Comparative immunohistochemical study of the presence of

mast cells in apical granulomas and periapical cysts:

Possible role of mast cells in the course of human
periapical lesions
Camila de Oliveira Rodini, DDS,
a
Aline Carvalho Batista, DDS, MSc,
b
and Vanessa Soares
Lara, DDS, MSc, PhD,
c
Sao Paolo, Brazil
UNIVERSITY OF SA

O PAOLO
Cells other than macrophages and lymphocytes have recently been shown capable of producing cytokines
and mediators. Among these are mast cells, a cell population now recognized for its immunoregulatory properties.
Little is known about the complex interactions between cells, cytokines, and other inammatory elements in periapical
lesions. The objective of this investigation was to determine the immunohistochemical pattern of expression of mast
cells tryptase in periapical lesions based on study of 20 apical granulomas and 20 periapical cysts. Microscopic
analysis revealed mast cells to be present in greater numbers in periapical cysts than in apical granulomas, and in
cysts were more numerous in regions of active inammation. Mast cells tended to be more common in the peripheral
regions of both periapical lesions, and were often found in close proximity to lymphocytes. These ndings lead us to
propose a functional relationship between these two cell populations that may facilitate elicitation of an immune
response contributory to the pathogenesis of periapical lesions. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod
2004;97:59-63)
Periapical lesions occur in response to chronic irritation
in periapical tissue, generally resulting from an infected
root canal. A great variety of bacterial antigens may
stimulate host immune responses, including antigen-
specic immunologic responses and nonspecic in-
ammatory reactions. Specic etiological agents of in-
duction, participating cell populations, and growth
factors associated with maintenance and resolution of
periapical lesions are incompletely understood.
Among the cells found of periapical lesions, mast
cells have been detected in the inammatory inltrates
of granulomas and cysts, suggesting a role for mast
cells in the inammatory mechanisms of these le-
sions.
1-7
The contributions of mast cells in host defense
as effector cells in innate immunity and in responses to
allergic, chemical, and biological factors such as mi-
croorganisms and parasites, has been extensively inves-
tigated.
8-11
There is increased awareness of the poten-
tial range of mast cell functions and interactions in the
immune response, including expanding understanding
of the role of mast cells in modulating immune re-
sponse humoral and cellular events. Although recog-
nized as a source of histamine, serotonin, and other
vasoactive amines believed to control vascular tone and
permeability,
12
mast cells also play important roles in
the immunopathology of immediate- and delayed-type
hypersensitivity reactions and in the pathogenesis of
chronic inammation, contributing to initiation and am-
plication of host defense mechanisms against bacterial
infections.
11,13
It has become clear that mast cells are
able to produce a wide range of mediators, creating
linkages between early mast cell activation and subse-
quent T-lymphocyte stimulation. Mast cells appear to
modulate CD8 T-cell proliferative responses and cy-
tokine production,
14-15
and to present antigens to T
cells in vitro.
11,16-19
Mast cells may also prime T cells in
vivo for efcient T-cell as well as antibody responses to
specic antigens.
11
In cell-mediated inammatory dis-
eases, including periapical lesions, inammatory reactions
driven by T cellmast cell interactions can be expected to
have a high degree of redundancy in terms of mediators.
19
This work was supported by grants from the Fundacao de Amparo a`
Pesquisa do Estado de Sao Paulo (FAPESP - 98/13274-0 and 00/
10427-1). We thank Fatima Aparecida Silveira (Department of Sto-
matology, Bauru Dental School, University of Sao Paulo) for excel-
lent technical support, Jose Roberto Pereira Lauris (PhD) for his help
in statistical analysis, and Paul Taylor Shafee for his English lan-
guage services in copyediting the manuscript.
a
Postgraduate Student of Oral Pathology, Department of Stomatology
(Pathology), Bauru Dental School, University of Sao Paulo, Bauru,
Brasil.
b
Postgraduate Student of Oral Pathology, Department of Stomatol-
ogy, Federal University of Goias, and Postgraduate Student of Oral
Pathology, Department of Stomatology (Pathology), Bauru Dental
School, University of Sao Paolo.
c
Department of Stomatology (Pathology), Bauru Dental School, Uni-
versity of Sao Paulo, Brasil.
Received for publication Jan 19, 2003; returned for revision Mar 4,
2003; accepted for publication Jun 27, 2003.
1079-2104/$ - see front matter
2004, Elsevier Inc. All rights reserved.
doi:10.1016/S1079-2104(03)00378-0
59
The objective of our investigation was to examine
the immunohistochemical expression and localization
of mast cell tryptase in apical granulomas and periapi-
cal cysts in order to enhance understanding of inam-
matory and immunological phenomena associated with
the evolution of periapical lesions.
