and disease. They are present in healthy oral mucosa
and in larger numbers during pathologic processes.11
Macrophages are classified as M1 (proinflammatory)
and M2 (anti-inflammatory) according to the functions
of their effectors.12 It has been postulated that M1
macrophages present in OLP might contribute to
exacerbation of manifestations of the disease through
the production of proinflammatory agents, such as
tumor necrosis factor alpha and interleukin-1b.13
Due to their phagocytic capacity, macrophages
perform necessary housekeeping functions in healthy
tissues, clearing away apoptotic cells and debris to
maintain tissue homeostasis.14 They participate as
central sentinels in the detection of infection and tissue
damage and importantly in immune surveillance (the
process by which the immune system identifies
cancerous and/or precancerous cells and eliminates
them before they can cause harm).15 A protective role
in tumorigenesis has been described for M1
macrophages, which activate tumor-killing mecha-
nisms and antagonize the suppressive activities of
tumor-associated macrophages, myeloid-derived sup-
pressor cells, M2 macrophages, regulatory macro-
phages, and immature myeloid cells, which have all
been shown to suppress adaptive tumor-specific immune
responses and to promote tumor growth, invasion,
metastasis, stroma remodeling, and angiogenesis.16
Recent evidence shows significant alterations in
macrophages phenotypes and functions during carci-
nogenesis, including a shift from antitumor to protumor
functions.16-18 Our finding that smoking reduced mac-
rophages expression in OLL may represent one of these
alterations, and this may, at least theoretically, promote
malignant transformation in OLL.
To date, the possible interaction between smoking and
OLL has not been well addressed. In the few reports that
attempted to explore this interaction, the main focus was
to study the correlation between tobacco smoking and the
malignant potential of OLL from an epidemiologic
perspective (i.e., smoking habits among patients with
OLP who developed oral cancer).6,7,19 Based on studies
that did not exclude smokers from OLP populations,
epidemiologic data showed that smoking habits are less
common among patients with OLP who developed oral
squamous cell carcinoma compared to both matched OLP
controls and patients with oral squamous cell carcinoma
but without OLP.19,20 Therefore, there is lack of epide-
miologic evidence on malignant transformation of OLL
driven by smoking.
Apart from the possible interaction between OLL and
known carcinogens, it has been suggested that the
malignant transformation of OLL may be related to, or
dependent on, a series of molecular stimuli originating
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ORIGINAL ARTICLE
Alrashdan et al. 55
in the inflammatory infiltrate, rather than on external
carcinogens.9 Chronic inflammation has been shown to
be associated with various types of cancer,21,22 and it
has been widely reported that the inflammatory infiltrate
can be a strong risk factor for cancer development in
ulcerative colitis, atrophic gastritis, and Barret esopha-
gitis, among other diseases.23,24
Mignogna et al.9 proposed that OLP represents an oral
model for such a link between chronic inflammation and
malignancy and described, with relevant evidence, the
possible role of each type of inflammatory cells in the
malignant transformation of OLP. According to this
theory, macrophages, mast cells, lymphocytes, and
fibroblasts can contribute to the process of
carcinogenesis in OLP by secreting cytokines,
chemokines, matrix metalloproteinases, and regulated
upon activation normal T cell expressed and secreted
(RANTES) molecules, which have been postulated to
cause DNA damage; bypass p53 tumor suppression
function; induce immortalization; and influence the
growth, survival, angiogenesis, and invasion of cancer
cells. To the best of our knowledge, this theory has not
been experimentally validated.
