1. Epithelium- Role in defense
Dr. Vamsi Lavu
Dept of Periodontics
Sri Ramachandra Dental College
2. • In the past- gingiva was considered as a
“physical barrier”.
• Current findings in periodontal research-
Paradigm shift to
“deterministic role in host immune
response to bacterial challenge”
4. Review of some studies
Authors Study Result
Takata et al 1988 Colloidal gold labelled
Concavalin A- JE of rats
JE cells- phagoctic activity.
Possible first line defense
Krisanaprakornkit S et al
1998
HBD-1 expression in
gingival epithelial cells
culture and tissue- PCR
Constitutive expression in
epithelial cells but not
fibroblasts
Tonetti et al 1998 IL-8 mRNA and ICAM-1
expression in JE of healthy
gingiva
Gradient in expression of
ICAM -1 and IL-8
Seguier S et al 1999 Immunohistologic and
morphometric analysis for
cytotoxic lymphocytes in
gingivitis
Detected Intra-epithelial
lymphocytes which are CD
3+ve and CD 8 +ve.
5. Review of studies- Contd
Authors Study Result
Miyauchi M et al 2001 Expression of TNF alpha, Il-
1 by JE cells-
(Immunohistochemistry)
Demonstrated expression
of the Pro-inflammatory
cytokines
Ren L et al 2004 Expression profile of LBP in
gingival epithelium
(peptide and m-RNA)
LBP expression decreases
from coronal to apical
aspect (ie) from sulcular to
junctional epithelium.
Found in cytoplasm of
granular and cornified
layers.
Re-think of the concept of the epithelium as a
physical barrier.
6. • The role of epithelium in active defense is
outlined below:
– Expression of toll-like receptors.
– Production of antimicrobial peptides.
– Antigen presenting cells.
– Expression of cell adhesion molecules.
– Pro-inflammatory cytokine and chemokine
production.
8. • Toll gene – discovered in 1985- implicated in
development- Drosophila.
• Drosophila- Toll protein- mediated immune
response to fungal infection.
• Toll like receptors- recognize specific
conserved microbial components- “Pathogen
associated molecular patterns”.
• Constitute an important part of innate
immune response.
12. TLR’s in gingival epithelium
• Kisumoto Y et al 2004, constitutive expression
of TLR-2- TLR-6 and TLR-9.
• Ckhara et al 2002, low levels of TLR-4
expression .
• TLR-2 distribution in the epithelium (Kisumoto
et al 2004), TLR-2 expression is denser in the
spinous layers than basal epithelial layers
13. • Asai et al 2001, demonstrated that Il-8 and
MCP-1 production by gingival epithelial cells in
response to P.gingivalis fimbriae and Staph
aureus peptidoglycan was mediated through
TLR-2.
• TLR-3 and TLR 9 have also been demonstrated
and these are specific for viruses.
14. SUMMARY:
TLR’S ARE EXPRESSED ON GINGIVAL EPITHELIAL
CELLS.
GINGIVAL EPITHELIUM ACTS AS ALERT SENTINELS-
PRODUCE CHEMOKINES REQUIRED FOR INNATE
IMMUNE CELL MIGRATION WHEN THE TLR’S ON
EPITHELIAL CELLS RECOGNIZE PAMP’S.
15. Why is there no continuous inflammation
of the gingiva??
• Oral gingival epithelium- exposed to several
hundred bacterial species.
• How is it that gingiva mediates the response
to microbes through TLR signalling and avoids
severe chronic inflammation – “Question of
Debate”.
16. • Hajishengallis G et al, Genco RJ et al 2004,
Mutukuru et al 2005 have suggested that there is
a development of tolerance in the gingival
epithelium that is mediated through down-
regulation of TLR expression and inhibition of
intracellular signalling in the oral gingival
epithelium.
• This hypothesis is based on the findings in the gut
epithelium which have a similar environment
(Abreu MT 2003).
18. • Four different families of anti-microbial
peptides:
– Alpha defensins.
– Beta defensins.
– Cathellicidins.
– Saponins.
Others include calprotectin, histatin 5, hepcidin,
lactoferrin, cathepsin G, adrenomedullin.
19. Defensins
• Best characterized antimicrobial proteins in
humans.
• Localized to 8p23 chromosome.
• Beta defensins are phylogenetically older.
20. Structure of defensins:
• They have an amphipathic organization- allows
clustering of the hydrophobic , hydrophilic and the
cationic sections of the molecule into separate areas.