MATERIAL AND METHODS
Tissue
Biopsy specimens from human patients were se-
lected from the les of the Anatomical Pathology Lab-
oratory, Department of Stomatology, Bauru Dental
School, University of Sao Paulo. A total of 20 cases
each of apical granuloma and periapical cyst were
included in this study. Cysts generally showed well-
dened cavities lined by stratied squamous epithelium
of variable thickness, with capsules displaying a mod-
erate to intense inltration of inammatory cells. Peri-
apical granulomas generally showed severe inltration
of inammatory cells and foamy macrophages, with no
evidence of epithelium lining. Samples were selected
based on microscopic examination of slides stained
with hematoxylin and eosin. We prioritized the most
recent cases and most representative lesions with re-
spect to integrity and tissue arrangement. Each sub-
jects rights were protected, with valid informed con-
sent obtained from each patient.
Immunohistochemistry
Sections, 3-m thick, were cut from tissue blocks,
collected on Silane-coated glass slides (DAKO, S3003,
Glostrup, Denmark), and processed in the standard
manner for immunohistochemistry, using an immuno-
peroxidase (avidin-biotin-peroxidase) method. Sections
were deparafnized via immersion in xylol and alcohol,
followed by incubation with 3% hydrogen peroxide
diluted with phosphate-buffered saline (PBS) solution
for 40 minutes. Sections were promptly incubated at
room temperature in 3% normal serum diluted with
distilled water for 20 minutes, and were sequentially
incubated in murine antihuman mast cell tryptase
(M7052; DAKO) monoclonal antibodies, diluted with
1% PBS-BSA (bovine serum albumin) at 1:3000, at
4C overnight. The sections were next washed with
PBS with Triton X-100 pa (Mallinckrodt, USA) and
incubated in biotinylated mouse anti-IgG (immuno-
globulin-G) (K0492; DAKO) antibodies in 1% PBS-
BSA for 60 minutes at room temperature. Sections
were next incubated in avidin-biotin complex (K0492;
DAKO) for 45 minutes at room temperature and were
incubated in a solution of 5-mg 3,3-diaminobenzidin
(D4293; SIGMA, USA) diluted with 10 mL of PBS
containing 180 mL of H2O2 (20 vol) for 5 minutes at
room temperature. Following washing with distilled
water, sections were counterstained with Mayers he-
matoxylin (Merck, Germany) for 5 minutes. Negative
controls consisted of sections in which primary anti-
bodies were omitted and replaced with either nonim-
mune murine serum (X 0910; DAKO) or with 1%
PBS-BSA.
Assessment and quantication of immunostaining
were analyzed by 2 investigators (C.O.R., V.S.L.). The
number of positively stained cells in periapical lesions
was counted in 10 consecutive microscopic high-power
elds (100) in a representative section of each spec-
imen. Mast cell immunostaining data were expressed as
mean count of positive cells. Quantication of positive
mast cells was calculated as the proportion of the total
number of inammatory mononuclear cells. Statistical
analysis was performed using the Mann Whitney non-
parametric test, where signicance was established as P
.05.
RESULTS
Quantitative microscopic analysis of the sections re-
vealed mast cells to be more numerous in periapical
cysts (n 20) than in apical granulomas (n 20) (P
.046). The mean standard deviation (SD) of tryptase-
positive mast cells was 22.30% 8.23% in periapical
cysts and 17.44% 4.34% in apical granulomas. Gen-
eral and qualitative microscopic analysis indicated mast
cells to be present in active inammatory areas as well
as in peripheral regions of both periapical lesions,
around nerve bers, and among polymorphonuclear
leukocytes, foamy macrophages, and broblasts. In the
apical cyst group, mast cells were also located just
beneath the cystic epithelium, in the connective tissue,
and in intraepithelial areas. We observed mast cells in
close contact with lymphocyte-like cells, rather than
adjacent to blood vessels, in both periapical cysts and
granulomas. Negative controls used for each immuno-
histochemical reaction did not show any staining, con-
rming the specicity of the procedure.
DISCUSSION
Expression of mast cells was evaluated using an
immunoperoxidase staining procedure with antitryptase
monoclonal antibodies, a technique considered highly
specic and sensitive in the detection of mast cells in
routinely processed tissues.
20
Tryptase is considered a
specic mast cell marker, based on reaction restricted
entirely to the granules of mast cells. The longstanding
procedure for mast cell identication based on
methachromatic staining for heparin is less reliable, as
the technique may stain cells such as macrophages and
broblasts due to released mast cell granules from
phagocytosis, and it may fail to stain immature mast
cells.
21
Based on immunohistochemical analysis, we
60 Rodini et al ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY
January 2004
observed periapical cysts to exhibit greater numbers of
mast cells than apical granulomas. This nding has
raised our interest concerning possible contributions of
mast cells and their mediators in chronic inammation,
as well as the functional relationship of mast cell and
immunocompetent cell populations in periapical le-
sions, as already reported in other immune cellmedi-
ated diseases.
19
The recent discovery that mast cells produce and
release a variety of multifunctional cytokines points to
new mechanisms by which mast cells might signi-
cantly inuence either IgE-dependent or IgE-indepen-
dent responses, extending their potential functions from
proinammatory effector cells to regulatory compo-
nents of the immune system, thus contributing to de-
velopment and amplication of specic and nonspecic
inammatory responses.