An early IHC study assessing lymphocytic infiltrate in
OLP showed this to be composed mainly of T cells, and
the majority of T cells within the epithelium and adjacent
to damaged basal keratinocytes to be activated CD8þ
lymphocytes.25 CD8þ T cells were also co-localized
with apoptotic keratinocytes in OLP lesions.26 The
majority of intraepithelial lymphocytes in OLP are
CD8þ cytotoxic T cells, and most lymphocytes in the
lamina propria were reported to be CD4þ helper T
cells.25,27,28 The dominant role of CD8þ T cells and
CD4þ T cells in OLP pathogenesis was further
confirmed by the expression of the chemokines CCR5
and CXCR3, known to be selectively expressed by such
cells in OLP infiltrate.29 Activated cytotoxic CD8þ T
cells are thought to trigger keratinocytes apoptosis via
the release of tumor necrosis factor alpha.8
The role of antigen-presenting cells in OLL has also
been well investigated.8,13 Among these cells, Langer-
hans cells have gained much attention, and a high
number of Langerhans cells have been shown to be
present in the basal layer of the epithelium in OLP.30
These cells reside in the suprabasal layers of the
stratified epithelium of the skin and oral mucosa, and
their function is to capture and present antigens.31 It
is thought that Langerhans cells play an important
role in the pathogenesis of OLP by presenting
antigens to the T lymphocyte through class II major
histocompatibility complex molecules, introducing not
only an initial sensitivity to the antigen (primary
immune response) but also a subsequent secondary
immune response, which causes the appearance of the
clinical signs of disease.13
ORAL AND MAXILLOFACIAL PATHOLOGY
56 Alrashdan et al.
The present study assessed the key inflammatory
cells in OLL (T lymphocytes [CD3þ], helper [CD4þ]
and cytotoxic [CD8þ] subpopulations, Langerhans
cells [CD1aþ], and macrophages [CD68þ]) and
showed that the clinical variables of OLL (site and
pattern) may influence the components of the inflam-
matory infiltrate to some degree. The higher expression
and epithelial infiltrate of T lymphocytes (CD3þ) in the
buccal mucosa, compared with other sites, may reflect a
classic OLP inflammatory process in a classic site. The
Koebner phenomenon, known to exacerbate OLP,2 may
also help explain the overexpression of T lymphocytes
as a sign of trauma-induced exacerbation of inflam-
mation in this particular site. However, this finding did
not apply to the reticular pattern, which showed a lower
expression of CD3þ cells, indicating a milder inflam-
matory process that was not influenced by trauma.
In agreement with our findings, several other studies
have shown that different OLP clinical types show
different expression of components of the inflammatory
infiltrate.32,33 Rodriguez- Nunez et al.32 suggested that
such differences may even reflect different mechanisms
of the disease in different OLP forms. However, this
conclusion has not been experimentally proven. In a
recent IHC study performed on OLP histologic
specimens, it has been shown that a significant
correlation exists between OLP red lesions (erosive
and bullous) and the expression of CD3þ, CD4þ,
CD56þ, and CD8þ cells. However, white lesions
(reticular and plaque-like) showed no correlation and
showed lower expression of these cell populations.34
The finding in the present study that plaque-like
OLL are significantly associated with higher expres-
sion of Langerhans cells (CD1aþ) indicates a poten-
tially different behavior of the inflammatory infiltrate in
this particular OLL pattern. According to the hypothesis
that Langerhans cells, as key antigen presenting cells,
are able to activate CD4þ helper T cells and subse-
quently CD8þ cytotoxic T cells, causing more
recruitment and migration of such T cells,8 it would be
expected to find a concomitant increase in T-cell
expression when the Langerhans cells are
overexpressed (as in the case with plaque-like OLP);
however, our results did not show this to be true.
It is well known that the buccal mucosa is the most
common site for OLP, followed by the tongue and
gingivae, whereas the palate, floor of the mouth, and
upper lip are uncommon sites of OLP.5,35,36 In a series of
700 patients with OLP, gingival lesions were diagnosed
in 48% of cases, usually associated with diffuse oral
involvement, without age predilection.37 A separate
study that involved 62 Brazilian patients with OLP
found that age and gender did not affect the clinical
presentation of OLP, including gingival involvement,
which affected 29% of the cases.38 Our results,
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OOOO
January 2016
however, indicated that OLL affecting the gingivae
were found in 17% of the patients and that they were
significantly associated with older age. Whether older
age can affect the clinical presentation of OLL, as we
have demonstrated, requires further epidemiologic
studies involving larger populations for confirmation.
OLL signs and symptoms can range from patients
being unaware of the diseasedwith lesions that are
completely asymptomatic, as commonly seen in retic-
ular OLLdto those experiencing mucosal sensitivity,
burning, and debilitating pain, classically with
erythematous and erosive/ulcerative OLL.2 Our findings
did not deviate from these clinical facts; white lesions
(reticular and plaque-like OLL) were associated with
less clinical symptoms, whereas patients with erythem-
atous and erosive OLL were more commonly symp-
tomatic. The hyperkeratosis commonly seen with white
OLL patterns (reticular and plaque-like) is likely to
provide protection for the mucosa from erosion and
ulceration, and hence less clinical symptoms are usually
associated with such OLL patterns.
CONCLUSIONS
We have shown that the inflammatory infiltrate in OLL
may show some variations, depending on the clinical
pattern of the disease as well as the intraoral sites
affected. Moreover, we have demonstrated that smok-
ing can alter the macrophage cellular component of the
inflammatory infiltrate in patients with OLL. Consid-
ering the crucial role of macrophages, especially in
immune surveillance, it seems plausible that the inter-
action between smoking and OLL may lead to changes
in the inflammatory process, thus altering the risk of
malignant transformation. However, further studies
with larger populations are required to investigate other
potential interactions between the inflammatory infil-
trate in OLL and external factors (in particular smoking
and alcohol drinking) and how such interactions may
influence the clinical behavior and malignant potential
of OLL.
We would like to thank Mr. Dennis Rowler and Ms. Emily
Szycman for their technical support during the immunohis-
tochemistry procedures.
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Reprint requests:
Michael McCullough, BDSc, MDSc, PhD
Department of Oral Anatomy
Medicine and Surgery
Melbourne Dental School
The University of Melbourne
720 Swanston Street
Melbourne 3010
Australia
m.mccullough@unimelb.edu.au