• Some antimicrobial peptides are linear in structure but
assume this organization after entering the microbial
membrane, whereas others maintain this structure
with anti-parallel beta sheet arrangement that is
constrained by the number of disulfide bonds.
21. • Basket like structure – due to combination of
disulfide crosslinks with amphiphilic nature of
the peptides.
• The top of the basket consists of N and C
terminal residues giving a polar positively
charged region.
• The hydrophobic regions are grouped at the
bottom of the basket.
23. • Alpha and beta defensins differ in their
location of di-sulfide bonds between cysteine
residues:
– Beta defensins- (C1-5,C2-4,C3-6).
– Alpha defensins- (C1-6, C2-4, C3-5).
The alpha defensins are found as full protein in the
neutrophils and are termed as HNP1-4.
The HNP-5,6 are found in the Paneth cells of the gut
as the proform
25. Mechanism of the antimicrobial peptide action
• SHAI MATSUZAKI- HUANG MODEL
• Bacterial cell wall – negative charge due to
phospholipid layer with negatively charged
phosphate towards the outer surface.
• The defensins have a positive charged end (top of
the basket) which is attracted towards the
negatively charged phospholipid layer.
• The initial electrostatic attraction is then followed
by displacement of the membrane lipids in a
wedge like manner.
27. • The mechanisms after this stage probably differ
depending on the molecule and the bacterial cell
wall, resulting in a leakage of the cytoplasmic
contents due to a new osmotic equilibrium.
• In some cases the defensins have specific
intracellular targets such as inhibition of ATPase
actions and preventing chaperone assisted
protein folding (KragolG et al 2001).
28. Beta defensins
• Beta defensins are expressed in gingiva,
tongue, salivary glands, mucosa.
• Gingiva and oral mucosa express Beta
defensin 1,2,3 (Krisanaprakornkit et al 1998,
2000).
• hBD-1- constitutive.
• hBD-2- inducible.
29. Beta defensin expression:
• Associated with differentiation of the
epithelium.
Normal gingiva:
• mRNA expression is strong in the spinous layer
of the tissue while the peptides are detected
in the upper granular and cornified layer.
31. • The strongest expression is at the gingival margin,
adjacent to the region of plaque formation on the
tooth surface and in the inflammed sulcular
epithelium (Dale et al 2001).
• HBD-1 and hBD-2 are not detectable in the JE.
• Lui et al 2002, proposed that differentiation is a
pre-requisite for beta defensin expression in
epidermis. This appears to hold good in the oral
epithelium also as evidenced by the lack of
expression in the JE.
33. • HBD-2 is expressed in the normal and
inflamed human gingiva, unlike in other
epithelia such as skin, gut epithelium (Dale et
al 2001).
• Krisanaprakornkit et al 2000 have suggested
that this hBD-2 expression is in response to
oral commensals and it indicates a heightened
state of readiness.
34. Epithelial receptors for hBD-2 induction
Two families have been implicated:
• TLR family.
• PAR family- Proteinase activated receptors.
• TLR-2,4 have been shown to be involved in
bacterial sensing and intracellular signalling.
There is however no evidence for available for
TLR induced hBD expression.
35. PAR family:
• PAR’s are seven trans-membrane domain G
protein coupled receptors.
• PAR activation involves proteolytic cleavage of
the extracellular domain resulting in a new
amino terminus that acts as a tethered ligand
that binds to one of the extracellular loops of
the receptor (Coughlin and Camires 2003).
36. • PAR-2 is activated by various trypsin like enzymes
including mast cell tryptase, neutrophil
proteinase 3 as well as P.gingivalis proteinases.
(Lourbakes et al 1998).
• Chung WO, Dale A et al 2004, demonstrated the
involvement of PAR-1 receptor in upregulation of
hBD-2 mRNA expression in gingival epithelial cells
stimulated by P.gingivalis proteinases.
38. • They include
– bactericidal/ permeability increasing protein like
protein.
– Calprotectin.
– Adrenomedullin.
– The neutrophils migrating through the epithelium
also contribute Alpha defensins and LL-37
although they are strictly of not epithelial origin.
39. Bactericidal/permeability increasing protein like
protein
• BPIP like protein is an antibacterial protein with
selectivity for gram negative bacteria.
• BPIP like protein are either about 250 amino
acids in length or more than 450 amino acids.
Structure:
• It consists of BPIP domains BPI1 and BPI2.
• They have a disulfide bridge between cysteine
residues.
40. The different types of BPIP like protein found
include:
• Parotid secretory protein. (Madsen HO, Hjorth
JP, 1985).