22-24
Mast cell activation has
been reported to induce T-cell migration either directly
by the release of exossomes
24
and chemokines such as
lymphotactin, interleukin-16 (IL-16), and MIP-1, or
indirectly by induction of adhesion molecule expres-
sion on endothelial cells.
12,25
Mast cells have a long life
span and can produce a large array of cytokines, re-
ecting possible effects of mast cells on T-cell surviv-
al.
19
A functional relationship between mast cells and T
lymphocytes has been suggested, as activated mast
cells are often found in close apposition to T cells in
some inammatory processes,
25,26
including apical
granuloma.
27
T-cellmast cell interactions have been
shown to be bidirectional, fullling mutually regulatory
and/or modulatory roles, including inuences on cellu-
lar processes such as growth, proliferation, activation,
migration, and antigen presentation.
11,16-19,28,29
Also,
T-cell-derived mediators, such as -chemokines, di-
rectly induce mast cell degranulation.
25
Our results are
in accordance with the concept of a functional relation-
ship between mast cells and T cells, as in both apical
granulomas and periapical cysts, mast cells reside in
close physical proximity to T cells rather than blood
vessels.
Concerning mast cellT-cell interactions, mast cells
can secrete Th2-type
14-15,27,30-34
and Th1-type
35,36
cy-
tokines, inuencing T-cell differentiation.
37,38
In both
mouse
38
and human systems, mast cells are able to
modulate proliferation and cytokine production re-
sponses of CD8 T cells.
14,39,40
Thus, mast cells may
be involved in generation of cytotoxic T cells
25,41
and
T-suppressor cells producing a negative feedback
mechanism (suppression) of lymphocyte immune re-
sponse.
27,30-32
We previously suggested participation of CD8
cells in cytotoxicity mechanisms in periapical lesions,
particularly in cysts, or immunosuppressive phenome-
na.
42
In the present study, we observed tryptase-posi-
tive cells in apical inammatory lesions, with localiza-
tion of positive cells in the zones of inammatory cell
accumulation including central areas of the granulomas
and sub-epithelial regions of periapical cysts. These
immunohistochemical results are similar to those pre-
viously observed when we used anti-CD8 antibodies,
42
although tryptase-positive cells were also noted in pe-
ripheral regions of periapical lesions. In comparing
current ndings to those in our previous study, we
suggest that the presence of mast cells in periapical
lesions may be related to the regulation of cellular
immune mechanisms driven by CD8 cells. However,
the exact nature of the immunoregulation involving
these cell populations remains to be established. Fur-
ther, mast cells in periapical inammatory lesions may
act as antigen-presenting cells for T lymphocytes. Sub-
sequent T-cell activation would lead to mast cell acti-
vation, producing both degranulation and release of
cytokines such as TNF-, with proinammatory and
prosecretory effects on mast cells and other cell types.
Interestingly, the mast cell is the only cell type identi-
ed from which TNF- is immediately released from
preformed stores within 10 to 20 minutes after chal-
lenge.
8-9,43
The presence of TNF- has been reported in
human periapical lesions.
7,44-47
Tissue effects of this
proinammatory cytokine include stimulation of oste-
oclastic bone resorption, increase of local vascular re-
sponse,
46
and promotion of chronic inammation in
human periapical lesions.
7
Other products of mast cells,
such as tryptase and metalloproteinase-9, may degrade
basement membrane structural proteins, resulting in
membrane breaks permitting access of T cells, includ-
ing CD8 cells.
19
Smith et al have indicated that mast cell production
and secretion of histamine may contribute to expansion
of the periapical cyst, as histamine vasoactive proper-
ties would result in release of plasmatic proteins.
4
Un-
der conditions of poor lymphatic drainage, released
plasmatic proteins would tend to diffuse into the lumi-
nal uid of periapical cysts, increasing osmotic pres-
sure and generating cyst expansion. Activation or sup-
pression of the immune response may also occur. Mast
cell release of prostaglandins during degranulation may
have a role in bone resorption, thus promoting cyst
growth. Teronen et al observed that mast cells were
typically degranulated when situated at the periphery of
odontogenic cysts near the surrounding bone, suggest-
ing that mast cells and mast cell tryptase contribute to
bone resorption during cyst growth.
48
We also observed
tryptase-positive cells in the periphery of the periapical
lesions, including periapical cysts.
The interactions of mast cells and other immune cells
involved in the pathogenesis of inammatory periapical
lesions have not been fully elucidated. Thus, it has been
Rodini et al 61 ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY
Volume 97, Number 1
difcult to determine the role of mast cells and their
products in the expansion of the cystic lesions. In vivo
studies in our laboratory are in progress to dene the
participation of mast cells and their products in exper-
imentally induced periapical lesions in rats. As knowl-
edge of in vivo modulatory mechanisms of mast cells in
periapical lesions is increased, perhaps clinical appli-
cation of agents blocking mast cell secretion will pro-
vide a new therapeutic strategy.
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Reprint requests:
Vanessa Soares Lara, DDS, MSc, PhD
Rua Servio Tulio Carrijo Coube, 3-33, apto 91-C, Edif cio
TRIANON
17012-632
vanessa@fob.usp.br
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