• Palate, lung, nasal epithelium carcinoma
associated protein. (Wetson WM et al 1999).
• Bovine salivary protein. (Rajan GH et al 1996)
• von Ebner’s minor salivary gland protein.
41. Expression of BPIP like proteins by gingival
epithelium:
• Are expressed in human gingival keratinocytes
where their expression is regulated by the oral
bacterium P. gingivalis and the pro-
inflammatory cytokine tumor necrosis factor
alpha. (Shiba H et al 2005, Gorr et al
manuscript in prepn).
42. Functions of BPIP like protein:
• Parotid secretory protein structure: block the binding
of LPS to lipopolysaccharide binding protein and blocks
the LPS stimulated secretion of TNF alpha from
macrophages.
• PSP is also antibacterial to Pseudomonas aeruginosa.
• The other epithelial secreted BPIP like proteins also
have antimicrobial proteins.
43. Calprotectin:
• Also called calgranulin.
• Is a calcium and zinc binding protein also
referred to as S100 A8 and S100 A9. It is
constitutively expressed in cells of stratified
oral epithelia and in cultured gingival
epithelial cells (Ross & Herzberg, 2001).
44. MOA of calprotectin:
• Competition for zinc, a growth requirement for
many microbial species (Brandtzaeg et al 1995).
• Calprotectin expression confers protection from
bacterial binding and invasion and may
contribute to resistance of gingival cells to
invasion by P.gingivalis, a gram negative
periodontal pathogen (Nisapakultorn et al 2001).
45. Adrenomedullin:
• It is a multifunctional peptide that was initially
characterized for vasodilatory action.
• Allaker & Kapas 2003, have demonstrated that it
has anti-bacterial function against g+ve and g-ve
bacteria but not anti-fungal activity.
• It is constitutively expressed and secreted by oral
epithelial cells and expression is increased in
response to live oral bacteria, IL-1, TNF alpha
(Kapas et al 2001a, Kapas et al 2001b).
46. • Adrenomedullin has structural homology with
calcitonin gene related peptide and functional
homology with beta defensins.
LL-37:
• Is a cathellicidin with conserved pro-region
which keeps it inactive till proteases cleave
the protein after the protein is secreted
(Zanetti et al 2008).
47. Functions of LL-37:
• It is a chemoattractant for neutrophil, monocytes and T
cells (Chertov et al 1996).
• It also stimulates mast cells and alters macrophage gene
expression to upregulate chemokines and their receptors
resulting in greater responsiveness to the environment
(Scott et al 2000).
• LL-37 has the greatest activity against A.a and
Capnocytophaga species.
48. Alpha defensins :
• are relatively ineffective against most oral microbes
tested except C.ochracea and C.albicans.
• HNP 1-3 also inhibit HIV virus.
• Alpha defensins also are involved in modulation of
adaptive immune response by selectively attracting
naïve CD4 T cells and immature dendritic cells via a G
protein coupled receptor (Yang et al 2000).
49. Defensins- Link between innate and adaptive immunity
Alpha defensins
• Activate the classical complement pathway (Panyutich AV
et al 1994).
• Upregulate IL-8 production by epithelial cells which in turn
increase neutrophil recruitment. (Van Wetering S et al
1997).
• Are chemotactic for CD 8 and CD4/CD45 RA T cells and
immature dendritic cells (Chertov O et al 2000).
• In vitro they enhance the release of TNF alpha and IL-1
from activated monocytes (Chertov O et al 2000).
• Decrease VCAM-1 expression from TNF alpha activated
endothelium without affecting ICAM-1 expression (Chaly
YV et al 2000).
50. Defensins- Link between innate and adaptive immunity
Beta defensins
• Chemoattractant for immature dendritic
cells, CD 4/CD 45 RO (memory) T cells and CD
8 cells via binding to chemokine receptor
CCR6 (Yang D et al 1999).
52. Antigen presenting cells
Introduction
• They represent a unique family of cells that
are involved in antigen capture, processing
and presentation to the T cells.
• They represent a bridge between innate and
adaptive immunity.
54. Studies done in localization of dendritic cells in the
gingiva and the PDL with markers used
Study Dendritic cell &
markers
Tissues Disease
studied
Major finding
Cutler &
Jotwani 2004
Langerhans
cells
Immature
dendritic cell
Lysosomal
dendritic cell,
Mature
dendritic cell
Langerin, Cd1a
DC-LAMP, CD
83
Periodontitis Lysosomal
bearing
dendritic cells,
C 83 +ve,
mature
dendritic cells
engaged with T
cells
Cirrincione et
al 2002
Langerhans
cells
Co-stimulatory
markers
CD 1a, CD 80,
CD 86
MHC II, CD 54
Periodontitis DC are
abundant in
lamina propria
of pocket
epithelium,
expression of
MHC II
55. Jotwani et al Langerhans
cells , mature
and immature
dendritic cells
LAG,Cd1a,CD
83
Periodontitis,
gingivitis
Increased LC in
the epithelium
of
periodontitis
Seguier et al
2000
Langerhans
cells
CD1a Periodontitis,
gingivitis
Morph of LC
changed in
upper vs lower
epithelia of
gingivitis and
pditis.
Seguier et al
2000
Langerhans
cells
CD1a Periodontitis,
gingivitis,
healthy
LC decreased in
epithelia of
gingivitis and
pditis,
intraepithelial
lymphocytes
are increased
56. Jotwani et al
2001
Langerhans
cell, mature
and immature
Dendritic cell
LAG, CD 1a, CD
83
Periodontitis,
gingivitis
Increased Lc in
epithelium of
periodontitis
and CD 83+ve
dendritic cells
in lamina
propria
forming Oral
lymphoid
follicle
Lundqvist and
Hammarstrom
1993
Langerhans
cells
Cd1a,b,c Inflamed
gingiva
Mostly CD1a
cells found in
epithelium.
CD1b cells not
found, CD 1c
cells found in
basal
epithelium
57. Conclusion from the studies listed:
• CD1a langerhans cells are the primary leukocytes
involved in the response of oral epithelium to
infectious , dysplastic, atopic diseases.
• Langerhans cells have been shown to increase in
number in epithelium with gingivitis,
experimental gingivitis and periodontitis.
• The identity of the precursors of the langerhans
cells that infiltrate the oral/gingival epithelium is
unclear at present.
58. Concept of oral lymphoid follicle
• The mature dendritic cells play a role of in the
formation of the “Oral lymphoid follicle” or
lymphoid foci that develop inter-proximally
around the teeth in periodontitis.
• In gingivitis stage- migration of langerhans
cells or their progenitors homing to the
epithelium in response to the “pathogen
molecular patterns” from the oral biofilm.
61. Role in antigen recognition and capture
• The dendritic cells are localized to the gingival
epithelium where they play a role in recognizing
and capturing conserved sequences of the
microbes called as “pathogen associated
molecular patterns”.
• This is achieved by a an array of receptors that
the dendritic cells possess
– Toll like receptors.
– C-lectin receptors.
In addition the dendritic cells also possess phagocytosis
receptors for antibody and complement.
62. • Signal transduction is mediated by IL-1
receptor and MyD88 and ultimately results in
release of NF-kB which translocates to the
nucleus.
• This translocation is followed by activation of
genes involved in dendritic cell maturation
and upregulation of the expression co-
stimulatory molecules.
65. Conclusion : Role of epithelium in defense.
• Epithelium plays a deterministic role through:
– Production of defensins.
– Expression of TLR’s
– Through chemokine and cytokine production.
production.
– Tolerance when required.
– Dendritic cell influx and emigration.
66. References
1. Toll like receptors and their role in periodontal
disease. Mahanonda R and Pichyangkul.
Periodontology 2000, 2007.
2. Antimicrobial peptides in the oral environment:
Expression and function in health and disease.
Dale BA, Federicks LP. Curr issues Mol Biol 2005
July;7(2): 119-133.
3. Periodontal epithelium- A newly recognized role
in health and disease. Dale BA. Periodontology
2000,2002.
67. References (contd)
4. Human variability in innate immunity. Kinane DF
et al. Periodontology 2000,2007.
5. Oral mucosal dendritic cells and periodontitis.
Cutler and Teng. Periodontology 2000,2007.
6. Modulation of the innate immune response in
the periodontium. Douglas et al. Periodontology
2000,2004.
7. The junctional from health to disease. Bosshardt
and Lang. Critical reviews in Oral Biology and
Medicine. J Dent Res 2005;84(1):9-20.
68. ACKNOWLEDGMENT
• Professor R.Suresh, Dean, Head of
Department, Department of Periodontics, Sri
Ramachandra Dental College.
• All my senior staff and colleagues of my
department.
• My postgraduate students and undergraduate
students.
• The Management – for the excellent online
library facility.
69. Thank you
“The past is known, the
present is happening and
the future is unknown”
